- Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to metarhodoposin
Phototransduction in flies is the fastest known G protein-coupled signaling cascade, but how this performance is achieved remains unclear. This study investigated the mechanism and role of rhodopsin inactivation. The lifetime of activated rhodopsin (metarhodopsin = M*) was determined in whole-cell recordings from Drosophila photoreceptors by measuring the time window within which inactivating M* by photoreisomerization to rhodopsin could suppress responses to prior illumination. M* was inactivated rapidly (τ ~20 ms) under control conditions, but ~10-fold more slowly in Ca2+-free solutions. This pronounced Ca2+ dependence of M* inactivation was unaffected by mutations affecting phosphorylation of rhodopsin or arrestin but was abolished in mutants of calmodulin (CaM) or the CaM-binding myosin III, NINAC. This suggests a mechanism whereby Ca2+ influx acting via CaM and NINAC accelerates the binding of arrestin to M*. These results indicate that this strategy promotes quantum efficiency, temporal resolution, and fidelity of visual signaling (Liu, 2008).
This study exploited the bistable nature of invertebrate rhodopsins to measure the lifetime of activated metarhodopsin in Drosophila. The approach measures the time window during which photoreisomerization of M* can suppress the response to light. The relative lack of overlap of the R and M spectra in UV opsins has been exploited by recording from the UV-sensitive photoreceptors in Limulus median ocelli. This strategy was adapted for Drosophila by using flies engineered to express the UV opsin Rh3; the effective M* lifetime was found to be very short (τdec ≈20 ms) under physiological conditions. Strikingly, M* lifetime was prolonged ~10-fold in the absence of Ca2+ influx, indicating that M-Arr2 binding is Ca2+ dependent and that M* lifetime is the rate-limiting step in response deactivation in Ca2+-free solutions. Further experiments led to proposal of a mechanism for Ca2+-dependent M* inactivation by Arr2, mediated by calmodulin (CaM) and myosin III NINAC (Liu, 2008).
Photoisomerization of rhodopsin (R) by short-wavelength light (480 nm for Rh1 or 330 nm for Rh3) generates active metarhodopsin (M*). M* continues to activate Gq until it binds arrestin (Arr2) or is reconverted to R by long-wavelength illumination (570/460 nm). M is serially phosphorylated by rhodopsin kinase (RK), but this is not required for M* inactivation or Arr2 binding. CaMKII-dependent phosphorylation of Arr2 at Ser366 and photoreconversion of Mpp to Rpp is required for the release of Arrp. Phosphorylation of Arr2 also prevents endocytotic internalization of M-Arr2. In Arr2S366A or mutants defective in CamKII, photoreconversion fails to release Arr2. Finally, Rpp is dephosphorylated by the Ca-CaM-dependent rhodopsin phosphatase (rdgC) to recreate the ground state, R. The results suggest that under low-Ca2+ conditions Arr2 is prevented from rapid binding to M* because it is sequestered by NINAC or a NINAC-regulated target; however, Ca2+ influx acting via CaM rapidly releases Arr2. Each microvillus contains ~70 Arr2 molecules, ensuring rapid quenching of M* once they are free to diffuse. The role of M* phosphorylation remains uncertain but may be involved in Rh1 internalization by the minor arrestin, Arr1 (Liu, 2008).
Ca2+ dependence of M* lifetime had not previously been demonstrated in an invertebrate photoreceptor, and the consensus from data in Drosophila suggested no obvious mechanism by which M* lifetime could be regulated by Ca2+. The finding that M* inactivation is strongly Ca2+ dependent prompted a re-examination of possible roles of Rh1 and Arr2 phosphorylation as well as CaM. Although M* lifetime remained strongly Ca2+ dependent in mutants defective in rhodopsin and arrestin phosphorylation, the Ca2+ dependence of M* inactivation was effectively eliminated in hypomorphic cam mutants. This requirement for CaM appeared to be mediated by the myosin III NINAC protein, since the Ca2+ dependence of M* inactivation was effectively abolished in both the null ninaCP235 mutant and an allele (ninaCKD) in which CaM levels in the microvilli were unaffected. NINAC, which is the major CaM-binding protein in the photoreceptors, has long been known to be required for normal rapid response deactivation (Porter, 1993b), but the mechanistic basis remained unresolved. These results now strongly suggest that it is specifically required for the Ca2+- and CaM-dependent inactivation of M* by Arr2 (Liu, 2008).
How might NINAC regulate the Ca2+-dependent inactivation of M*? A clue comes from the finding that Arr2 levels were substantially reduced in ninaC mutants. After taking this into account, the lack of Ca2+ dependence of M* inactivation in ninaC mutants was in fact associated with a very pronounced acceleration of response inactivation under Ca2+-free conditions. This was most clearly revealed in ninaCKD, which appears to be specifically defective only in Ca2+-dependent M* inactivation and does not show the additional response defects of the null ninaC phenotype (e.g., Hofstee, 1996). This suggests a disinhibitory mechanism whereby Ca2+-dependent inactivation of M* may be achieved, at least in part, by the NINAC-dependent prevention of Arr2-M* binding under low-Ca2+ conditions. Specifically, it is suggested that in Ca2+-free solutions, or in the low-Ca2+ conditions prevailing during the latent period of the quantum bump under physiological conditions, Arr2 in the microvilli is predominantly bound to NINAC or a NINAC-regulated target, thus restricting its access to M*. However, following Ca2+ influx, CaCaM would bind to NINAC, causing NINAC to release Arr2, which, as a soluble protein, could then rapidly diffuse to encounter and inactivate M* (Liu, 2008).
Interestingly, a recent study reported that NINAC can interact with Arr2 in a phosphoinositide-dependent manner (Lee, 2004a). This interaction was described in the context of a role of NINAC in light-induced translocation of Arr2, which was reported to be disrupted in ninaC mutants. However, involvement in translocation was challenged by a subsequent study reporting that Arr2 translocation was unaffected in ninaC mutants (Satoh, 2005). It will be interesting to see whether the Arr2-NINAC interactions described by Lee (2004a) reflect a role in the CaCaM- and NINAC-dependent inactivation of M* reported in this study (Liu, 2008).
It has long been known that responses under Ca2+-free conditions decay ~10-fold more slowly than in the presence of Ca2+. The current results establish that the inactivation of M* by Arr2 is the rate-limiting inactivation step in such Ca2+-free responses, with a time constant of ~200 ms in wild-type photoreceptors. Following inactivation of M* by photoreisomerization under Ca2+-free conditions, the response decayed with a time constant of ~80 ms. This also provides a unique and direct measure of the time constant(s) of the downstream mechanisms of inactivation, which presumably include GTP-ase activity of the Gq-PLC complex and removal of DAG by DAG kinase. It will be interesting so see whether Ca2+ also accelerates these inactivation mechanisms (Liu, 2008).
By contrast, the failure to accelerate response decay by overexpressing Arr2 in the presence of Ca2+ indicates that inactivation of M* is not rate limiting under physiological conditions. This can be understood by recognizing that the macroscopic kinetics are determined by the convolution of the bump latency distribution and bump waveform, the latter probably terminated by Ca2+-dependent inactivation of the light-sensitive channels. Until the Ca2+ influx associated with the quantum bump, the phototransduction machinery in each microvillus is effectively operating under Ca2+-free conditions. The results suggest that it is the Ca2+ influx associated with each quantum bump that promotes M* inactivation, and hence the timing of M* inactivation will be determined by the bump latency distribution and not vice versa. This leads to the, perhaps counterintuitive, concept that response termination is rate limited, not by any specific inactivation mechanism, but rather by the time course with which the cumulative probability of bump generation approaches 100% (Liu, 2008).
Clearly, rapid quenching of M* is essential to maintain the fidelity and high temporal resolution of phototransduction. In wild-type cells, an effectively absorbed photon generates only one quantum bump, but never (or extremely rarely) two or more; yet the multiple bump trains observed in arr2, cam, and ninaC mutants show that additional bumps are readily generated within 50–100 ms if M* fails to be inactivated. To prevent such multiple bumps without Ca2+-dependent feedback would require such a high rate of Arr2 binding that many M* molecules would be inactivated before they had a chance to activate sufficient G proteins to generate a quantum bump. This would result in an effective reduction in sensitivity, as is directly illustrated by the phenotype of p[Arr2] flies overexpressing Arr2. These show not only a 5-fold reduction in quantum efficiency. but also a reduction in bump amplitude and even an increase in bump latency, which is attributed to a decreased rate of second messenger generation. The mechanism proposed in this study provides an elegant solution to this dilemma. The analysis suggests that in the low-Ca2+ environment prior to Ca2+ influx, much of the Arr2 in the microvillus is bound to NINAC (or NINAC-regulated target), thus allowing M* to remain active long enough to activate sufficient G proteins to guarantee production of a full-sized quantum bump with high probability. Only after the bump has been initiated does Ca2+ influx accelerate the inactivation of M* by releasing Arr2, thus ensuring that only one bump is generated. This strategy is complemented and enabled by the ultracompartmentalization afforded by the microvillar design, which ensures that the Ca2+ rise is both extremely rapid and largely confined to the affected microvillus (Liu, 2008).
- Protein Interactions
- Regulatory arrestin cycle secures the fidelity and maintenance
of the fly photoreceptor cell
Excitation of fly photoreceptor cells is initiated by photoisomerization of rhodopsin to metarhodopsin, the active form of rhodopsin that stimulates signaling via G-proteins. Fly metarhodopsin is thermostable, does not bleach, and does not regenerate spontaneously to rhodopsin. For this reason, the activity of metarhodopsin must be stopped by an effective termination reaction. In contrast, there is also a need to restore the inactivated photopigment to an excitable state in order to keep a sufficient number of photopigment molecules available for excitation. The following findings reveal how these demands are met. The photopigment undergoes rapid phosphorylation upon photoconversion of rhodopsin to metarhodopsin and an efficient Ca2+ dependent dephosphorylation upon regeneration of metarhodopsin to rhodopsin. Phosphorylation decreases the ability of metarhodopsin to activate the guanine nucleotide-binding protein. Binding of 49-kDa arrestin (Arr2) further quenches the activity of metarhodopsin and protects it from dephosphorylation. Light-dependent binding and release of 49-kDa arrestin from metarhodopsin- and rhodopsin-containing membranes, respectively, directs the dephosphorylation reaction toward rhodopsin. This ensures the return of phosphorylated metarhodopsin to the rhodopsin pool without initiating transduction in the dark. Assays of rhodopsin dephosphorylation in the Drosophila retinal degeneration C (rdgC) mutant, a mutant in a gene previously cloned and predicted to encode a serine/threonine protein phosphatase, reveal that phosphorylated rhodopsin is a major substrate for the RdgC phosphatase. It is proposed that mutations resulting in either a decrease or an improper regulation of rhodopsin phosphatase activity bring about degeneration of the fly photoreceptor cells (Byk, 1993)
- Arrestin binding determines the rate of inactivation of the G protein-coupled receptor rhodopsin in vivo
G protein-coupled receptor inactivation is a crucial feature of cellular signaling systems; this process determines the catalytic lifetime of the activated receptor and is necessary for response termination. Although previous work has indicated a class of models in which several sequential steps are required for receptor inactivation, the rate-limiting event is still unclear. In this paper, a theory is developed that describes the kinetics of inactivation of the G protein-coupled receptor rhodopsin based on the rate of arrestin binding and the theory is tested using a combination of genetic and electrophysiological techniques in Drosophila photoreceptors. The theory quantitatively describes the inactivation kinetics of activated rhodopsin in vivo and can be independently tested with molecular and spectroscopic data. The results demonstrate that the rate of arrestin binding determines the kinetics of receptor inactivation in vivo and thus is the event that controls signal amplification at the first step of this G protein-coupled transduction cascade (Ranganathan, 1995).
Two arrestin genes, arrl and arr2, have been isolated
in Drosophila (Smith, 1990; Hyde, 1990; LeVine, 1990; Yamada, 1990), both of which are expressed
in all photoreceptors (R1-R8) in the eye (Dolph, 1993) and whose protein products share extensive amino acid identity (51%). These arrestins have at least
partially redundant functions in phototransduction (Dolph, 1993), although the Arr2 protein is severalfold more
abundant than art1 (LeVine, 1990; Matsumoto, 1991). In Drosophila, as in most invertebrates, the inactive arrestin-bound metarhodopsin species is a
stable complex in the dark, but can absorb another photon
to isomerize to phosphorylated rhodopsin, thereby releasing
arrestin (Byk, 1993). Phosphorylated
rhodopsin is subsequently dephosphorylated to regenerate
functional receptor molecules (Ranganathan, 1995 and references therein).
The stoichiometric requirement of arrestin in vivo for the
inactivation of metarhodopsin was demonstrated through
the isolation and characterization of Drosophila mutants
defective in arrestin function (Dolph, 1993). Hypomorphic
alleles were isolated in both arrestin genes, and
electrophysiological analysis of these mutant photoreceptors
revealed that a significant loss of arrestin function
leads to defective metarhodopsin inactivation, resulting in
abnormally slow deactivation of the light response. Abnormal
metarhodopsin inactivation in arrestin mutants was
demonstrated by a reduced threshold for induction of a
pathological state of the Drosophila photoreceptor known
as the prolonged depolarizing afterpotential (PDA). A PDA
is a sustained photoresponse triggered in wild-type cells
by substantial photoconversion (>20%) of rhodopsin to
metarhodopsin. During a PDA, photoreceptors
are refractory to further light stimuli and are said to
be inactivated. A PDA can be terminated by the photoconversion
of metarhodopsin back to rhodopsin, indicating
that unregulated metarhodopsin activity sustains the afterpotential.
Thus, a PDA represents the reversible saturation
of metarhodopsin-inactivation mechanisms in the cell. The
analysis of arrestin mutant photoreceptors shows that
the amount of rhodopsin isomerization required to induce
a PDA matches the amount of arrestin in the cell. Thus,
the generation of excess metarhodopsin over free arrestin
represents the basis of the PDA, and arrestin is required
stoichiometrically for metarhodopsin inactivation in vivo (Ranganathan, 1995).
What is the functional significance of the binding of arrestin
for the rate of receptor inactivation? Indeed, the
current view of metarhodopsin inactivation presented
above suggests a multistep inactivation process, in which
one of several events may set the inactivation rate. For
example, phosphorylation of metarhodopsin, arrestin binding,
the dissociation of Galpha, or conformational changes
induced in metarhodopsin by any of these steps could
each represent rate-determining events. The resolution of
this issue has important functional consequences, since
the lifetime of activated rhodopsin determines the gain in
the first step of phototransduction and thus influences the
overall sensitivity of the photoreceptor cell. In addition,
the rate of metarhodopsin inactivation contributes to determining
the temporal resolution of phototransduction (Ranganathan, 1995).
The rate-limiting process for receptor inactivation would
therefore represent an important control mechanism driving
photoreceptor adaptation and deactivation.
This paper formalizes a model for metarhodopsin
inactivation in which the stoichiometric binding of
arrestin determines the receptor inactivation rate. This
simple bimolecular reaction scheme leads to specific predictions
about the dependence of the kinetics of receptor
inactivation on free arrestin concentrations. These predictions are fully supported by electrophysiological
measurements of metarhodopsin inactivation in dissociated
Drosophila photoreceptors, where cytosolic arrestin
levels are manipulated using both genetic and
physiological techniques. In addition, a further test of the
model through independent measurement of internal parameters
using molecular and spectroscopic techniques
demonstrates a good fit with the electrophysiological data. These results show that arrestin binding determines the
kinetics of metarhodopsin inactivation in vivo (Ranganathan, 1995).
- Calcium/calmodulin-dependent kinase II phosphorylates Arrestin 1
Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca2+ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, Arrestin 1 (Phosrestin 1), is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. The identity of the protein kinase responsible for the phosphorylation of phosrestin I was sought, to link the transmembrane signaling with the light-adaptive inactivation response. Type II Ca2+/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. Partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca2+/calmodulin-dependent kinase II; the kinase activity exhibits regulatory properties that are
unique to Ca2+/calmodulin-dependent kinase II, such as Ca2+ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca2+/calmodulin-dependent kinase II autoinhibitory domain. Ca2+/calmodulin-dependent kinase KII activity is present in Drosophila eye preparations. These results are consistent with the hypothesis that Ca2+/calmodulin-dependent kinase
II phosphorylates phosrestin I. It is concluded that Ca2+/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation (Kahn, 1997).
- A role for the light-dependent phosphorylation of visual arrestin
Arrestins are regulatory proteins that participate in the
termination of G protein-mediated signal transduction. The major arrestin in the Drosophila visual system, Arrestin 2 (Arr2), is phosphorylated in a light-dependent manner by a
Ca2+/calmodulin-dependent protein kinase and
has been shown to be essential for the termination of the visual
signaling cascade in vivo. Nine alleles of the Drosophila photoreceptor
cell-specific arr2 gene have been isolated. Flies carrying each of these
alleles undergo light-dependent retinal degeneration and display
electrophysiological defects typical of previously identified arrestin
mutants, including an allele encoding a protein that lacks the major
Ca2+/calmodulin-dependent protein kinase
site. The phosphorylation mutant has very low levels of phosphorylation
and lacks the light-dependent phosphorylation observed with wild-type
Arr2. Interestingly, the Arr2 phosphorylation mutant is
still capable of binding to rhodopsin; however, it is unable to
release from membranes once rhodopsin has converted back to its
inactive form. This finding suggests that phosphorylation of arrestin
is necessary for the release of arrestin from rhodopsin. It is proposed
that the sequestering of arrestin to membranes is a possible mechanism
for retinal disease associated with previously identified rhodopsin
alleles in humans (Alloway, 1999).
Although it has been known for quite some time that Arr2 is
phosphorylated in a light-dependent manner, it is unclear just
what role this phosphorylation serves. The invertebrate
phototransduction cascade results in an increase in intracellular
calcium. It has been proposed that the calcium- and light-dependent
phosphorylation of Arr2 acts as the signal to bind and inactivate
metarhodopsin, and, therefore, Arr2 phosphorylation serves to modulate
the inactivation of the signaling cascade. However, this study has found that arrestin is able to bind to activated rhodopsin (metarhodopsin) in the
absence of phosphorylation, arguing against this feedback-regulation model. Invertebrate Arr2 is a very basic molecule with a
pKa of ~8.7. This characteristic may allow
it to rapidly interact with an exposed acidic surface on activated
rhodopsin in the absence of any covalent modification. Instead,
posttranslational modification is required to remove arrestin from
rhodopsin. Phosphorylation of Arr2 when it is bound to rhodopsin may
trigger a conformational change or add negative charge that enables the
release of Arr2 (Alloway, 1999).
The finding that the phosphorylation of Arr2 is required for proper
function brings up an apparent discrepancy: previously, the C terminus
of Arr2 was determined to be nonessential. In a previous study (Dolph, 1993),
a truncated form of Arr2 that lacked the last 45 aa of Arr2, including
the serine at position 366, was generated; this mutated form of Arr2
was phenotypically normal. An identical situation occurs in the bovine
system, where deletion of the C terminus of arrestin yields a protein
that still binds to rhodopsin but has lost its binding specificity,
binding to both photoactivated and nonphotoactivated rhodopsin. As such, it is believed that the truncated form of Arr2 is not defective in
release from rhodopsin but instead binds indiscriminately to both the
active and inactive forms of rhodopsin. The truncation mutant is
phenotypically normal; it still has a higher binding affinity for
the active form of rhodopsin (Alloway, 1999).
In the missense alleles generated from the screen, Arr2 is found
associated with membranes under all light conditions. This finding
clearly explains the null phenotype of these alleles. Because rhodopsin
is approximately five times more abundant than arrestin, all of
the Arr2 becomes bound to membranes, and no soluble Arr2 is available
to bind and inactivate metarhodopsin. However, it is unclear whether
the titration of arrestin or the formation of arrestin/rhodopsin
complexes is the primary cause of retinal degeneration in these
alleles. Interestingly, many recently characterized human dominant
rhodopsin alleles that are associated with retinitis pigmentosa and
stationary night blindness also have defects in arrestin binding. These alleles show continuous activity of rhodopsin in
vitro; however, both in vitro and in vivo,
these aberrant rhodopsin proteins are constitutively bound to arrestin.
One possible mechanism for retinal degeneration in these dominant
alleles is the titration of arrestin caused by its increased affinity
for the aberrant rhodopsin proteins. In this way, photoreceptor cells
undergo degeneration because of insufficient levels of soluble arrestin
to quench newly formed metarhodopsin (Alloway, 1999).
- The formation of stable rhodopsin-arrestin complexes induces apoptosis and photoreceptor cell degeneration
Although many different mutations in humans and Drosophila cause retinal degeneration, in most cases, a molecular mechanism for the degeneration has not been found. This study demonstrates the existence of stable, persistent complexes between rhodopsin and its regulatory protein arrestin in several different retinal degeneration mutants. Elimination of these rhodopsin-arrestin complexes by removing either rhodopsin or arrestin rescues the degeneration phenotype. Furthermore, it is shown that the accumulation of these complexes triggers apoptotic cell death and that the observed retinal degeneration requires the endocytic machinery. This suggests that the endocytosis of rhodopsin-arrestin complexes is a molecular mechanism for the initiation of retinal degeneration. It is proposed that an identical mechanism may be responsible for the pathology found in a subset of human retinal degenerative disorders (Alloway, 2000).
This paper demonstrates the existence of a novel mechanism to explain the light-dependent retinal degeneration that is observed in a subset of Drosophila visual system mutants. Mutations in three distinct genetic loci, norpA, arr2, and rdgB, result in the light-dependent formation of stable rhodopsin-arrestin complexes. Elimination of either member of this complex rescues the retinal degeneration in each of the three genetic backgrounds. In addition, it is shown that the formation of these stable rhodopsin-arrestin complexes triggers apoptotic cell death. Furthermore, endocytosis is essential for inducing cell death in norpA mutants, suggesting that the internalization of the rhodopsin-arrestin complexes is an early step in the initiation of apoptosis of retinal photoreceptors. It is possible that the excessive endocytosis saturates a downstream cellular function (for example, saturation of the early endosome or depletion of an endocytic protein), which signals the cell to undergo programmed cell death. Alternatively, the endocytosis may block a signal that protects the cell from apoptosis (Alloway, 2000)
The inactivation of the Drosophila phototransduction cascade is an extremely rapid event. Drosophila photoreceptors can shut off the light-activated currents in less than a 100 ms following termination of the light stimulus. One way in which photoreceptors have evolved to do this is at the level of the G protein-coupled receptor rhodopsin. Immediately upon activation, rhodopsin is multiply phosphorylated on its C terminus, and this phosphorylation greatly increases its affinity for the abundant soluble protein arrestin. Invertebrate Arr2 is a very basic molecule with a pKa of ~8.7 and, therefore, has a high affinity for phosphorylated rhodopsin. Thus, this interaction of activated rhodopsin and arrestin occurs very rapidly, and the receptor is quickly inactivated. It is tempting to speculate that the posttranslational modifications of arrestin and rhodopsin are essential to eliminate these complexes. In such a model, it is necessary to phosphorylate Arr2, thereby making it less basic, as well as to dephosphorylate rhodopsin, thereby making it less acidic. Thus, the system is designed such that the influx of calcium activates CamKII, the kinase that phosphorylates arrestin, and rdgC, the phosphatase that dephosphorylates rhodopsin. Both of these steps are crucial, as evidenced by the fact that rdgC mutants and arr2(S366A) mutants both undergo rapid light-dependent retinal degeneration. Evidence suggests that, in the absence of these posttranslational modifications, stable rhodopsin-arrestin complexes persist in the cell and are instrumental in the pathology of retinal degeneration (Alloway, 2000)
The retinal degeneration induced by rhodopsin-arrestin complexes can be partially rescued by a temperature-sensitive allele of dynamin. This observation suggests that the rhodopsin-arrestin complexes are being removed from the photoreceptor rhabdomere by the endocytic machinery. A similar receptor internalization pathway occurs for the vertebrate β-adrenergic receptor. β-arrestin is involved in the inactivation and internalization of the β-adrenergic receptor. A small domain near the carboxyl terminus of β-arrestin directly interacts with clathrin and targets the β-adrenergic receptor-β-arrestin complex for internalization. This clathrin binding site is curiously missing in the visual arrestins. However, in spite of the absence of the clathrin-interacting motif, these complexes seem to have the ability to interact with the endocytic machinery. One attractive model is that visual arrestin serves as an AP2-like adaptor, just like β-arrestin, but recruits clathrin by a different mechanism. The question of whether the endocytic proteins are recognizing motifs in rhodopsin or arrestin awaits further studies. Many models have been proposed to explain retinal degeneration in humans, including constitutive activity of the phototransduction cascade, improper trafficking of photoreceptor cell components, and defects in the recycling of rhodopsin. The results described in this study implicate the endocytic pathway in retinal degeneration and suggest that excessive endocytosis can be a trigger for apoptotic cell death (Alloway, 2000)
The internalization of rhodopsin-arrestin complexes via receptor-mediated endocytosis could partly explain the membrane association of arrestin in certain mutant backgrounds. The enhanced membrane affinity of arrestin could in part be due to the rhodopsin-arrestin complexes rapidly interacting with the endocytic machinery. It is possible that, once the complex is associated with clathrin cages, the arrestin would be locked in the membrane-associated state. In support of this model, it has recently been demonstrated that the unphosphorylated form of arrestin directly interacts with clathrin in vitro. Therefore, it is possible that the role of arrestin phosphorylation is to block interactions with clathrin, and any mutant background that fails to phosphorylate arrestin yields complexes due to clathrin interactions. However, it is still essential that a stable rhodopsin-arrestin complex be formed initially to allow for the assembly of the endocytic proteins (Alloway, 2000)
A large number of mutations in the human rhodopsin gene have been isolated that are responsible for retinal disease. Interestingly, there are several mutations in human rhodopsin that form stable rhodopsin-arrestin complexes. These include a mutation in a highly conserved lysine in the seventh transmembrane domain that has been shown to cause autosomal-dominant retinitis pigmentosa. This mutant form of rhodopsin is found both in vivo and in vitro to be constitutively phosphorylated and tightly bound to arrestin. It had been hypothesized that the complexes between rhodopsin and arrestin may be instrumental in the retinal degeneration process. Possibly, as in Drosophila, these stable human rhodopsin-arrestin complexes are removed from the rod outer segments by the endocytic machinery, and this excessive endocytosis is the direct cause of apoptotic retinal degeneration (Alloway, 2000)
One question yet to be addressed is why photoreceptor cells have a mechanism for eliminating rhodopsin-arrestin complexes if, under nonpathological conditions, these complexes do not persist in the cell. One possible explanation is that arrestin has a secondary function to eliminate defective rhodopsin molecules from the photoreceptor cell. A rhodopsin molecule that becomes photochemically damaged may render the receptor nonfunctional or constitutively active. The presence of a constitutively active rhodopsin molecule could potentially be very detrimental and, therefore, necessitates its removal from the photoreceptor cell. Presumably, any rhodopsin molecule that becomes constitutively active will bind arrestin and generate a stable rhodopsin-arrestin complex. The stabily bound arrestin would target the dysfunctional rhodopsin molecule for endocytosis and degradation. In this manner, arrestin could function as a surveillance protein, eliminating defective rhodopsin molecules from the photoreceptor cell. However, in certain mutant backgrounds, the regulation of rhodopsin-arrestin complex formation is defective, and a large number of complexes are formed. The increased endocytosis of these complexes initiates apoptosis and retinal degeneration (Alloway, 2000).
- A molecular pathway for light-dependent photoreceptor apoptosis in Drosophila
Light-induced photoreceptor apoptosis occurs in many forms of inherited retinal degeneration resulting in blindness in both vertebrates and invertebrates. Though mutations in several photoreceptor signaling proteins have been implicated in triggering this process, the molecular events relating light activation of rhodopsin to photoreceptor death are yet unclear. This study uncovers a pathway by which activation of rhodopsin in Drosophila mediates apoptosis through a G protein-independent mechanism. This process involves the formation of membrane complexes of phosphorylated, activated rhodopsin and its inhibitory protein arrestin, and subsequent clathrin-dependent endocytosis of these complexes into a cytoplasmic compartment. Together, these data define the proapoptotic molecules in Drosophila photoreceptors and indicate a novel signaling pathway for light-activated rhodopsin molecules in control of photoreceptor viability (Kiselev, 2000).
A combination of genetic and biochemical methods was used to reveal a novel light-dependent but G protein-independent signaling pathway regulating photoreceptor cell viability in Drosophila. In addition to its established function in inactivation of metarhodopsin (M), Arr2 acts as a clathrin adaptor protein to mediate the internalization of Arr2-M complexes. The analysis of rdgC306 mutants (that are unable to dephosphorylate metarhodopsin and display light-dependent retinal degeneration) shows that the Arr2-dependent internalization process is necessary for photoreceptor cell apoptosis and that the triggering condition for apoptosis is the long-term accumulation of Arr2-M-p complexes in an internal compartment. These findings are in strong agreement with work that show that formation and internalization of stable Arr2-M complexes are associated with photoreceptor apoptosis in Drosophila. This study shows that mutations in several phototransduction genes show Arr2-dependent retinal degeneration, suggesting that the Arr2-dependent internalization pathway is likely to be a common mechanism for triggering apoptotic photoreceptor cell death (Kiselev, 2000).
How does this pathway function in wild-type photoreceptor cells and how is apoptosis suppressed under normal physiological conditions? Two phosphorylation events control this process: (1) the phosphorylation of M promotes apoptosis through an unknown mechanism downstream of the internalization process and the RdgC phosphatase counteracts this process by efficient dephosphorylation of M, and (2) the phosphorylation of Arr2 suppresses apoptosis by disrupting clathrin interaction and preventing internalization of the receptor complexes. The net flux of the proapoptotic Arr2-M-p complexes through the internalization pathway is therefore determined by the quantity of metarhodopsin created upon light exposure, the activity of the RdgC phosphatase, and the phosphorylation status of Arr2. Interestingly, both the activity of the RdgC phosphatase and the phosphorylation of Arr2 are upregulated by the light-dependent increase in intracellular calcium that occurs during visual signaling in Drosophila photoreceptors. Thus, phototransduction appears to promote the survival of photoreceptor cells by reducing the accumulation of internalized Arr2-M-p complexes (Kiselev, 2000).
These results help clarify a number of unexplained and mysterious observations in the study of the photoreceptor signaling in Drosophila. For example, mutations that eliminate the phosphorylation domain of either rhodopsin or arrestin have no effect on the sensitivity or kinetics of light transduction. Nevertheless, these phosphorylation events occur rapidly upon light activation and require the investment of substantial metabolic resources since rhodopsin and arrestin are among the most abundant of photoreceptor proteins. It is suggested that these phosphorylation events are dedicated regulatory processes that apply primarily to the control of cell viability. The large commitment of metabolic energy in these steps presumably highlights the concept that like all cells, photoreceptors need to suppress the latent apoptotic machinery for survival (Kiselev, 2000).
How can internalized Arr2-M-p complexes trigger apoptosis? Several studies support for the idea that internalized, arrestin-bound GPCRs can trigger additional signaling events. For example, β-arrestin1 recruits c-Src to form three-protein complexes with activated β2 adrenergic receptors, and mediates the internalization of the entire complex through direct interaction with clathrin. Upon internalization, the complex triggers stimulation of mitogenic signaling through activation of the Ras-MAP kinase pathway. Cell survival has been shown to be promoted by this pathway through both suppression of the apoptotic machinery and transcriptional activation of prosurvival factors, and β-arrestin- and receptor internalization-dependent activation of MAP kinases is reported to be the basis for the antiapoptotic effects of substance P. Thus GPCRs can trigger a second round of signaling through arrestin-mediated internalization and may generally promote the survival of cells through activation of mitogenic signaling. One possibility is that Arr2-mediated internalization of dephosphorylated metarhodopsin in Drosophila photoreceptors plays a similar role in triggering a prosurvival signaling pathway and that phosphorylated M-Arr2 complexes may trigger the apoptotic machinery by disruption of this process (Kiselev, 2000).
The conservation of arrestin-mediated internalization between Drosophilarhodopsin and mammalian β receptors highlights the similarity in functional mechanisms throughout the GPCR superfamily. The stimulus-dependent phosphorylation at the carboxyl terminus is no exception; this property is shared by most GPCRs and suggests that RdgC-like receptor phosphatases may also be found in other signaling systems. Two mammalian homologs of RdgC, PPEF-1 and PPEF-2, have been cloned that show tissue distributions consistent with a role in several sensory signaling processes. Indeed, PPEF-2 is found exclusively in the retinal rod photoreceptors and in the pineal gland, although the role of this protein in regulating function of vertebrate rhodopsin is not yet established. However, retinal degeneration in transgenic mice expressing a constitutively active form of rhodopsin (K296E) does not result from excessive stimulation of the visual signaling cascade; instead the mutant rhodopsin is found in a constitutively phosphorylated form, tightly bound to arrestin molecules. The corresponding mutation in human rhodopsin is associated with one form of retinitis pigmentosa, suggesting that stable phosphorylated GPCR-arrestin complexes may also be proapoptotic in humans (Kiselev, 2000).
An interesting issue of the apoptotic mechanism in rdgC306 photoreceptors is the long but apparently reliable time delay in the commitment to cell death. For example, DPP analysis of a population of rdgC306 flies exposed to light shows no individuals with retinal degeneration until the fourth day, but then nearly all animals show complete degeneration over the next 2 days. Such fidelity in the timing of apoptosis in many independent animals is reminiscent of many human age-dependent macular degenerations in which affected individuals show a similar age of onset of symptoms, and argues that the timing of degeneration must be a well-regulated feature of the commitment process and not a simple, randomly distributed event. Since formation and internalization of the Arr2-M-p complexes proceeds with a time course of hours, and commitment to apoptosis requires days, it is concluded that the mechanism controlling the decision for cell death must reside in the multivesicular bodies (MVB) that represent the stable pool of internalized membranes in Drosophilaphotoreceptors. The detailed characterization of commitment to apoptosis in rdgC306 photoreceptors should provide insight into understanding the factors controlling programmed timing of apoptosis (Kiselev, 2000).
- Loss of the phospholipase C induces massive endocytosis of rhodopsin and arrestin in Drosophila photoreceptors
A subset of visual transduction mutants in Drosophila melanogaster induce the formation of stable complexes between rhodopsin and arrestin. One such mutant is in a visual system-specific phospholipase C (PLC). The rhodopsin/arrestin complexes generated in PLC mutants induce massive retinal degeneration. Both arrestin and rhodopsin undergo light-dependent endocytosis in a PLC mutant background. Interestingly, the internalized rhodopsin is rapidly degraded, but the arrestin is fully stable. The data are discussed with respect to mechanisms of arrestin-mediated endocytosis and human retinal disease (Orem, 2002).
- Light adaptation through phosphoinositide-regulated translocation of Drosophila visual arrestin
Photoreceptor cells adapt to bright or continuous light, although the molecular mechanisms underlying this phenomenon are incompletely understood. This paper reports a mechanism of light adaptation in Drosophila, which is regulated by phosphoinositides (PIs). Light-dependent translocation of arrestin is defective in mutants that disrupt PI metabolism or trafficking. Arrestin binds to PIP(3) in vitro, and mutation of this site delays arrestin shuttling and results in defects in the termination of the light response, which is normally accelerated by prior exposure to light. Disruption of the arrestin/PI interaction also suppresses retinal degeneration caused by excessive endocytosis of rhodopsin/arrestin complexes. These findings indicate that light-dependent trafficking of arrestin is regulated by direct interaction with PIs and is required for light adaptation. Since phospholipase C activity is required for activation of Drosophila phototransduction, these data point to a dual role of PIs in phototransduction (Lee, 2003).
A curious but longstanding observation is that visual arrestin undergoes a dramatic light-dependent translocation from the inner segment to the outer segment of rods and cones. However, the function of this dynamic movement has not been described. Drosophila visual arrestin, Arr2, also undergoes light-dependent shuttling between the cell bodies and the phototransducing portion of the photoreceptor cells, the rhabdomeres (Kiselev, 2000). This observation has provided the potential for using a genetic approach to address two unresolved issues: (1) the mechanisms regulating this movement and (2) the function for this light-driven translocation. Due to the slow time course of the light-induced movements, which occur over a few to many minutes, an interesting possibility is that the trafficking of arrestin could contribute to long-term adaptation, since the concentration of arrestin has been proposed to limit response termination Ranganathan, 1995). One well-known illustration of long-term adaptation is experienced upon entering and leaving a darkened room, such as a movie theater. Interestingly, this delay occurs over a similar time course as the light-dependent translocation of visual arrestins (Lee, 2003).
Consistent with the proposal that the regulated movement of arrestin contributes to slow adaptation, it was found that a reduction in the rate of Arr2 translocation has a major impact on a light-dependent component of the photoresponse. In wild-type, it was found that the rate of termination of the photoresponse is significantly faster in flies that have had prior exposure to light; however, this adaptation feature is virtually eliminated in arr2 null mutant flies. Of particular significance, termination of the ERG response is much slower in flies that have defects in the light-dependent movement of Arr2. Based on these findings indicating that light-dependent movement of Arr2 contributes to long-term adaptation in Drosophila, it is proposed that dynamic movements of mammalian visual arrestin may contribute to long-term adaptation in humans, in addition to other established mechanisms, such as chromophore regeneration (Lee, 2003).
Transducin also undergoes a light-driven translocation between the rod outer and inner segments, and this movement out of the outer segment is correlated with a reduction in the amplitude of the photoresponse. Similar light-dependent movement of the Gqα occurs in fly photoreceptor cells, and this translocation is dependent on the presence of the Gβγ. However, the effect of this translocation on adaptation has not been addressed. Recently, light-regulated translocation of the TRPL channel has been suggested as a novel mechanism for reducing the sensitivity to increasing intensities of light. Therefore, Drosophila appears to have at least two long-term light-adaptation mechanisms: light-dependent movement of Arr2 into the rhabdomeres for increasing the speed of termination of the photoresponse and light-dependent movement of TRPL into the cell body for enabling the photoreceptor cells to adjust the amplitude of their response to background light (Lee, 2003).
A second central question concerning the light-driven changes in the spatial distribution of Arr2 concerns the underlying mechanism. Several observations support the conclusion that the movements of Arr2, to and from the rhabdomeres, are regulated by PIs. The rate of Arr2 translocation is much slower as a result of alterations in the levels of expression of gene products, such as a PI-transfer protein (RDGB), CDS, and PTEN, that affect the metabolism or distribution of PIs. Furthermore, Arr2 binds to PIs in vitro, and mutations in this binding site causes translocation defects. In addition, endocytosis of β arrestin is mediated in vitro through interaction with PIs, though such a mode of regulation has not been demonstrated in vivo. In this report, evidence is provided that the movement of visual arrestin is regulated by PIs in photoreceptor cells in both directions, in and out of the rhabdomeres (Lee, 2003).
An issue raised by the current experiments is the identity of the PI or IP that interacts with and regulates the movement of Arr2 in vivo. It seems more likely that Arr2 binds to PIs than IPs, since the shuttling defects, which are observed in mutants affecting the PI-transfer protein or cds, are similar to those resulting from mutation of the PI/IP binding site in Arr2. PIP3 is a prime candidate for regulating Arr2, since the IC50 is lowest for PIP3. Furthermore, overexpression of the phosphatase that hydrolyzes PIP3 (PTEN) results in impairment of Arr2 translocation to the rhabdomeres but not shuttling to the cell bodies. These results support the conclusion that PIP3 facilitates shuttling of Arr2 to the rhabdomeres but also indicate that PIP3 does not affect movement of Arr2 from the rhabdomeres to the cell bodies. Given that translocation of Arr2 is disrupted by mutation of the PI binding site in Arr2, it is proposed that another PI, which remains to be identified, is required for this latter movement. The defect in Arr2 shuttling to the cell bodies in the PTEN null might result from an increased level of PIP3, which competes with another PI required for movement to the cell bodies (Lee, 2003)
The demonstration that translocation of Arr2 is regulated by PIs addresses a lingering question concerning potential roles of PIs in photoreceptor cells. The Drosophila visual transduction cascade is among the most intensively studied GPCR cascades. During the last 30 years, many proteins and mutations have been identified that perturb PI signaling; however, the targets and mechanisms directly regulated by PIs have not been previously described (Lee, 2003).
The regulation of Arr2 shuttling by PIs occurs on the order of a few to many minutes. This is in contrast to the millisecond time scale, which operates in the activation of phototransduction. Although the specific activation mechanism involved in Drosophila phototransduction remains elusive, it is established that it depends on a PLCβ (NORPA). Thus, PLC-mediated hydrolysis of PIP2 leads to rapid activation of the light-sensitive channels through the millisecond generation of PIP2 metabolites or reduction in PIP2 levels. Since adaptation occurs over a much slower timescale, regulation of this latter phenomenon exclusively by direct effects of second messengers on protein activities might be too rapid. Rather, regulation of adaptation by the translocation of signaling proteins provides a mechanism whereby changes in second messengers, such as PIP3, result in delayed effects on the magnitude and the kinetics of signaling. Therefore, PIs appear to have the capacity to serve a dual role in activation and adaptation by modulating the activities and localization of signaling proteins (Lee, 2003).
A major unresolved issue in mammalian vision is the function of PIs in rods and cones, since cGMP rather than lipid second messengers mediate activation of mammalian phototransduction. This question arises in part from the observation that several enzymes regulating PIs, including p110 PI3-kinase and DAG kinase, are activated in mammalian photoreceptor cells in a light-dependent manner. Furthermore, rods and cones express homologs of many eye-enriched proteins that function in Drosophila phototransduction. These include a PLCβ4 and M-rdgB2. However, the functions of these genes in rods and cones have not been identified, despite the generation of mouse knockouts (Lee, 2003).
It is proposed that PIs are excellent candidates for regulating the intracellular translocation of mammalian photoreceptor proteins in general and visual arrestin in particular. Consistent with this proposal are the observations that mammalian visual arrestin undergoes a light-dependent translocation, which appears to occur through an active mechanism rather than via passive diffusion. Moreover, mammalian visual arrestin binds to IPs in vitro, although an interaction with PIs was not tested. If mammalian visual arrestin binds PIs, then disruption of PI metabolism may interfere with adaptation in rods and cones, similar to the defects in arr23K/Q, rdgB, cds, and PTEN mutant flies. Thus, it would be interesting to reevaluate the PLCβ4 and m-rdgB2 knockout mice for effects on arrestin translocation and light adaptation (Lee, 2003).
Previous reports have shown that stable Arr2/rhodopsin complex formation leads to retinal degeneration in norpA or rdgC flies (Alloway, 2000; Kiselev, 2000). Removal of the arr2 gene in a norpA or rdgC background partially suppresses the photoreceptor cell death. This partial suppression could be due to elimination of Arr2/rhodopsin complexes, reduction in endocytosis of rhodopsin, or disruption of some other Arr2 function. In this work, Arr2/rhodopsin binding and PI-regulated trafficking of Arr2 were uncoupled by expressing Arr23K/Q, which is defective in movement but not rhodopsin binding. Since the retinal degeneration in norpA is largely rescued in arr23K/Q flies, these results suggest that apoptosis in norpA results from endocytosis of Arr2/rhodopsin complexes rather than a defect in Arr2/rhodopsin binding. This conclusion is further supported by the finding that there was even greater suppression of the norpA degeneration in an arr23K/Q than in an arr25 null background (Lee, 2003).
It is possible that endocytosis of stable arrestin/rhodopsin complexes may contribute to certain types of retinal degenerations in humans, as appears to be the case in Drosophila. If so, the findings in the current report that retinal degeneration is suppressed by interfering with the PI/Arr2 interaction raise the intriguing possibility that application of drugs that suppress PI production in the rods and cones or PI binding to visual arrestin may be an effective approach to suppress certain types of human retinal dystrophies (Lee, 2003).
- Light-dependent translocation of visual arrestin regulated by the NINAC myosin III
To address whether NINAC is required for light-dependent trafficking of Arr2, the major visual arrestin, Arr2 spatial distribution was examined in wild-type and null ninaC mutant flies (ninaCP235) at several time points after light stimulation. In dark-adapted wild-type and ninaCP235 flies, Arr2 is dispersed throughout the photoreceptor cells, with a greater overall proportion of the Arr2 pool in the cell bodies than in the rhabdomeres. After a 5 min exposure of wild-type flies to blue light, which stably converts rhodopsin to light-activated metarhodopsin, Arr2 is concentrated in the rhabdomeres. However, in ninaCP235 flies, Arr2 is not concentrated in the rhabdomeres, even after stimulating the flies for 1 hr with light. Elimination of NINAC does not have a global effect on rhabdomeral localization, since the spatial distributions of other rhabdomere-enriched proteins are similar in wild-type and ninaCP235 photoreceptor cells. An exception is calmodulin; NINAC is the major retinal calmodulin binding target, and elimination of NINAC results in instability of calmodulin in both the cell body and rhabdomeral compartments (Porter, 1993a; Porter, 1995). In contrast to calmodulin, the overall concentration of Arr2 is unaffected in ninaCP235 photoreceptor cells (Lee, 2004a).
To address the requirements for the individual NINAC isoforms for light-dependent shuttling of Arr2 into the rhabdomeres, immunostaining was performed on photoreceptor cells obtained from flies that expressed only p174 (ninaCΔ132) or p132 (ninaCΔ174) (Porter, 1992). After a 5 min pulse of blue light, the relative concentration of Arr2 in the ninaCΔ132 rhabdomeres increased, although to a smaller extent than in wild-type. Following exposure of ninaCΔ132 to blue light for 1 hr, Arr2 was enriched in the rhabdomeres. Thus, the kinetics of Arr2 trafficking in ninaCΔ132 are retarded rather than eliminated as in the ninaCP235 null mutant. Light-induced movement of Arr2 into the rhabdomeres is not impaired detectably in ninaCΔ174; however, p174 appears to contribute to some extent to Arr2 translocation as elimination of both proteins in ninaCP235 causes a more severe phenotype than disruption of p132 alone. These data indicated that p132 is the NINAC isoform most critical for light-dependent trafficking of Arr2 into the rhabdomeres. In contrast to these results, elimination of p132 has no effect on the dark-associated retrograde movement of Arr2 from the rhabdomeres to the cell bodies (Lee, 2004a).
The Arr2 movement could be due to a requirement for either the protein kinase or myosin domain. However, it could not be tested whether mutations in the myosin domain disrupt Arr2 translocation since deletion of this domain or point mutations in conserved residues, which are critical for the activity of other myosins, cause instability of the NINAC proteins (Porter, 1993b). Therefore, to determine whether the protein kinase domain was required, the corresponding lysine in NINAC (residue 45), which is required for ATP binding in other kinases, was changed to an arginine. It was found that both NINAC isoforms are expressed at the same levels in the transgenic flies, ninaCK45R, as in wild-type. In addition, on the basis of electroretinograms (ERGs), which measure the summed light responses of all retinal cells, there is no apparent defect in activation or termination of the photoresponse. Of importance here, light-dependent translocation of Arr2 and long-term adaptation are indistinguishable between ninaCK45R and wild-type flies. These data indicate that the protein kinase domain is dispensable for trafficking of Arr2 (Lee, 2004a).
The translocation of Arr2 is required for long-term light adaptation (Lee, 2003). Therefore, ninaC mutants that show defects in Arr2 trafficking should display reductions in light adaptation. To test this proposal, electroretinogram recordings, were performed. In wild-type flies, termination of the light response is relatively slow in flies that have been dark adapted for 4 hr, much faster after a 10 min pre-exposure to light, and further accelerated after 1 hr of prior light exposure. In contrast, it was found that ninaCP235 flies display a defect in long-term adaptation; acceleration of the rate of termination of the photoresponse by pre-exposure to light is much less pronounced than in wild-type. The data further support the conclusion that NINAC plays a crucial role in Arr2 translocation and in long-term light adaptation (Lee, 2004a).
To determine whether there is a correlation between long-term light adaptation and light-dependent translocation of Arr2 in ninaCΔ132 and ninaCΔ174 flies, ERGs were performed using the long-term adaptation paradigm described above. It was found that the rates of termination of the photoresponse were similar between wild-type and ninaCΔ174, while the rate of the termination of the photoresponse in ninaCΔ132, after a 10 min pre-exposure to light, was intermediate between wild-type and the null ninaCP235 flies. These results indicate that the rates of long-term adaptation are consistent with the light-dependent changes in Arr2 distribution in these flies (Lee, 2004a).
The preceding results raise the question as to how NINAC regulates the trafficking of Arr2. One possibility is that Arr2 can bind directly to NINAC. However, Arr2 and NINAC did not appear to associate directly in vitro or to co-immunoprecipitate from fly heads. Therefore, whether there might be an indirect interaction between NINAC and Arr2, which might be disrupted by the conditions used for the immunoprecipitations, was considered. To address this question, advantage was taken of the observation that the two NINAC isoforms are the major retinal calmodulin binding proteins, while Arr2 does not bind calmodulin. Therefore, whether Arr2 from fly heads might associate with calmodulin-agarose beads in a NINAC-dependent manner was tested. To perform this assay, pull-down assays were employed using extracts prepared from wild-type, ninaCΔ132, ninaCΔ174, and ninaCP235 fly heads. Arr2 was detected in the bound fraction from wild-type, ninaCΔ132, and ninaCΔ174 but not from extracts prepared from null ninaCP235 fly heads. Gα did not bind to calmodulin-agarose in either the presence or absence of NINAC, while a known retinal calmodulin binding protein, RDGC, bound to calmodulin-agarose in a NINAC-independent manner. These data indicated that Arr2 interacts with both isoforms of NINAC, since the binding to calmodulin-agarose is dependent on the presence of NINAC (Lee, 2004a).
Candidate molecules that could potentially contribute to Arr2/NINAC interactions and which are disrupted by amphiphilic detergents, such as TritonX, include PIs. PIs are especially attractive candidate intermediates linking Arr2 and NINAC, since Arr2 binds PIs and the Arr2/PI interaction is crucial for its light-dependent movement and for long-term light adaptation (Lee, 2003). If the Arr2/NINAC interaction requires PIs, then it should be disrupted using a derivative of Arr2 with three lysine to glutamine substitutions (arr23K/Q), which severely disrupt PI binding (Lee, 2003). It was found that using arr23K/Q extracts, Arr2 binding to the beads decreases dramatically (Lee, 2004a).
To provide a second assay to characterize the Arr2/NINAC interaction, pull-down assays were employed using NINAC proteins partially purified from fly heads and GST fused to the C-terminal portion of Arr2 (Arr2C-wt; amino acids 204-401), which contains the main Arr2/PI binding site (Lee, 2003). It was found that NINAC associates with GST-Arr2C-wt, but not to GST alone. Moreover, there was relatively little association between NINAC and GST-Arr2C3K/Q. Consistent with the reciprocal pull-down assays using NINAC bound to beads, the interaction between GST-Arr2C-wt and NINAC was disrupted by addition of TritonX-100 to the buffer. The combination of the above data suggested that PIs mediates the association between Arr2 and NINAC (Lee, 2004a).
To directly test whether PIs promote the Arr2/NINAC interaction, whether addition of PIs enhance binding of partially purified NINAC to GST-Arr2-wt was considered. Phosphatidylinositol-4,5-bisphosphate (PIP2) was used for these assays since PIP2 is among the most abundant PIs in vivo and is among the PIs that bind most efficiently to Arr2 (Lee, 2003). While 0.1% TritonX-100 disrupts the Arr2/NINAC interaction, it was found that addition of 30 μM or more PIP2 increases the association between both NINAC isoforms and GST-Arr2C-wt. Phosphatidylinositol (PIns, an uncharged phosphoinositide) or inositol hexaphosphate (IP6, a highly charged inositol phosphate without a lipid moiety) do not augment the Arr2/NINAC interaction (Lee, 2004a).
The data indicating that PIs promote the physical association of Arr2 and NINAC raise the possibility that NINAC also binds PIs. Therefore, binding assays were performed using PIP2 and PIP3 beads. It was found that both partially purified isoforms of NINAC bind to PIP2 and PIP3 beads but not to control beads. Since NINAC associated with Arr2, the PI/NINAC interaction could potentially have been mediated through Arr2. However, it was found that NINAC partially purified from either null arr25 or arr23K/Q flies bind effectively to either the PIP2 or PIP3 beads (Lee, 2004a).
- Rhodopsin kinase activity modulates the amplitude of the visual response in Drosophila: The GPRK1 might have a phosphorylation-independent role affecting Rh1/Arr2 binding
A feature shared between Drosophila rhodopsin and nearly all other G protein-coupled receptors is agonist-dependent protein phosphorylation. Despite extensive analyses of Drosophila phototransduction, a determination of the identity and function of the rhodopsin kinase (RK) has been elusive. This study provides evidence that G protein-coupled receptor kinase 1 (GPRK1), which is most similar to the ß-adrenergic receptor kinases, G protein-coupled receptor kinase 2 (GRK2) and GRK3, is the fly RK. GPRK1 is enriched in photoreceptor cells, associates with the major Drosophila rhodopsin, Rh1, and phosphorylates the receptor. As is the case with mammalian GRK2 and GRK3, Drosophila GPRK1 includes a C-terminal pleckstrin homology domain, which binds to phosphoinositides and the Gßgamma subunit. To address the role of GPRK1, transgenic flies were generated that expressed higher and lower levels of RK activity. Those flies with depressed levels of RK activity display a light response with a much larger amplitude than WT. Conversely, the amplitude of the light response is greatly suppressed in transgenic flies expressing abnormally high levels of RK activity. These data point to an evolutionarily conserved role for GPRK1 in modulating the amplitude of the visual response (Lee, 2004b).
Phosphorylation of Rh1 has been suggested to stabilize arrestin/Rh1 complexes (Kiselev, 2000). Therefore, an examination was made to determine whether the association and/or dissociation of the major arrestin (Arr2) with Rh1 was affected in ogprk1 and ogprk1K220R flies that exhibit high and low Rh1 phosphorylation, respectively. To characterize the Arr2/Rh1 interaction, an arrestin pelleting assay was used. As expected, in wild type most of the Arr2 binds to Rh1 after exposure to blue light, which stabilizes the active form of Rh1 and promotes the Rh1/Arr2 interaction. After an identical blue light treatment, slightly less Arr2 binds to Rh1 in ogprk1K220R than in WT flies. Surprisingly, the proportion of bound Rh1/Arr2 is even lower in ogprk1 than in either wild type or ogprk1K220R. Because the effects on the level of Rh1 phosphorylation are opposite in ogprk1 and ogprk1K220R flies, these data suggested that the GPRK1 might have a phosphorylation-independent role affecting Rh1/Arr2 binding. In contrast to these results, no differences between fly strains in the dissociation of the Rh1/Arr2 complexes promoted by exposure to orange light (Lee, 2004b).
- Suppression of constant-light-induced blindness but not retinal degeneration by inhibition of the rhodopsin degradation pathway
Continuous exposure to light, even at relatively low intensities, leads to retinal damage and blindness in wild-type animals. However, the molecular mechanisms underlying constant-light-induced blindness are poorly understood. It has been presumed that the visual impairment resulting from long-term, continuous exposure to ambient light is a secondary consequence of the effects of light on retinal morphology, but this has not been addressed. To characterize the mechanism underlying light-induced blindness, a molecular genetic approach was applied using Drosophila. The temporal loss of the photoresponse was found to be paralleled by a gradual decline in the concentration of rhodopsin. The decline in rhodopsin and the visual response are suppressed by a C-terminal truncation of rhodopsin, by mutations in arrestin, and by elimination of a lysosomal protein, Tetraspanin 42Ej (Sunglasses). Conversely, the visual impairment is greatly enhanced by mutation of the rhodopsin phosphatase, rdgC. Surprisingly, the mutations that suppressed light-induced blindness do not reduce the severity of the retinal degeneration resulting from constant light. Moreover, mutations known to suppress retinal degeneration did not ameliorate the light-induced blindness. These data demonstrate that the constant light-induced blindness and retinal degeneration result from defects in distinct molecular pathways. The results support a model in which visual impairment caused by continuous illumination occurs through an arrestin-dependent pathway that promotes degradation of rhodopsin (Lee, 2004c).
It is a longstanding observation that exposure of wild-type animals to constant light leads to retinal degeneration. In the current work, attempts were made to determine if constant light causes phototoxicity in Drosophila and, if so, whether the mechanism could be characterized further by using a genetic approach. As a sensitive and quantitative assay for phototoxicity, focus was placed on the effects of continuous light on the photoresponse by performing ERGs. It was found that flies maintained under continuous ambient light for many days gradually lose their visual response and eventually go blind (Lee, 2004c).
An important unanticipated finding was that the mechanism of light-induced blindness is distinct from that underlying light-induced retinal degeneration. Mutations such as arrestin2 and sun that suppress the light-induced blindness do not suppress the retinal degeneration resulting from exposure to the identical light conditions. In fact, a C-terminal deletion of rhodopsin (Rh1Δ356), which significantly reduces the severity of visual impairment by continuous illumination, actually accelerates the morphological damage resulting from constant ambient light. The lack of suppression of the low-light-induced retinal degeneration in wild-type flies is striking because the rh1Δ356 and the arr23K/Q alleles analyzed in this study greatly suppress certain genetically induced retinal degenerations (Lee, 2004c).
The results in the current study demonstrate that the loss of the photoresponse due to continuous light is not simply a secondary consequence of the retinal degeneration, which occurs in parallel with the visual impairment. Rather, light-induced apoptosis and blindness result from perturbations in different processes. This point is further illustrated by the findings that mutations in or overexpression of proteins known to suppress apoptosis in flies and other organisms do not ameliorate the light-induced blindness. Furthermore, although disruption of arrestin2 suppresses the visual defects caused by continuous illumination, mutations in arrestin actually cause retinal degeneration in the presence of cyclic light in both flies and the mouse (Lee, 2004c and references therein).
The combination of results presented in this study indicates that the low-light-induced blindness is due to a decline in rhodopsin levels. An indication that this is the case is that Rh1 is the only protein that declines in parallel with the visual impairment. Furthermore, mutations that either decrease or increase the severity of the Rh1 degradation cause a comparable suppression or enhancement of the visual impairment. However, genetic suppression of the light-induced decline in Rh1 levels does not reduce the retinal degeneration resulting from constant light. Thus, the decrease in the concentration of rhodopsin did not appear to underlie the retinal degeneration (Lee, 2004c).
A key question concerns the mechanism through which continuous low light causes a large reduction in Rh1 levels. The Rh1 degradation resulting from acute exposure to very bright light occurs through an arrestin-independent pathway, which remains to be defined. In contrast to these results, mutations in arrestin2 suppress the low-light-induced loss of Rh1. The differences in the mechanisms underlying bright- versus low-light-induced blindness are somewhat reminiscent of a recent mouse study demonstrating that retinal degeneration caused by extended exposure to low light is caused by a different mechanism than that for retinal degeneration caused by brief exposure to very bright light. However, the similarities between this recent report and the current study are limited because the light-induced loss of the ERG is not due to retinal degeneration. Although elimination of the trimeric G protein suppresses the retinal degeneration in the mouse, a hypomorphic allele of the Gαq does not reduce the severity of low-light-induced blindness in the fly, although the amplitude of the ERG is reduced as a result of a decreased concentration of the G protein (Lee, 2004c).
Arrestin was originally characterized as a regulatory protein that functions in the inactivation of rhodopsin and other G protein-coupled receptors (GPCRs). Arrestin has been shown to bind clathrin and, under some conditions, to participate in endocytosis of GPCRs. The interaction between rhodopsin and arrestin is usually transient and typically does not lead to endocytosis of rhodopsin. However, in mutant backgrounds that remove the rhodopsin phosphatase (RDGC) or phospholipase C (NORPA), which is the effector for the G protein, the Drosophila rhodopsin Rh1 is stably bound to arrestin, leading to endocytosis. Once internalized through endocytosis, GPCRs are either recycled to the plasma membrane or degraded. In the case of the norpA and rdgC mutant flies, it is not known if the internalized Rh1 is ultimately recycled or degraded. Moreover, stable rhodopsin/arrestin complexes had not previously been observed in wild-type photoreceptor cells (Lee, 2004c).
The results in this work support a molecular model in which constant light leads to blindness through a multi-step process initiated by the formation of stable rhodopsin/arrestin complexes and culminating with the loss of the light receptor, Rh1. A continuous low or moderate level of illumination, in the absence of any mutation, promotes the formation of stable rhodopsin/arrestin complexes. The concentration of Rh1 gradually declines, through a process involving the photoreceptor cell enriched lysosomal protein Sunglasses (Lee, 2004c).
The preceding pathway underlying low-light-induced blindness is supported by genetic evidence. Deletion of the C terminus of Rh1 prevents the formation of stable rhodopsin/arrestin complexes, which result from certain genetic perturbations that dramatically disrupt phototransduction. In wild-type flies exposed to constant illumination, the truncated Rh1 (Rh1Δ356) also interferes with the formation of Rh1/Arr2 complexes and greatly suppresses light-induced blindness. In addition, arrestin2 mutations suppress the light-induced decline in Rh1 and the impairment in the photoresponse. Elimination of the photoreceptor cell enriched lysosomal protein Sun also reduces the severity of the light-induced blindness, but to a lesser extent than in rh1Δ356 or arr23K/Q mutant backgrounds (Lee, 2004c).
These data suggest that the formation of rhodopsin/arrestin complexes is the key step determining the extent of Rh1 degradation and visual impairment in response to constant light. Additional evidence in support of this model is that the harmful effect of continuous light on the photoresponse is accelerated significantly in a genetic background, rdgC, which increases Rh1/arrestin complexes and Rh1 degradation (Lee, 2004c).
It is proposed that mammalian visual impairment, which results from exposure to continuous but low-intensity light, may also occur through an arrestin-dependent mechanism and reductions in rhodopsin levels. According to this model, stable rhodopsin/arrestin complexes and endocytosis/degradation of rhodopsin do not normally occur to any significant extent in wild-type animals. Rather, as a result of continuous light, the rhodopsin concentration gradually decreases through an arrestin-dependent pathway. It will be interesting to determine whether mutations that affect arrestin trafficking in mammals also suppress visual impairment resulting from constant light (Lee, 2004c).
The model presented here differs from the 'equivalent-light hypothesis', which proposes that phototoxicity and retinal degeneration resulting from continuous light are due to constitutive activation of signaling by rhodopsin or other phototransduction molecules. Although there is compelling evidence that the equivalent-light hypothesis applies to certain forms of morphological degeneration in the retina, the data indicate that the light-induced blindness occurs through an increase in stable rhodopsin/arrestin complexes and degradation of rhodopsin. This conclusion is also supported by the observation that the hypomorphic allele of the Gαq does not suppress the visual impairment in flies. Although it remains to be determined whether rhodopsin/arrestin complexes occur in wild-type mammals in response to continuous low light, it has been shown that intense levels of light cause a decline in rhodopsin levels in vertebrates. It will be interesting to address whether high- and low-light-induced degradation of mammalian rhodopsin occur through arrrestin-independent and arrestin-dependent mechanisms, respectively, as is the case in Drosophila (Lee, 2004c).
A relevant question is why a mechanism exists for formation of stable rhodopsin/arrestin complexes and degradation of rhodopsin if this phenomenon has negative consequences for the visual response. It has been suggested that endocytosis and degradation of stable rhodopsin/arrestin complexes may normally occur at very low levels and provide a quality control mechanism for eliminating photodamaged rhodopsins, which might otherwise accumulate in photoreceptor cells and have deleterious effects. Thus, the constant-low-light-induced blindness in wild-type animals would appear to be a pathological consequence resulting from excessive activity of a quality control mechanism, which is normally protective (Lee, 2004c).
- A dominant rhodopsin mutation triggers two mechanisms of retinal degeneration and photoreceptor desensitization
A variety of rod opsin mutations result in autosomal dominant retinitis pigmentosa and congenital night blindness in humans. One subset of these mutations encodes constitutively active forms of the rod opsin protein. Some of these dominant rod opsin mutant proteins, which desensitize transgenic Xenopus rods, provide an animal model for congenital night blindness. In a genetic screen to identify retinal degeneration mutants in Drosophila, a dominant mutation was identified in the ninaE gene (NinaEpp100) that encodes RH1, the rhodopsin that is expressed in photoreceptors R1-R6. Deep pseudopupil analysis and histology showed that the degeneration is attributable to a light-independent apoptosis. Whole-cell recordings revealed that the NinaEpp100 mutant photoreceptor cells are strongly desensitized, which partially masks their constitutive activity. This desensitization primarily results from both the persistent binding of arrestin (ARR2) to the NINAEpp100 mutant opsin and the constitutive activity of the phototransduction cascade. Whereas mutations in several Drosophila genes other than ninaE induce photoreceptor cell apoptosis by stabilizing a rhodopsin-arrestin complex, NinaEpp100 represents the first rhodopsin mutation that stabilizes this protein complex. Additionally, the NinaEpp100 mutation leads to elevated levels of Gqalpha in the cytosol, which mediates a novel retinal degeneration pathway. Eliminating both Gqalpha and arrestin completely rescues the NinaEpp100-dependent photoreceptor cell death, which indicates that the degeneration is entirely dependent on both Gqalpha and arrestin. Such a combination of multiple pathological pathways resulting from a single mutation may underlie several dominant retinal diseases in humans (Iakhine, 2004).
- Arrestin1 mediates light-dependent rhodopsin endocytosis and cell survival while Arrestin2 quenches rhodopsin signaling
Arrestins are pivotal, multifunctional organizers of cell responses to GPCR stimulation, including cell survival and cell death. In Drosophila norpA and rdgC mutants, endocytosis of abnormally stable complexes of rhodopsin (Rh1) and fly photoreceptor Arrestin2 (Arr2) trigger cell death, implicating Rh1/Arr2-bearing endosomes in pro-cell death signaling, potentially via arrestin-mediated GPCR activation of effector kinase pathways. In order to further investigate arrestin function in photoreceptor physiology and survival, Arr2’s partner photoreceptor arrestin, Arr1, was studied in developing and adult Drosophila compound eyes. Arr1, but not Arr2, was shown to be essential for normal, light-induced rhodopsin endocytosis. Also distinct from Arr2, Arr1 is essential for light-independent photoreceptor survival. Photoreceptor cell death caused by loss of Arr1 is strongly suppressed by coordinate loss of Arr2. It was found that Rh1 C-terminal phosphorylation is essential for light-induced endocytosis and also for translocation of Arr1, but not Arr2, from dark-adapted photoreceptor cytoplasm to photosensory membrane rhabdomeres. In contrast to a previous report, no requirement was found for photoreceptor myosin kinase NINAC in Arr1 or Arr2 translocation. This study concludes that the two Drosophila photoreceptor arrestins mediate distinct and essential cell pathways downstream of rhodopsin activation. It is proposed that Arr1 mediates an endocytotic cell-survival activity, scavenging phosphorylated rhodopsin and thereby countering toxic Arr2/Rh1 accumulation; elimination of toxic Arr2/Rh1 in double mutants could thus rescue arr1 mutant photoreceptor degeneration (Satoh, 2005).
Arrestins mediate and regulate cell responses to G protein-coupled receptor (GPCR) signaling. Recruited to activated GPCRs marked by conformational change and phosphorylation, arrestins uncouple G proteins from active receptors and promote receptor endocytosis, desensitizing cells. Endocytosed receptors, in complex with arrestins, initiate a second, non-G protein branch of GPCR signaling, scaffolding and activating kinases to 'signaling endosomes' that particularly engage cytoplasmic pathways. Arrestin-scaffolded kinases notably include c-Src family and mitogen-activated protein kinases (MAPKs), and their activation impacts numerous cell activities including chemotaxis, cell motility, and cell survival. In Drosophila photoreceptors, stable complexes of visual arrestin Arr2 with phosphorylated, active Rh1 metarhodpsin (M-p) cause massive, fatal endocytosis. While mechanisms of this cell death remain to be detailed, disruption of MAPK signaling is a likely suspect. In order to better understand how arrestins cooperate in cell physiology and survival, Arr2's partner visual arrestin, Arr1 was studied in developing and adult Drosophila photoreceptors (Satoh, 2005).
Upon stimulation, like GPCRs generally, both Drosophila Rh1 and vertebrate rhodopsins activate rhodopsin kinase (RK), which phosphorylates the receptor's C terminus. Vertebrate rhodopsin phosphorylation decreases signaling and recruits visual arrestin, whose binding further quenches activity. Elimination of C-terminal phosphorylation sites in a mouse rhodopsin truncation mutant prolongs photoresponses. Mutations of human RK cause Oguchi disease, a congenital stationary night blindness (Satoh, 2005).
The significance of Drosophila Rh1 phosphorylation for photoresponse deactivation is less clear. A phosphoregulated cycle of arrestin binding to, and release from, activated Rh1 recycles the receptor for another round of photodetection. However, flies lacking Rh1 C-terminal phosphorylation sites show normal photoresponse deactivation and Arr2 binds activated Rh1, metarhodopsin (M), without requiring phosphorylation. Indeed, Arr2 binds to M prior to phosphorylation, and hyperphosphorylation of Rh1 by rhodopsin kinase decreases Arr2 binding. Rh1 C-terminal phosphodeficient mutants rescue photoreceptor degeneration in norpA mutants lacking effector phospholipase C and rdgC mutants lacking rhodopsin phosphatase, but a role for Rh1 C-terminal phosphorylation in normal cell physiology remains to be determined (Satoh, 2005).
Like Drosophila photoreceptors, vertebrate rods express two visual arrestins: arrestin, with a long C terminus resembling fly Arr2, and p44, a splice variant whose truncated C terminus resembles the shorter Arr1. Like Arr1, p44 is a minority arrestin, present at levels approximately 10% those of full-length arrestin. Like Arr2, p44 binds unphosphorylated, as well as phosphorylated, active rhodopsin. Upon illumination, p44 redistributes to 'lipid raft' membrane microdomains, while arrestin does not. It has been proposed that the two rod visual arrestins mediate receptor shutoff in differing light regimes, p44 operating in dim illumination normal for rod cell function and arrestin acting in bright illumination. Like RK, mutations of human arrestin cause Oguchi disease, likely resulting from constant, low-level activation of the phototransduction cascade. Constitutive phototransduction cascade activity, 'equivalent light' that entrains the pathophysiology of intense, damaging illumination, may underlie several forms of retinal degeneration (Satoh, 2005).
Vertebrate rhodopsin is unusual among GPCRs in that receptor activation and arrestin recruitment does not promote endocytosis. Mammalian nonvisual cells commonly express two arrestins, β-arrestin1 and β-arrestin2, that target activated receptors for endocytosis at coated pits. Similar to mammalian nonvisual GPCRs, invertebrate rhodopsins are normally endocytosed upon activation; illuminated compound eye photoreceptors accumulate long-lived, rhodopsin-bearing endosomes. Arrestin has been localized to rhodopsin-bearing endosomes of Limulus photoreceptors, and Arr2 has been demonstrated to mediate the massive Rh1 endocytosis that kills norpA and rdgC mutant photoreceptors, but a functional requirement for arrestin in normal light-induced endocytosis has not been demonstrated (Satoh, 2005).
Arrestins translocate from resting cell cytoplasm to activated GPCRs at the plasma membrane; visual arrestins translocate to illuminated photosensory membrane organelles, vertebrate outer segments, and invertebrate rhabdomeres. Normal arrestin translocation in rods of mice lacking phosphorylated rhodopsin rules out simple diffusion to a light-dependent phospho-rhodopsin arrestin binding 'sink'. Translocation fails in photoreceptors deficient for the microtubule motor kinesin-II subunit, KIF3A, consistent with active arrestin transport to outer segments via the connecting cilium. However, recent experiments show arrestin translocation is energy independent, supporting translocation via diffusion to light-activated Rh* rhodopsin. Light-induced Arr2 translocation to Drosophila rhabdomeres promotes adaptation and has been reported to require the photoreceptor Myosin-III, NINAC (Satoh, 2005).
In the present work, Arr1 has been characterized in Drosophila photoreceptors. Arr1 is 7-fold less abundant than Arr2, and Arr1 loss by itself had no reported phenotype; its only reported phenotype is to prolong the photoresponse 10-fold in arr1;arr2 double mutants. This study finds that Arr1 is necessary for normal light-dependent endocytosis and that Arr1 loss causes light-independent photoreceptor cell death. Rh1 C-terminal phosphorylation is essential for Arr1 translocation to stimulated rhabdomeres and for light-induced Rh1 endocytosis. Unexpectedly, it was also found that both Arr1 and Arr2 translocate robustly to rhabdomeres of NINAC null ninaCP235 flies. Elimination of Arr2 rescues photoreceptor death caused by loss of Arr1. These results demonstrate a vital role for Arr1 in rhodopsin endocytosis and cell survival (Satoh, 2005).
This study has found that the majority arrestin, Arr2, quenches rhodopsin signaling, while Arr1 promotes light-induced rhodopsin endocytosis. Both functions are cell essential. Arr2 loss leads to light-dependent cell death, and results of this study show that Arr1 loss blocks light-dependent rhodopsin endocytosis and causes light-independent cell death. Photoreceptor cell death following endocytosis of abnormally stable Arr2/Rh1 complexes suggests that they signal pro-cell death activity. Results in this study show that Arr2 removal rescues photoreceptor death caused by Arr1 loss. It is proposed that Arr1 normally captures phospho-rhodopsin and targets it to endocytic removal, inhibiting Arr2/Rh1 accumulation and its toxic endocytosis, thereby effecting a vital prosurvival activity (Satoh, 2005).
Results here showing Rh1 C-terminal phosphorylation promotes Arr1 binding and light-dependent endocytosis reveal a new role for Drosophila Rh1 phosphorylation. It is speculated that Rh1 phosphorylation promotes strong Arr1 binding, perhaps mediated by simultaneous engagement of activation and phosphorylation recognition domains as described for vertebrate visual arrestin. Durable Arr1/Rh1 complexes could capture phosphorylated Rh1 for endocytic removal, perhaps downregulating signaling that exceeds rdgC phosphatase capacity and/or promoting Rh1 turnover. It is notable that in normal flies, RLV immunofluorescence shows low levels of Arr2 along with consistently stronger Arr1 staining, suggesting that Arr1 may facilitate Arr2/Rh1 complex endocytosis as well as inhibit its accumulation (Satoh, 2005).
Drosophila photosensory membrane turnover is poorly understood. Unlike vertebrate outer segments, which are constantly renewed at their base as older, distal tip membrane is shed and phagocytosed by apposed retinal pigment epithelium (RPE), Drosophila photoreceptors do not show the circadian shedding of rhabdomere microvilli observed in some compound eyes, and fly eyes lack a phagocytic RPE equivalent. Drosophila Arr1 may mediate rhodopsin turnover, directing phospho-Rh1 to endocytosis and removal under basal, dark conditions as well as in response to light (Satoh, 2005).
A requirement for Arr1 in endocytosis is unexpected, given that its unusually short C-terminal lacks motifs that bind AP-2 and clathrin to promote clathrin-mediated endocytosis (CME). It remains to be determined if Arr1 targets Rh1 to already-nucleated clathrin-coated pits, or if Arr1 contains novel endocytosis-promoting domains. At 364 amino acids in length, Arr1 lacks a Ser366, whose phosphorylation in Arr2 decreases Rh1 binding, but sequence alignment suggests that Arr1 Ser361 may provide a comparable phospho-regulatory site. A third Drosophila arrestin, Kurtz, contains canonical C-terminal clathrin and AP-2 binding domains, but Kurtz is not expressed in the fly eye. GFP-clathrin light chain and α-adaptin localize at the rhabdomere base, suggesting that Rh1 is internalized by CME. Dynamin mediates scisson of invaginated-coated pits from the plasma membrane and dynamin loss inhibits CME. Restrictive temperatures decrease light-induced Rh1 endocytosis in temperature-sensitive dynamin shits mutant flies, suggesting that CME participates in Rh1 endocytosis (Satoh, 2005).
Results of this study show that Arr2 is dispensable for light-induced endocytosis. Arr2 can promote endocytosis when norpA or rdgC mutants stabilize Arr2/Rh1 complexes, but in normal flies, these complexes are transient, destabilized by Arr2 phosphorylation that rapidly follows photostimulation. Arr2 phosphorylation following photostimulation also inhibits clathrin binding, further diminishing endocytic participation. Provocatively, although vertebrate arrestin-rhodopsin complexes are not normally endocytosed, a Retinitis Pigmentosa mutant rhodopsin forms stable arrestin complexes that are endocytosed and disrupt normal cell function (Satoh, 2005).
It is speculated that endocytic Arr2/M-p signaling kills arr1 mutant photoreceptors. Several observations show constitutive, light-independent Rh1 endocytosis: fluorescence microscopy of 7-day-old dark-reared adult wild-type photoreceptors reveals a low level of small Rh1-immunopositive large vesicles (RLVs). In the electron microscope, dark-reared photoreceptors show occasional multivesicular bodies (MVBs) and coated pits at the rhabdomere base. GFP-clathrin light chain and α-adaptin localize to the rhabdomere base of both dark- and light-exposed photoreceptors. Spontaneous rhodopsin activation has been observed in Drosophila, and, once formed, Drosophila M is stable for at least 40 min. It is speculated that Arr1 targets spontaneously activated M-p to basal endocytosis. Either alone or in competition with Arr2, Arr1 constantly scavenges M-p, and, by 3 days without this surveillance, Arr2/M-p complexes, stable in the absence of light-induced Arr2 phosphorylation, reach levels that cause massive and toxic endocytosis. Confocal immunofluorescence detects Arr2 in endosomes of degenerating arr1 mutant photoreceptors. Elimination of endocytosis in arr1;arr2 double mutants may thus rescue arr1 mutant degeneration. Constant surveillance over a rhabdomere's approximately 100 million receptors and timely capture and removal of inappropriately signaling receptors may be essential for normal photoreceptor physiology (Satoh, 2005).
Photoreceptor cell death in norpA mutants does not depend on apoptosis pathway proteins Rpr, Hid, Grim, and Dronc caspase, and expression of apoptosis inhibitor p35 does not rescue norpA or rdgC degeneration. Resemblance between Arr2-mediated Drosophila photoreceptor cell death and nonapoptotic, autophagic cell death of mouse striatal cells in response to neurokinin-1 receptor activation has been noted. Observations made in this study that the morphology of photoreceptor cell death in Arr1 mutants does not simply resemble apoptosis, that expression of antiapoptotic p35 gives only modest photoreceptor rescue, and a failure to detect activated caspase 3 or caspase 7 in dying photoreceptors, suggest that nonapoptotic, autophagic pathways may participate in arr1 mutant retinal degeneration. Arrestin-mediated GPCR signaling pathways intersect pathways of autophagy-regulated cell survival, potentially including AKT/PKB prosurvival signaling (Satoh, 2005).
The observation that Arr2 translocates normally in photoreceptors lacking NINAC differs from that of Lee and Montell (2004b), who report loss of Arr2 translocation in ninaCP235 null mutant photoreceptors. This study finds, in contrast, robust light-induced translocation of Arr1 and Arr2 in ninaCP235 photoreceptors. Some contribution of NINAC to Arr2 translocation may be evidenced by the increased frequency of Arr1 staining in ninaCP235 rhabdomeres; if Arr2 competes with Arr1 and Arr2 delivery is decreased in mutants, Arr1 may be advantaged (Satoh, 2005).
Differences in genetic strain, illumination levels, and age have been ruled out as the reason for Arr2 translocation in ninaCP235 observed here but not in Lee and Montell. The different results may be attributable in part to different immunolocalization methods; whole-mount confocal immunofluorescence shows excellent subcellular detail. These observations are otherwise in good agreement with previous reports of arrestin translocation. Results presented in this study are consistent with diffusion-based arrestin translocation, paralleling recent observations that mouse arrestin translocation does not require energy (Satoh, 2005).
Drosophila Arr1 and Arr2 share hallmark arrestin capacities: both bind and quench activated receptor signaling, and both can promote endocytosis. However, in normal cells, each has emphasized one of these two activities: Arr2 specializes in Rh1 signal quenching while Arr1 mediates endocytosis of activated Rh1. The mechanistic bases for this specialization remain to be determined, but the normal balance of both operations is cell essential. Arrestin pathways are broadly conserved across eukaryotes, and Drosophila photoreceptors offer a useful window into endocytosis and signaling, an 'inseparable partnership' that impacts virtually all cell physiology (Satoh, 2005).
- An essential role for endocytosis of rhodopsin through interaction of visual arrestin with the AP-2 adaptor
A class of retinal degeneration mutants have been identified in Drosophila in which the normally transient interaction between arrestin2 (Arr2) and rhodopsin is stabilized and the complexes are rapidly internalized into the cell body by receptor-mediated endocytosis. The accumulation of protein complexes in the cytoplasm eventually results in photoreceptor cell death. The endocytic adapter protein AP-2 is essential for rhodopsin endocytosis through an Arr2-AP-2ß interaction, and mutations in Arr2 that disrupt its interaction with the ß subunit of AP-2 prevent endocytosis-induced retinal degeneration. If the interaction between Arr2 and AP-2 is blocked, this also results in retinal degeneration in an otherwise wild-type background. This indicates that the Arr2-AP-2 interaction is necessary for the pathology observed in a number of Drosophila visual system mutants, and suggests that regular rhodopsin turnover in wild-type photoreceptor cells by Arr2-mediated endocytosis is essential for photoreceptor cell maintenance (Orem, 2006).
The results demonstrate that Drosophila Arr2 plays a role as a mediator of rhodopsin endocytosis by interacting with the AP-2 adaptor complex. The AP-2 adaptor complex is required for the endocytosis of rhodopsin during certain pathological conditions and the disruption of this complex rescues norpA-mediated retinal degeneration. In addition, flies with a single point mutation in the AP-2 binding domain of Arr2, norpA-induced endocytosis of stable rhodopsin-arrestin complexes and the subsequent retinal pathology is blocked. Internalization of rhodopsin by an Arr2-AP-2 interaction is essential for photoreceptor cell viability (Orem, 2006).
Certain Arr2 variants bind tightly to rhodopsin. This results in the recruitment of the endocytic machinery and cell death via excessive rhodopsin endocytosis. In this study, an Arr2 variant (arr2R393A) was found that binds more tightly to rhodopsin than wild-type Arr2. However, this mutant does not trigger extensive retinal degeneration. This is further evidence for the essential role of the Arr2-AP-2 interaction in receptor internalization. In this Arr2 background complexes are formed between Arr2 and Rh1, but since Arr2 cannot interact with AP-2 they are not internalized and no photoreceptor cell death is induced (Orem, 2006).
These data point to an essential role for the endocytosis of rhodopsin through Arr2 in the maintenance of photoreceptor cells. Previous work has implicated an essential role for Drosophila arrestins in endocytosis; however, these studies used mutations that either blocked all endocytosis in the photoreceptor cell or utilized loss-of-function mutants that could have other effects on the photoreceptor. Photoreceptor cell degeneration can be induced by inhibiting dynamin function with a dominant negative mutation or by blocking AP-2 function, as described in this study. However, it is possible that a global inhibition of endocytosis may halt the internalization of compounds essential for cell viability. Therefore, cell death may be unrelated to defects in the phototransduction cascade or the internalization of rhodopsin. In addition, retinal degeneration in Arr1 mutants may be due to the defect in endocytosis but pleiotropic affects associated with the loss of Arr1 may also contribute to the aberrant retinal morphology. By using Arr2 variants that are unable to internalize rhodopsin, the endocytosis of one protein (rhodopsin) was selectively blocked while leaving general endocytosis intact. Therefore, the retinal degeneration observed in this study is due solely to defects in rhodopsin internalization through its interaction with Arr2 (Orem, 2006).
One interesting question concerns the purpose of the essential role for rhodopsin-Arr2 endocytosis. One possibility is that this may be a mechanism to remove damaged rhodopsin from the cell. If rhodopsin is photochemically damaged in such a way that it becomes constitutively active, it would be deleterious to the cell, and would need to be removed. Presumably constitutively active rhodopsin would form a stable complex with Arr2 and be targeted for endocytosis through the interaction of arrestin with the AP-2 adaptor complex. This would provide a surveillance mechanism for the cell, whereby defective rhodopsin molecules are quickly and efficiently removed. Second, it may be an adaptive mechanism. In high light conditions the amount of activated rhodopsin may exceed the ability of arrestin to quickly decouple the metarhodopsin from the phototransduction pathway. This would lead to a loss of visual temporal resolution and be detrimental to cell viability. However, under high light conditions at any given time a higher percentage of the cellular arrestin will be bound to rhodopsin and increase the likelihood that the Arr2 bound to rhodopsin will interact with AP-2 and drive internalization of rhodopsin. This would serve to lower the concentration of rhodopsin available to activate the phototransduction cascade and thereby reduce sensitivity under conditions of intense illumination (Orem, 2006).
- Light-induced translocation of Drosophila visual Arrestin2 depends on Rac2
Photoreceptor cells are remarkable in their ability to adjust their sensitivity to light over a wide range of intensities. Rapid termination of the photoresponse is achieved in part by shuttling proteins in and out of the light-transducing compartment of the photoreceptor cells. One protein that undergoes light-dependent translocation is the rhodopsin regulatory protein arrestin. However, the mechanisms coupling rhodopsin to arrestin movement are poorly understood. This study shows that light-dependent shuttling of the major arrestin in Drosophila photoreceptor cells, Arrestin2 (Arr2), occurs independently of known elements of the phototransduction cascade. Disruptions of the trimeric G protein, phospholipase Cβ, the TRP channel, or the Na+/Ca2+ exchanger did not influence Arr2 localization. Rather, it was found that loss of the small GTPase Rac2 severely impaired Arr2 movement and prolonged the termination of the photoresponse. These findings demonstrate that light-induced translocation of Arr2 occurs through a noncanonical rhodopsin/Rac2 pathway, which is distinct from the classical phototransduction cascade (Elsaesser, 2010).
Activity-dependent shuttling of signaling proteins between the cell surface and intracellular compartments is a widespread phenomenon which contributes to the magnitude and duration of signaling in neurons and many other cell types. One of the earliest demonstrations of activity-dependent translocation of signaling proteins from one cell compartment to another was the light-induced translocation of visual arrestin from the inner to the outer segments of rod photoreceptor cells over the course of a few minutes. Light-dependent shuttling of signaling proteins is an evolutionarily conserved phenomenon, as photostimulation also triggers the movement of the Drosophila visual arrestins from the cell bodies to the fly counterpart to rod outer segments, the rhabdomeres. The trimeric G proteins that function in mammalian and Drosophila phototransduction undergo light-dependent translocation as well, as does the Drosophila transient receptor potential-like (TRPL) channel. However, in contrast to the arrestins, these latter proteins shuttle out of the outer segments and rhabdomeres in response to light. The movements of these signaling proteins have important physiological consequences, as they contribute to light adaptation and termination of the photoresponse and thus are crucial for the ability of photoreceptor cells to adjust their sensitivity to the surrounding light conditions (Elsaesser, 2010).
The mechanisms and signaling pathways controlling the translocation of the Drosophila arrestins, G protein, and TRPL proteins have been explored but are incompletely understood. The light-dependent movement of the major visual arrestin, referred to as Arrestin2 (Arr2), requires interaction with PIP3. In addition, the NINAC myosin III has been reported to contribute to the spatial reorganization of Gq, TRPL, and Arr2, although Arr2 depends on NINAC only under blue but not white light. Because light triggers the translocations, they would be expected to require activity of the phototransduction cascade. In flies, light-activated rhodopsin engages a heterotrimeric G protein, Gq, leading to stimulation of a phospholipase C (PLC) and opening of the TRP and TRPL cation channels. Visual arrestin binds to rhodopsin and attenuates signaling by dislodging the heterotrimeric G protein associated with the light-activated rhodopsin. Indeed, movement of TRPL requires Gq and PLC, although the light-dependent shuttling of Gq has been reported to occur independently of PLC, TRP, or TRPL (Elsaesser, 2010).
The current work found that the dynamic spatial redistribution of Arr2 from the cell bodies to the rhabdomeres required rhodopsin, but did not depend on any of the other known components of the phototransduction cascade. These include Gq, PLC, TRP, TRPL, the Na+/Ca2+ exchanger (CalX), and protein kinase C. Rather, it was found that the small GTPase Rac2 interacts with rhodospsin and is essential for the translocation of Arr2 into the rhabdomeres. As is the case with photoreceptor cells expressing Arr2 derivatives that do not translocate efficiently, mutations in rac2 cause a defect in termination of the photoresponse. These data indicate that the light-dependent movement of Arr2 depends on a parallel phototransduction cascade that is initiated by coupling of rhodopsin to Rac2 (Elsaesser, 2010).
Rhodopsin is the archetypal G-protein-coupled receptor, which defines a family of highly related visual pigments. Before the current work, all light-activated pathways known to be physiologically required in photoreceptor cells functioned through heterotrimeric G proteins. Alternative candidate effectors for rhodopsin were small GTPases, because mammalian rhodopsin interacts with a Rho family member and activates this small GTPase in a light-dependent fashion. However, no role has been ascribed for transducing light activity through a rhodopsin/small GTPase pathway (Elsaesser, 2010).
This study found that the light-induced movement of Arr2 from the cell bodies to the rhabdomeres was strictly dependent on Rac2. Moreover, termination of the photoresponse was severely impaired in rac2δ flies. Thus, the findings indicate a signaling mechanism underlying one form of light adaptation which entails transduction of the light signal through a small GTPase, rather than engaging Rh1 with the Gq/PLC/TRP cascade (Elsaesser, 2010).
In addition to its role as a light receptor, rhodopsin plays a structural role during photoreceptor cell morphogenesis. This light-independent function of rhodopsin is mediated by Rac1. In contrast, the requirement for Rac2 for Arr2 movement did not appear to be due to a morphological defect because the ultrastructures of wild-type and rac2δ null mutant photoreceptor cells were indistinguishable. Thus, although the roles of Rac1 and Rac2 are often considered interchangeable, Rac2 is specifically required for light-dependent translocation of Arr2 translocation in adult photoreceptor cells, whereas Rac1 has a structural role during development. Other nonredundant functions of Rac2 have been described in the Drosophila cellular immune response (Elsaesser, 2010).
It appears that two phototransduction pathways contribute to light-dependent movements of signaling proteins in Drosophila photoreceptor cells. The classical pathway is required for shuttling of TRPL because it is dependent on most elements of the phototransduction cascade including the PLC. However, the movement of Arr2 depends on a second noncanonical, Rac2-dependent pathway. Light-dependent shuttling of the Gαq may also function through this second pathway because it occurs normally in mutants missing PLC, protein kinase C, or TRP (Elsaesser, 2010).
The current work raises questions concerning the nature of the proteins that function in concert with Rh1/Rac2 signaling in photoreceptor cells. Finally, because the mammalian Rac1, which is the GTPase most related to Drosophila Rac2, is activated in a light-dependent manner, it is proposed that rhodopsin/Rac signaling may be an evolutionarily conserved mechanism controlling light-induced arrestin translocation and light adaptation in mammalian rods and cones (Elsaesser, 2010).
- Regulation of arrestin translocation by Ca2+ and myosin III in Drosophila photoreceptors
Upon illumination several phototransduction proteins translocate between cell body and photosensory compartments. In Drosophila photoreceptors arrestin (Arr2) translocates from cell body to the microvillar rhabdomere down a diffusion gradient created by binding of Arr2 to photo-isomerized metarhodopsin. Translocation is profoundly slowed in mutants of key phototransduction proteins including phospholipase C (PLC) and the Ca(2+)-permeable transient receptor potential channel (TRP), but how the phototransduction cascade accelerates Arr2 translocation is unknown. Using real-time fluorescent imaging of Arr2-green fluorescent protein translocation in dissociated ommatidia, this study shows that translocation is profoundly slowed in Ca(2+)-free solutions. Conversely, in a blind PLC mutant with ~100-fold slower translocation, rapid translocation was rescued by the Ca(2+) ionophore, ionomycin. In mutants lacking NINAC (calmodulin [CaM] binding myosin III) in the cell body, translocation remained rapid even in Ca(2+)-free solutions. Immunolabelling revealed that Arr2 in the cell body colocalizes with NINAC in the dark. In intact eyes, the impaired translocation found in trp mutants was rescued in ninaC;trp double mutants. Nevertheless, translocation following prolonged dark adaptation was significantly slower in ninaC mutants, than in wild type: a difference that was reflected in the slow decay of the electroretinogram. The results suggest that cytosolic NINAC is a Ca(2+)-dependent binding target for Arr2, which protects Arr2 from immobilization by a second potential sink that sequesters and releases arrestin on a much slower timescale. It is proposed that rapid Ca(2+)/CaM-dependent release of Arr2 from NINAC upon Ca(2+) influx accounts for the acceleration of translocation by phototransduction (Hardie, 2012).
Photoreceptors are highly polarized cells with membrane-rich photosensory compartments separated from the rest of the cell body. It has recently become widely recognized that several phototransduction proteins translocate between these compartments in response to light, representing a form of long-term light and dark adaptation. One of the best-studied examples is arrestin, which terminates the light response by binding to photo-isomerized rhodopsin (metarhodopsin). In dark-adapted photoreceptors most arrestin localizes to the cell body in both vertebrate and insect photoreceptors, but on illumination translocates to the photosensory compartmen. In fly photoreceptors the photosensory compartment is represented by the rhabdomere, a light-guiding, rod-like stack of ~30,000 densely packed apical microvilli loaded with rhodopsin and proteins of a phototransduction cascade mediated by heterotrimeric Gq protein, phospholipase C (PLC), and Ca2+-permeable 'transient receptor potential' (TRP) channels (Hardie, 2012).
Although the possible role of active transport by molecular motors remains debated, recent evidence in both vertebrate rods and Drosophila microvillar photoreceptors supports an essentially passive diffusional model of arrestin translocation, down gradients established by light-regulated 'sinks'. Recent studoes provided evidence that metarhodopsin (M) is the major light-activated sink in fly rhabdomeres by showing that the dominant arrestin isoform (Arr2) translocated in a 1:1 stoichiometric relationship to the number of rhodopsin photo-isomerizations (Satoh, 2010). This study also showed that Arr2 translocation was very rapid (τ ~10 s), but profoundly slowed in mutants of various phototransduction proteins including Gq, phospholipase C (PLC) (norpA), and the major Ca2+-permeable TRP channel (Satoh, 2010). The evidence suggested that Ca2+ influx via the light-sensitive channels was required to accelerate Arr2 translocation, possibly by releasing Arr2 from a Ca2+-dependent cytosolic sink (Satoh, 2010). However, direct evidence for the role of Ca2+ was lacking, while the identity of the putative cytosolic sink and the mechanism(s) mediating the acceleration remained unresolved (Hardie, 2012).
This study shows directly that Ca2+ is both necessary and sufficient to accelerate Arr2 translocation and provides evidence that the Ca2+-regulated cytosolic sink is the cytosolic isoform of NINAC, a calmodulin (CaM) binding myosin III. The evidence also suggests the existence of another potential Ca2+ dependent cytosolic sink, which sequesters and releases arrestin on a much slower timescale, and that NINAC protects Arr2 from sequestration and immobilization by this site. The data support a mechanism for the Ca2+-dependent translocation of Arr2 that is remarkably similar to a previously proposed disinhibitory mechanism of Ca2+-dependent inactivation of M (Liu, 2008) required for rapid termination of the light response (Hardie, 2012).
Previously studies have proposed that Ca2+ influx via the light-sensitive TRP channels is required for rapid Arr2 translocation, because the slow translocation in trp mutants could be rescued by genetic elimination of Na+/Ca2+ exchanger activity. The present study confirmed the role of Ca2+ directly by imaging Arr2-GFP translocation in dissociated ommatidia, and showing that extracellular Ca2+ is required for rapid Arr2 translocation. Ca2+ was not only required, but also sufficient to enable rapid translocation without any products of PLC activity, since the Ca2+ ionophore, ionomycin, fully rescued translocation in blind norpA mutants lacking PLC. Significantly, it was found that the requirement of Ca2+ for rapid translocation was obviated in null mutants of NINAC (CaM binding MyoIII), with the cytosolic p132 isoform of NINAC alone being sufficient to slow down translocation in Ca2+-free conditions. This suggests that cytosolic NINAC p132 acts as a Ca2+/CaM-dependent 'brake' on translocation by binding Arr2, releasing it in response to Ca2+ influx associated with the photoresponse. This conclusion was further supported by finding that the ninaC mutation rescued rapid translocation in trp mutants (in ninaC;trp double mutants). However, the failure to rescue translocation in norpA;ninaC mutants, except under special conditions, and the demonstration of significant slowing of translocation following prolonged dark adaptation in ninaC mutants also indicated the existence of a second Ca2+-dependent cytosolic sink (Hardie, 2012).
Despite an earlier study reporting that Arr2 translocation was impaired in ninaC mutants, as shown here and previously (Satoh, 2005; Satoh, 2010), Arr2 translocation, whether of endogenous Arr2 or GFP-tagged Arr2, appears essentially intact in ninaC-null mutants. In fact, far from being impaired, the results indicate that translocation can be rescued by ninaC mutations under conditions where translocation is slowed down by reduced Ca2+ influx. Although translocation was significantly slower in ninaC mutants following prolonged dark adaptation, it was never prevented and full translocation was always achieved within ~2-3 min of appropriate illumination (Hardie, 2012).
A novel phenotype of ninaCP235-null mutants, and also ninaCΔ174 mutants lacking only the rhabdomeric p174 isoform, was the complete absence of an early rapid increase in fluorescence routinely observed during the first ~500 ms of measurements of Arr2-GFP fluorescence from the DPP of wild-type photoreceptors or dissociated ommatidia. With a time constant of ~260 ms, this rapid phase was ~40× faster than the overall translocation (τ ~10 s) itself the fastest protein translocation reported in a photoreceptor to date, and probably diffusionally limited. It therefore seems unlikely that the fast phase represents a 40x faster, ninaC-dependent movement of Arr2 from cell body into the rhabdomere. Instead we suggest that it represents a change in the fluorescence efficiency of Arr2-GFP as it is released (via Ca2+ influx) from the rhabdomeric p174 NINAC isoform. A lower fluorescence when bound to NINAC might reflect crowding of the GFP-fluorophore, or could be due to some other feature of the nano-environment of the fluorophore when Arr2 is bound to NINAC in the microvilli. This interpretation was supported by the ability to eliminate the rapid phase by pre-illumination with long wavelength light, which induces Ca2+ influx without net change in M. The rapid phase then re-emerged with a time constant of ∼3 s in the dark, presumably representing rapid rebinding of Arr2-GFP to NINAC (Hardie, 2012).
After more than a few minutes in the dark, Arr2 translocation into the rhabdomere became progressively slower, with clear functional consequences in a parallel slowing of the decay of the ERG. This gradual slowing was considerably more pronounced in ninaC-null mutants, where it si proposed that the slowing represents binding or sequestration of Arr2 via one or more NINAC-independent target(s) or compartment(s). Release from such sites also requires activation of the phototransduction cascade, and translocation could be accelerated back to levels typical of short dark-adaptation times by pre-illumination with bright orange light, which itself does not generate a net increase in M. It seems likely that the rise in Ca2+ is also responsible for release from this site; however, the involvement of other products of the phototransduction cascade cannot be excluded. The identity of this second site or compartment remains a subject for future investigation. Given previous reports that Arr2 can bind to phosphoinositides, negatively charged phosphoinositide species on endomembranes, which could be screened by Ca2+, might represent promising candidates. Drosophila Arr2 is an unusually basic (positively charged) protein and may thus have a strong tendency to bind to such sites. The finding that the slowing of translocation with dark adaptation was more pronounced in ninaC mutants suggests that one of the functions of cytosolic NINAC may be to prevent immobilization of Arr2 by this alternative potential sink. Because the Ca2+-dependent release of Arr2 from NINAC occurs on a subsecond timescale, this then allows more rapid translocation (and hence recovery of the electrical response) after a period in the dark (Hardie, 2012).
These results demonstrate that Arr2 translocation is accelerated by Ca2+ influx, and suggest that this is mediated by a disinhibitory mechanism, whereby NINAC p132 binds to Arr2 under low Ca2+ conditions in the dark, rapidly releasing it in response to Ca2+ influx associated with the photoresponse. Although inferred from essentially independent experiments, this mechanism is strikingly similar to one previously proposed for the rapid, Ca2+-dependent inactivation of M during the light response itself. In that study the time constant of M inactivation by Arr2 was found to be accelerated from ~200 ms under Ca2+-free conditions to ~20 ms following Ca2+ influx. This Ca2+ dependence was eliminated in both ninaC-null mutants, and in ninaCΔ174 mutants lacking the rhabdomeric p174 (but not in ninaCΔ132 mutants lacking cytosolic p132). The results also indicated a disinhibitory mechanism, leading to the proposal that Arr2 in the microvilli was bound to rhabdomeric NINAC p174 under low Ca2+ conditions in the dark, thus hindering its diffusional access to activated M. Ca2+ influx via the first activated TRP channels, then rapidly releases Arr2, allowing it to diffuse, bind to, and inactivate M (Hardie, 2012).
NINAC p132 and p174 share a common CaM binding site (CBS) and although p174 has a second CBS not found in p132, the pronounced slowing of translocation with dark adaptation in null ninaCP235 mutants was recapitulated in mutants lacking the common CBS. It is therefore suggested that essentially the same mechanism underlies the Ca2+-dependent rapid translocation of Arr2, but now acting via NINAC p132 rather than p174 and working over much larger distances (several micrometers as opposed to the nanometer dimensions of single microvilli) and hence slower timescales (Hardie, 2012).
The proposed interaction between NINAC and Arr2 finds some support from biochemical data reporting coimmunoprecipitation of Arr2 and NINAC in extracts from whole heads (Lee, 2004). However, that study also reported that both Arr2 and NINAC had significant in vitro affinity for phosphoinositides. It was proposed that the NINAC/Arr2 association was indirect and mediated by both Arr2 and NINAC binding to phosphoinositide-rich membrane. Although the current results clearly indicate that Ca2+-dependent modulation of Arr2 binding to M and Ca2+-dependent translocation of Arr2 are both dependent upon NINAC, the possibility cannot be excluded that the interaction is mediated indirectly via a NINAC-dependent target. Ultimate verification will require direct demonstration of NINAC/Arr2 binding and its dependence upon Ca2+/CaM (Hardie, 2012).
The regulated multisink model proposed in this study differs fundamentally from an earlier model in which NINAC was proposed as a molecular motor transporting Arr2 in phosphoinositide-rich vesicles. By contrast it shows strong parallels with current models for arrestin translocation in vertebrate rods. Here, phosphorylated rhodopsin represents the light-activated sink in the outer segments, while microtubules have been proposed as the cytosolic sink in the inner segments. There is also evidence indicating light-regulated acceleration of translocation in vertebrate rods. The mechanism is unclear; however, intriguingly a recent study has implicated roles for PLC and protein kinase C possibly stimulating release of arrestin from its cytosolic sink (Hardie, 2012 and references therein).
Such regulated-sink models have the advantage of simplicity: directed translocation requires no more than diffusion coupled with regulated binding, can rapidly transport virtually unlimited quantities of protein, and per se consumes essentially no energy. While it can be conveniently studied in photoreceptors with their distinctive polarized morphologies and high concentrations of transduction machinery, translocation according to the same general principles may represent a general and elegant solution to the problem of directed movements of signaling proteins (Hardie, 2012).
- Role of rhodopsin and arrestin phosphorylation in retinal degeneration of Drosophila
Arrestins belong to a family of multifunctional adaptor proteins that regulate internalization of diverse receptors including G-protein-coupled receptors (GPCRs). Defects associated with endocytosis of GPCRs have been linked to human diseases. Enhanced green fluorescent protein-tagged arrestin 2 (Arr2) was used to monitor the turnover of the major rhodopsin (Rh1) in live Drosophila. It was demonstrated that during degeneration of norpAP24 photoreceptors the loss of Rh1 is parallel to the disappearance of rhabdomeres, the specialized visual organelle that houses Rh1. The cause of degeneration in norpAP24 is the failure to activate CaMKII (Ca2+/calmodulin-dependent protein kinase II) and retinal degeneration C (RDGC) because of a loss of light-dependent Ca2+ entry. A lack of activation in CaMKII, which phosphorylates Arr2, leads to hypophosphorylated Arr2, while a lack of activation of RDGC, which dephosphorylates Rh1, results in hyperphosphorylated Rh1. How reversible phosphorylation of Rh1 and Arr2 contributes to photoreceptor degeneration was investigated. To uncover the consequence underlying a lack of CaMKII activation, ala1 flies were characterized in which CaMKII was suppressed by an inhibitory peptide, and it was shown that morphology of rhabdomeres was not affected. In contrast, it was found that expression of phosphorylation-deficient Rh1s, which either lack the C terminus or contain Ala substitution in the phosphorylation sites, was able to prevent degeneration of norpAP24 photoreceptors. This suppression is not due to a loss of Arr2 interaction. Importantly, co-expression of these modified Rh1s offered protective effects, which greatly delayed photoreceptor degeneration. Together, it is concluded that phosphorylation of Rh1 is the major determinant that orchestrates its internalization leading to retinal degeneration (Kristaponyte, 2012).