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)
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).
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).
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).
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