Visual arrestin plays a crucial role in the termination of the light response in vertebrate photoreceptors by binding selectively to light-activated, phosphorylated rhodopsin. Arrestin localizes predominantly to the inner segments and perinuclear region of dark-adapted rod photoreceptors, whereas light induces redistribution of arrestin to the rod outer segments. The mechanism by which arrestin redistributes in response to light is not known, but it is thought to be associated with the ability of arrestin to bind photolyzed, phosphorylated rhodopsin in the outer segment. In this study, it was shown that light-driven translocation of arrestin is unaffected in two different mouse models in which rhodopsin phosphorylation is lacking. It was further shown that arrestin movement is initiated by rhodopsin but does not require transducin signaling. These results exclude passive diffusion and point toward active transport as the mechanism for light-dependent arrestin movement in rod photoreceptor cells (Mendez, 2003).
Phosphorylation of activated G-protein-coupled receptors and the subsequent binding of arrestin mark major molecular events of homologous desensitization. In the visual system, interactions between arrestin and the phosphorylated rhodopsin are pivotal for proper termination of visual signals. By using high resolution proton nuclear magnetic resonance spectroscopy of the phosphorylated C terminus of rhodopsin, represented by a synthetic 7-phosphopolypeptide, it was shown that the arrestin-bound conformation is a well ordered helix-loop structure connected to rhodopsin via a flexible linker. In a model of the rhodopsin-arrestin complex, the phosphates point in the direction of arrestin and form a continuous negatively charged surface, which is stabilized by a number of positively charged lysine and arginine residues of arrestin. Opposite to the mostly extended structure of the unphosphorylated C-terminal domain of rhodopsin, the arrestin-bound C-terminal helix is a compact domain that occupies a central position between the cytoplasmic loops and occludes the key binding sites of transducin. In conjunction with other binding sites, the helix-loop structure provides a mechanism of shielding phosphates in the center of the rhodopsin-arrestin complex and appears critical in guiding arrestin for high affinity binding with rhodopsin (Kisselev, 2004).
The phosphorylated carboxyl terminus of rhodopsin is required for the stable binding of visual arrestin to the full length rhodopsin molecule. Phosphorylation of the carboxyl terminus has been shown to induce conformational changes in arrestin, which promote its binding to the cytoplasmic loops of rhodopsin. However, it has not been determined whether phosphorylation is also responsible for the direct binding of the rhodopsin carboxyl terminus to arrestin. To further investigate the role of rhodopsin phosphorylation on arrestin binding, surface plasmon resonance was used to measure the interaction between a synthetic phosphopeptide corresponding to the carboxyl terminus of rhodopsin and visual arrestin in real time. Synthetic peptides were generated that correspond to the phosphorylated and nonphosphorylated carboxyl terminus of bovine rhodopsin. These peptides were immobilized on a biosensor chip and their interaction with purified visual arrestin was monitored by surface plasmon resonance on a BIAcore 2000 or 3000. A synthetic peptide phosphorylated on residues corresponding to Ser-338, Thr-340, Thr-342 and Ser-343 of bovine rhodopsin is sufficient for direct binding to visual arrestin. In contrast, a second phosphopeptide phosphorylated on Thr-340 and Thr-342 and a nonphosphorylated synthetic peptide are not able to bind arrestin. A peptide fully substituted at all serine and threonine residues with glutamic acid is unable to substitute for phosphorylation. It is concluded that surface plasmon resonance is a sensitive method for detecting small differences in affinity. This technique was successful in detecting differences in the affinity of phosphorylated and nonphosphorylated rhodopsin peptides for visual arrestin. The data suggest that these are low-affinity interactions and indicate that phosphorylation is responsible for the direct binding of the rhodopsin carboxyl terminus to visual arrestin. Four phosphorylated residues are sufficient for this interaction. Because the affinity of the synthetic phosphopeptide for arrestin is substantially lower than the full length rhodopsin molecule, the cytoplasmic loops and rhodopsin carboxyl terminus appear to interact in a cooperative manner to stably bind arrestin (Liu, 2004).
The binding of visual arrestin to phosphorylated, activated rhodopsin serves as a model for studying the inactivation process of a large class of G-protein coupled receptor systems. In this study, the use of insertional mutagenesis, fluorescence labeling, and scanning alanine mutagenesis was combined to identify the surface of interaction between arrestin and rhodopsinThe ten amino acid myc tag (EQKLISEEDL) was inserted in eleven loop structures that connect ßstrands and the tagged arrestins were heterologously expressed in yeast. Binding competition assays were performed with these proteins, using an anti-myc monoclonal antibody. Site specific cysteines were also substituted in selected loop structures in arrestin. These cysteines were labeled with a fluorescent reporter to assess the proximity of the introduced cysteine with rhodopsin in the bound complex. Competitive inhibition of arrestin binding to light activated, phosphorylated rhodopsin with an anti-myc antibody showed that all competitive sites lay along a single surface encompassing the N- and C-terminal domains. Fluorescence labeling of these loop structures and subsequent interaction with rhodopsin indicates close apposition of loops 68-78 and 248-253 to rhodopsin in the receptor bound state. Scanning mutagenesis of loop 248-253 implicates Ser-251 and/or Ser-252 as a potential interaction point with rhodopsin. These results clearly suggest a surface of arrestin to which rhodopsin binds upon light activation and phosphorylation. This surface encompasses elements from both the N- and C-terminal domains of arrestin (Smith, 2004).
Arrestins selectively bind to phosphorylated activated forms of their cognate G protein-coupled receptors. Arrestin binding prevents further G protein activation and often redirects signaling to other pathways. The comparison of the high-resolution crystal structures of arrestin2, visual arrestin, and rhodopsin as well as earlier mutagenesis and peptide inhibition data collectively suggest that the elements on the concave sides of both arrestin domains most likely participate in receptor binding directly, thereby dictating its receptor preference. Using comparative binding of visual arrestin/arrestin2 chimeras to the preferred target of visual arrestin, light-activated phosphorylated rhodopsin (PRh*), and to the arrestin2 target, phosphorylated activated m2 muscarinic receptor (P-m2 mAChR*), the elements that determine the receptor specificity of arrestins were determined. It was found that residues 49-90 (ß-strands V and VI and adjacent loops in the N-domain) and 237-268 (ß-strands XV and XVI in the C-domain) in visual arrestin and homologous regions in arrestin2 are largely responsible for their receptor preference. Only 35 amino acids (22 of which are nonconservative substitutions) in the two elements are different. Simultaneous exchange of both elements between visual arrestin and arrestin2 fully reverses their receptor specificity, demonstrating that these two elements in the two domains of arrestin are necessary and sufficient to determine their preferred receptor targets (Vishnivetskiy, 2004).
The visual arrestins in rhabdomeral photoreceptors are multifunctional phosphoproteins. They are rapidly phosphorylated in response to light, but the functional relevance of this phosphorylation is not yet fully understood. The phosphorylation of Limulus visual arrestin is particularly complex in that it becomes phosphorylated on three sites, and one or more of these site are phosphorylated even in the dark. The purpose of this study was to examine in detail the light-stimulated phosphorylation of each of the three sites in Limulus visual arrestin in intact photoreceptors. It was found that light increases the phosphorylation of all three sites (S377, S381, and S396), that S381 is a preferred phosphorylation site, and that S377 and S381 are highly phosphorylated in the dark. The major effect of light was to increase the phosphorylation of S396, the site located closest to the C-terminal and very close to the adaptin binding motif. It is speculated that the phosphorylation of this site may be particularly important for regulating the light-driven endocytosis of rhabdomeral membrane (Sineshchekova, 2004).
The mechanism of visual arrestin release from light-activated rhodopsin was addressed using fluorescently labeled arrestin mutants. Two mutants, I72C and S251C, when labeled with the small, solvent-sensitive fluorophore monobromobimane, exhibit spectral changes only upon binding light-activated, phosphorylated rhodopsin. This analysis indicates that these changes are probably due to a burying of the probes at these sites in the rhodopsin-arrestin or phospholipid-arrestin interface. Using a fluorescence approach based on this observation, it was demonstrated that arrestin and retinal release are linked and are described by similar activation energies. However, at physiological temperatures, it was found that arrestin slows the rate of retinal release approximately 2-fold and abolishes the pH dependence of retinal release. Using fluorescence, EPR, and biochemical approaches, intriguing evidence was found that arrestin binds to a post-Meta II photodecay product, possibly Meta III. It is speculated that arrestin regulates levels of free retinal in the rod cell to help limit the formation of damaging oxidative retinal adducts. Such adducts may contribute to diseases like atrophic age-related macular degeneration (AMD). Thus, arrestin may serve to both attenuate rhodopsin signaling and protect the cell from excessive retinal levels under bright light conditions (Sommer, 2005).
G-protein coupled receptor signaling is terminated by arrestin proteins which preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind the active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, an extensive analysis was performed of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicate that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. Several additional phosphate binding elements were identified in the N-domain, and the active receptor was demonstrated to preferentially engage the arrestin C-domain. It was also found that the inter-domain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high-affinity receptor-binding state (Hanson, 2005).
Arrestins play a fundamental role in the regulation and signal transduction of G protein-coupled receptors. This paper described the crystal structure of cone arrestin at 2.3Å resolution. The overall structure of cone visual arrestin is similar to the crystal structures of rod visual and the non-visual arrestin-2, consisting of two domains, each containing ten ß-sheets. However, at the tertiary structure level, there are two major differences, in particular on the concave surfaces of the two domains implicated in receptor binding and in the loop between ß-strands I and II. Functional analysis shows that cone arrestin, in sharp contrast to its rod counterpart, binds cone pigments and non-visual receptors. Conversely, non-visual arrestin-2 binds cone pigments, suggesting that it may also regulate phototransduction and/or photopigment trafficking in cone photoreceptors. These findings indicate that cone arrestin displays structural and functional features intermediate between the specialized rod arrestin and the non-visual arrestins, which have broad receptor specificity. A unique functional feature of cone arrestin is the low affinity for its cognate receptor, resulting in an unusually rapid dissociation of the complex. Transient arrestin binding to the photopigment in cones may be responsible for the extremely rapid regeneration and reuse of the photopigment that is essential for cone function at high levels of illumination (Sutton, 2005).
Beta-arrestins have been shown to competitively inhibit G protein-dependent signaling and mediate endocytosis for many of the hundreds of non-visual rhodopsin-family G protein-coupled receptors (GPCR). An open question of fundamental importance concerning the regulation of signal transduction of several hundred rhodopsin-like GPCRs is how these receptors of limited sequence homology when considered in toto can all recruit and activate the two highly conserved ß-arrestin proteins as part of their signaling/desensitization process. Though the serine and threonine residues that form GPCR kinase (GRK) phosphorylation sites are common ß-arrestin associated receptor determinants regulating receptor desensitization and internalization, the agonist-activated conformation of GPCR probably reveals the most fundamental determinant mediating the GPCR and arrestin interaction. This study identified a ß-arrestin binding determinant common to rhodopsin-family GPCRs formed from the proximal ten residues of the second intracellular loop. It was demonstrated by both gain and loss of function studies for the serotonin 2C, ß2-adrenergic, alpha2a-adrenergic, and neuropeptide Y type 2 receptors that the highly conserved amino acids proline or alanine, naturally occurring in rhodopsin-family receptors six residues distal to the highly conserved second-loop DRY motif, regulate ß-arrestin binding and ß-arrestin mediated internalization. In particular, as demonstrated for the b2AR, this occurs independently of changes in GRK phosphorylation. These results suggest that a GPCR conformation directed by the second intracellular loop, likely using the loop itself as a binding patch, may function as a switch for transitioning ß-arrestin from its inactive form to its active receptor binding state (Marion, 2005).
ß-arrestins, originally discovered in the context of heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) desensitization, also function in internalization and signaling of these receptors. c-Jun amino-terminal kinase 3 (JNK3) has been identified as a binding partner of ß-arrestin 2 using a yeast two-hybrid screen and by coimmunoprecipitation from mouse brain extracts or cotransfected COS-7 cells. The upstream JNK activators apoptosis signal-regulating kinase 1 (ASK1) and mitogen-activated protein kinase (MAPK) kinase 4 were also found in complex with ß-arrestin 2. Cellular transfection of ß-arrestin 2 causes cytosolic retention of JNK3 and enhanced JNK3 phosphorylation stimulated by ASK1. Moreover, stimulation of the angiotensin II type 1A receptor activates JNK3 and triggers the colocalization of ß-arrestin 2 and active JNK3 to intracellular vesicles. Thus, ß-arrestin 2 acts as a scaffold protein, which brings the spatial distribution and activity of this MAPK module under the control of a GPCR (McDonald, 2000).
In budding yeast, Ste5 is a scaffold protein that forms a multicomponent complex with the Fus3 (Kss1) MAPK, Ste7 MAPKK, and Ste11 MAPKKK to facilitate the specific and efficient activation of the mating pheromone pathway. Also in yeast, Pbs2, which itself is a MAPKK, has been proposed as a possible scaffold protein in the HOG (high-osmolarity glycerol response) signal transduction pathway. An intriguing similarity between Ste5 and ß-arrestin 2 is that both are recruited to the plasma membrane as a consequence of agonist stimulation of a GPCR. In mammalian cells, a group of JNK-interacting proteins (JIP1, -2 and -3) have been identified as scaffolding proteins for specific JNK signaling modules (McDonald, 2000 and references therein).
Like members of the JIP family, ß-arrestin 2 associates with all three kinase components of a single MAPK cascade, thus enhancing signaling efficiency. However, unlike JIPs, the ß-arrestin 2-MAPK module is regulated by agonist stimulation of GPCRs. It is likely that individual JNK isoforms exhibit distinct patterns of regulation. For example, as demonstrated in this study, JNK3 activity appears to be specifically enhanced by ß-arrestin 2, whereas JNK1 activity is unaffected. Thus, formation of similar complexes may prevent inappropriate cross talk between the various MAPK pathways (McDonald, 2000).
JNK1 is activated in response to several GPCRs. The association of JNK3 with ß-arrestin 2 provides a mechanism whereby ß-arrestin 2 might localize JNK3 to specific subcellular compartments and/or target JNK3 to specific substrates in response to GPCR agonists. The results reported here add to a growing list of functions subserved by ß-arrestins in regulating signaling through heptahelical receptors. By acting as a scaffold for a specific MAPK pathway, ß-arrestin 2 provides a mechanism for bringing this signaling module under the control of such receptors. Other evidence (suggests that ß-arrestins may also play roles in organizing pathways leading from GPCRs to activation of the ERKs (McDonald, 2000).
Activation of 7TM receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors act via Gq/11 to stimulate the PLC/Ca2+/PKC cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other 7TM receptors (including non-mammalian GnRHRs) and are implicated in arrestin binding. This study compares ERK signaling by human (h) and Xenopus (X) GnRHRs. In HeLa cells XGnRHRs undergo rapid and arrestin-dependent internalization and cause arrestin/GFP translocation to the membrane and endosomes, whereas hGnRHRs do not. Internalized XGnRHRs co-localize with arrestin/GFP, whereas hGnRHRs do not. Both receptors mediate transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2/GFP) and for both, ERK phosphorylation is reduced by PKC inhibition but not by inhibiting EGF receptor autophosphorylation. In the presence of PKC inhibitor, Darrestin(319-418) blocks XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number is varied, hGnRHRs activate PLC and ERK more efficiently than XGnRHRs, but are less efficient at causing ERK2/GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both cause ERK2/GFP translocation to the nucleus but at low receptor number XGnRHRs cause ERK2/GFP translocation whereas hGnRHRs do not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (likely G protein-independent) GnRHR signalling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization (Caunt, 2005).
Beta-arrestin mediates desensitization and internalization of ß-adrenergic receptors (ßARs), but also acts as a scaffold protein in extracellular signal-regulated kinase (ERK) cascade. Thus, the role of ß-arrestin2 was examined in the ßAR-mediated ERK signaling pathways. Isoproterenol stimulation equally activates cytoplasmic and nuclear ERK in COS-7 cells expressing ß1AR or ß2AR. However, the activity of nuclear ERK is enhanced by co-expression of ß-arrestin2 in ß2AR- (but not ß1AR-) expressing cells. Pertussis toxin treatment and blockade of Gßgamma action inhibits ß-arrestin2-enhanced nuclear activation of ERK, suggesting that ß-arrestin2 promotes nuclear ERK localization in a Gßgamma dependent mechanism upon receptor stimulation. ß2AR containing the carboxyl terminal region of ß1AR has lost the ß-arrestin2-promoted nuclear translocation. Since the carboxyl terminal region is important for ß-arrestin binding, these results demonstrate that recruitment of ß-arrestin2 to carboxyl terminal region of ß2AR is important for ERK localization to the nucleus (Kobayashi, 2005).
Physiological effects of ß adrenergic receptor (ß2AR) stimulation have been classically shown to result from Gs-dependent adenylyl cyclase activation. A novel signaling mechanism has been demonstrated wherein ß-arrestins mediate ß2AR signaling to extracellular-signal regulated kinases 1/2 (ERK 1/2) independent of G protein activation. Activation of ERK1/2 by the ß2AR expressed in HEK-293 cells has been resolved into two components dependent, respectively, on Gs-Gi/protein kinase A (PKA) or ß-arrestins. G protein-dependent activity is rapid, peaking within 2-5 min, is quite transient, is blocked by pertussis toxin (Gi inhibitor) and H-89 (PKA inhibitor), and is insensitive to depletion of endogenous ß-arrestins by siRNA. ß-Arrestin-dependent activation is slower in onset (peak 5-10 min), less robust, but more sustained and shows little decrement over 30 min. It is insensitive to pertussis toxin and H-89 and sensitive to depletion of either ß-arrestin1 or -2 by small interfering RNA. In Gs knock-out mouse embryonic fibroblasts, wild-type ß2AR recruited ß-arrestin2-green fluorescent protein and activated pertussis toxin-insensitive ERK1/2. Furthermore, a novel ß2AR mutant (ß2ART68F,Y132G,Y219A or ß2ARTYY), rationally designed based on Evolutionary Trace analysis, is incapable of G protein activation but can recruit ß-arrestins, undergo ß-arrestin-dependent internalization, and activate ß-arrestin-dependent ERK. Interestingly, overexpression of GRK-5 or -6 increases mutant receptor phosphorylation and ß-arrestin recruitment, leads to the formation of stable receptor-ß-arrestin complexes on endosomes, and increases agonist-stimulated phospho-ERK1/2. In contrast, GRK2, membrane translocation of which requires Gßgamma release upon G protein activation, is ineffective unless it is constitutively targeted to the plasma membrane by a prenylation signal (CAAX). These findings demonstrate that the ß2AR can signal to ERK via a GRK5/6-ß-arrestin-dependent pathway, which is independent of G protein coupling (Shenoy, 2006).
Beta-arrestins are multifunctional adaptor proteins that mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and ß-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in ß-arrestins upon binding to the activated phosphorylated receptor. This study characterizes the conformational changes in ß-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and MALDI-TOF MS profiles of ß-arrestin 2 in the presence of a phosphopeptide (V2R-pp) derived from the C terminus of the vasopressin type II receptor (V2R) or the corresponding nonphosphopeptide (V2R-np). V2R-pp binds to ß-arrestin 2 specifically, whereas V2R-np does not. Activation of ß-arrestin 2 upon V2R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of ß-arrestin 2; however, heparin and V2R-pp have different binding site(s) and/or induce different conformational changes in ß-arrestin 2. Release of the C terminus from the rest of ß-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to ß-arrestin 2 by 10-fold. Thus, the V2R-pp can activate ß-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of ß-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-ß-arrestin interactions can be modeled in vitro (Xiao, 2004).
Expression levels of the chemokine receptor, CC chemokine receptor 5 (CCR5), at the cell surface determine cell susceptibility to HIV entry and infection. Cellular activation by CCR5 itself, but also by unrelated receptors, leads to cross-phosphorylation and cross-internalization of CCR5. This study addresses the underlying molecular mechanisms of homologous and heterologous CCR5 regulation. As shown by bioluminescence resonance energy transfer experiments, CCR5 forms constitutive homo- as well as hetero-oligomeric complexes together with C5aR but not with the unrelated AT(1a)R in living cells. Stimulation with CCL5 of RBL cells that co-express CCR5 together with an N-terminally truncated CCR5-DeltaNT mutant, results in both protein kinase C (PKC)- and G protein-coupled receptor (GPCR) kinase (GRK)-mediated cross-phosphorylation of the mutant unligated receptor, as determined by phosphosite-specific monoclonal antibody. Similarly, both PKC and GRK cross-phosphorylates CCR5 in a heterologous manner after C5a stimulation of RBL-CCR5/C5aR cells, whereas AT(1a)R stimulation results only in classical PKC-mediated CCR5 phosphorylation. Co-expression of CCR5-DeltaNT together with a phosphorylation-deficient CCR5 mutant that neither binds ß-arrestin nor undergoes internalization partially restores the CCL5-induced association of ß-arrestin with the homo-oligomeric receptor complex and augmented cellular uptake of (125)I-CCL5. Co-expression of C5aR, but not of AT(1a)R, promotes CCR5 co-internalization upon agonist stimulation by a mechanism independent of CCR5 phosphorylation. Co-internalization of phosphorylated CCR5 is also observed in C5a-stimulated macrophages. Finally, co-expression of a constitutively internalized C5aR-US28(CT) mutant leads to intracellular accumulation of CCR5 in the absence of ligand stimulation. These results show that GRKs and ß-arrestin are involved in heterologous receptor regulation by cross-phosphorylating and co-internalizing unligated receptors within homo- or hetero-oligomeric protein complexes (Huttenrauch, 2005).
The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to ß-arrestin after agonist exposure. To define the importance of receptor phosphorylation and ß-arrestin binding in desensitization, and to determine whether ß-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, TRH receptors were expressed in fibroblasts from mice lacking ß-arrestin-1 and/or ß-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing ß-arrestins, including a ß-arrestin mutant that did not permit receptor internalization. TRH causes extensive receptor endocytosis in the presence of ß-arrestins, but receptors remain primarily on the plasma membrane without ß-arrestin. ß-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous ß-arrestins reduced TRH-stimulated inositol phosphate production by 48% (ß-arrestin-1), 71% (ß-arrestin-2), and 84% (ß-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, is unaffected by ß-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without ß-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that ß-arrestin binding is critical for desensitization and internalization (Jones, 2005).
The neuronal Na(+)/H(+) exchanger NHE5 isoform not only resides in the plasma membrane but also accumulates in recycling vesicles by means of clathrin-mediated endocytosis. To further investigate the underlying molecular mechanisms, a human brain cDNA library was screened for proteins that interact with the cytoplasmic C-terminal region of NHE5 by using yeast two-hybrid methodology. One candidate cDNA identified by this procedure encoded ß-arrestin2, a specialized adaptor/scaffolding protein required for internalization and signaling of members of the G protein-coupled receptor superfamily. Direct interaction between the two proteins was demonstrated in vitro by GST fusion protein pull-down assays. Sequences within the N-terminal receptor activation-recognition domain and the C-terminal secondary receptor-binding domain of ß-arrestin2 confer strong binding to the C terminus of NHE5. Full-length NHE5 and ß-arrestin2 also associated in intact cells, as revealed by their coimmunoprecipitation from extracts of transfected CHO cells. Moreover, ectopic expression of both proteins causes a redistribution of ß-arrestin2 from the cytoplasm to vesicles containing NHE5, and significantly decreases the abundance of the transporter at the cell surface. Comparable results were also obtained for the ß-arrestin1 isoform. These data reveal a broader role for arrestins in the trafficking of integral plasma membrane proteins than previously recognized (Szabo, 2005).
There is considerable evidence for the role of carboxyl terminal serines 355,356,364 in GRK-mediated phosphorylation and desensitization of ß2-adrenergic receptors (ß2ARs). Receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) were used to determine the role of these sites in ß-arrestin-dependent ß2AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant does not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nM isoproterenol for 5 minutes induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with PKA site mediated desensitization. When exposed to 1 muM isoproterenol to trigger GRK site mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phosphosite-specific antibody, it was determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist induced internalization of the SD3 and SA3 mutants were 44% and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited EGFP-ß-arrestin2 to the plasma membrane while the SA3 mutant did not. EGFP-ß-arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent ß-arrestin1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355,356 in wild-type receptors. These data demonstrate (1) that the lack of GRK sites does not impair PKA site phosphorylation (2) that the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced ß-arrestin binding and receptor internalization, and (3) that serines 355,356,364 play a pivotal role in the GRK-mediated desensitization, ß-arrestin binding and internalization of ß2ARs (Vaughan, 2006).
G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). ß-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of ß-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with ß-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling (Penela, 2001).
These results are consistent with the notion that GRK2-dependent binding of ß-arrestin to GPCRs allows the recruitment of c-Src to the receptor signaling complex at the plasma membrane, leading to phosphorylation of GRK2 on tyrosine residues and its targeting for degradation. This model is in agreement with the rapid ß-arrestin and c-Src recruitment following ß2AR stimulation, and with the agonist-stimulated phosphorylation of GRK2 by c-Src. Under basal conditions, ß-arrestin recruitment to the plasma membrane would be promoted by the activated state of different endogenous GPCRs and/or by the reported basal activity of overexpressed ß2AR. In the presence of GPCR agonists, an acceleration of the GRK2 degradation rate is detected, consistent with a more efficient ß-arrestin and c-Src translocation to the receptor complex. Although detailed knowledge of the sequential assembly of these proteins in a multimolecular complex is lacking, and other molecular interactions may participate in c-Src binding to the receptor complex and GRK2 tyrosine phosphorylation, the proposed model is consistent with the co-immunoprecipitation of ß-arrestin and c-Src, of GRK2 and ß-arrestin and of GRK2 and c-Src. Disruption of the ß-arrestin-c-Src interaction with specific mutants or inhibition of the phosphorylation step by dominant-negative Src or a GRK2 mutant lacking critical phosphorylation sites results, as predicted by this model, in a marked reduction in GRK2 degradation (Penela, 2001).
Light sensitivity and adaptation, general characteristics of rod photoreceptor cell vision, allow rods to modulate their response depending on the lighting environment to which they are exposed. In dim light, rods are maximally sensitive, whereas, in bright light, rods are essentially inactive. In the retinas of dark-adapted mice, arrestin (an inhibitory protein) is located in the rod inner segment (RIS), and transducin (an activating protein) is located in the rod outer segment (ROS). In light-adapted retinas, the proteins have reciprocal localizations. In this study, the data demonstrate that the temporal and spatial changes in the subcellular localization of arrestin and beta-transducin are correlated with the amount of light to which the animals are exposed. By using the frog Xenopus laevis and immunofluorescence confocal microscopy, the results also show that in the dark-adapted retina some arrestin remains in the ROS. The data most dramatically demonstrate that this residual arrestin is highly concentrated in the connecting cilium, the axoneme, and the microtubules associated with the disc incisures. These data suggest a structure-function relationship between the light-dependent positional status of arrestin and the elements of the rod photoreceptor cytoskeleton. The massive, rapid, light-induced reciprocal changes in the subcellular concentrations of these proteins must directly affect phototransduction and appear to be a general phenomenon by which photoreceptor cells rapidly and transiently regulate the trafficking and subcellular concentration of a variety of signal transduction proteins within the RIS and ROS. Hereditary mutations in the components of the movement mechanism should lead to defects in vision and possibly blindness (McGinnis, 2002).
The light-dependent redistribution of phototransduction components in photoreceptor cells plays a role in light adaptation. Upon illumination, rod and cone arrestins (Arr and cArr) translocate from the inner to the outer segments while transducin subunits (Talpha, Tbetagamma) translocate in the opposite direction. The underlying translocation mechanisms are unclear. This study examines these translocations in mice with defective phototransduction. The distribution of Arr, cArr, Talpha, and Tbetagamma was examined using immunoblotting and immunocytochemistry in dark- and light-adapted single knockout mice lacking G-protein coupled receptor kinase 1 (Grk1-/-) and double knockout mice lacking GRK1 and transducin alpha subunit (Grk1-/-/Gnat1-/-), or lacking GRK1 and arrestin (Grk1-/-/Arr-/-). Arr redistributed in the light to the outer segments was studied in Grk1-/- mice as well as in Grk1-/-/Gnat1-/- double knockout retinas. Immunoblotting revealed that approximately 25-50% of Arr associates with the membrane in light-adapted wild-type, Grk1-/- and Gnat1-/-/Grk1-/- mouse retinas. In contrast, cArr does not stably associate with light-adapted membranes in either wild-type or Grk1-/- retinas under these experimental conditions, but redistributes to the cone outer segments in a light-dependent manner. The redistribution of transducin subunits to the inner segments in light occurs in both wild-type and Grk1-/-/Arr-/- double knockout photoreceptors. However, Tbetagamma subunits do not redistribute in the absence of Talpha, suggesting that transducin only translocates as an intact heterotrimer. It is concluded that in rods, Arr redistribution requires neither rhodopsin phosphorylation nor phototransduction, suggesting the presence of another light-dependent pathway to trigger translocation. In cones, the light-dependent movement of cArr appears to be independent of stable association with the cone pigments. The light-dependent translocations of Arr and transducin subunits in opposite directions appear to be based on independent mechanisms (Zhang, 2003).
To test whether kinesin-II is important for transport in the mammalian photoreceptor cilium, and to identify its potential cargoes, Cre-loxP mutagenesis was used to remove the kinesin-II subunit, KIF3A, specifically from photoreceptors. Complete loss of KIF3A caused large accumulations of opsin, arrestin, and membranes within the photoreceptor inner segment, while the localization of alpha-transducin is unaffected. Other membrane, organelle, and transport markers, as well as opsin processing appeared normal. Loss of KIF3A ultimately causes apoptotic photoreceptor cell death similar to a known opsin transport mutant. The data suggest that kinesin-II is required to transport opsin and arrestin from the inner to the outer segment and that blocks in this transport pathway lead to photoreceptor cell death as found in retinitis pigmentosa (Marszalek, 2000).
Light-driven protein translocation is responsible for the dramatic redistribution of some proteins in vertebrate rod photoreceptors. In this study, the involvement of microtubules and microfilaments in the light-driven translocation of arrestin and transducin was investigated. Pharmacologic reagents were applied to native and transgenic Xenopus tadpoles, to disrupt the microtubules (thiabendazole) and microfilaments (cytochalasin D and latrunculin B) of the rod photoreceptors. Quantitative confocal imaging was used to assess the impact of these treatments on arrestin and transducin translocation. A series of transgenic tadpoles expressing arrestin truncations were also created to identify portions of arrestin that enable arrestin to translocate. Application of cytochalasin D or latrunculin B to disrupt the microfilament organization selectively slows only transducin movement from the inner to the outer segments. Perturbation of the microtubule cytoskeleton with thiabendazole slows the translocation of both arrestin and transducin, but only in moving from the outer to the inner segments. Transgenic Xenopus expressing fusions of green fluorescent protein (GFP) with portions of arrestin implicates the C terminus of arrestin as an important portion of the molecule for promoting translocation. This C-terminal region can be used independently to promote translocation of GFP in response to light. These results show that disruption of the cytoskeletal network in rod photoreceptors has specific effects on the translocation of arrestin and transducin. These effects suggest that the light-driven translocation of visual proteins at least partially relies on an active motor-driven mechanism for complete movement of arrestin and transducin (Peterson, 2005).
Proper function of visual arrestin is indispensable for rapid signal shut-off in rod photoreceptors. Dramatic light-dependent changes in arrestin subcellular localization are believed to play an important role in light adaptation of photoreceptor cells. This study shows that visual arrestin binds microtubules. The truncated splice variant of visual arrestin, p44, demonstrates dramatically higher affinity for microtubules than the full-length protein (p48). Enhanced microtubule binding of p44 underlies its earlier reported preferential localization to detergent-resistant membranes, where it is anchored via membrane-associated microtubules in a rhodopsin-independent fashion. Experiments with purified proteins demonstrate that arrestin interaction with microtubules is direct and does not require any additional protein partners. Most important, arrestin interactions with microtubules and light-activated phosphorylated rhodopsin are mutually exclusive, suggesting that microtubule interaction may play a role in keeping p44 arrestin away from rhodopsin in dark-adapted photoreceptors (Nair, 2004).
Agonist-dependent internalization of G protein-coupled receptors via clathrin-coated pits is dependent on the adaptor protein ß-arrestin, which interacts with elements of the endocytic machinery such as AP2 and clathrin. For the ß2-adrenergic receptor (ß2AR) this requires ubiquitination of ß-arrestin by E3 ubiquitin ligase, Mdm2. Based on trafficking patterns and affinity of ß-arrestin, G protein-coupled receptors are categorized into two classes. For class A receptors (e.g., ß2AR), which recycle rapidly, ß-arrestin directs the receptors to clathrin-coated pits but does not internalize with them. For class B receptors (e.g., V2 vasopressin receptors), which recycle slowly, ß-arrestin internalizes with the receptor into endosomes. In COS-7 and human embryonic kidney (HEK)-293 cells, stimulation of the ß2AR or V2 vasopressin receptor leads, respectively, to transient or stable ß-arrestin ubiquitination. The time course of ubiquitination and deubiquitination of ß-arrestin correlates with its association with and dissociation from each type of receptor. Chimeric receptors, constructed by switching the cytoplasmic tails of the two classes of receptors (ß2AR and V2 vasopressin receptors), demonstrate reversal of the patterns of both ß-arrestin trafficking and ß-arrestin ubiquitination. To explore the functional consequences of ß-arrestin ubiquitination a yellow fluorescent protein-tagged ß-arrestin2-ubiquitin chimera was constructed that cannot be deubiquitinated by cellular deubiquitinases. This 'permanently ubiquitinated' ß-arrestin does not dissociate from the ß2AR but rather internalizes with it into endosomes, thus transforming this class A receptor into a class B receptor with respect to its trafficking pattern. Overexpression of this ß-arrestin ubiquitin chimera in HEK-293 cells also results in enhancement of ß2AR internalization and degradation. In the presence of N-ethylmaleimide (an inhibitor of deubiquitinating enzymes), coimmunoprecipitation of the receptor and ß-arrestin is increased dramatically, suggesting that deubiquitination of ß-arrestin triggers its dissociation from the receptor. Thus the ubiquitination status of ß-arrestin determines the stability of the receptor-ß-arrestin complex as well as the trafficking pattern of ß-arrestin (Shenoy, 2003).
Angiotensin II type 1a (AT1a), vasopressin V2, and neurokinin 1 (NK1) receptors are seven-transmembrane receptors (7TMRs) that bind and co-internalize with the multifunctional adaptor protein, ß-arrestin. These receptors also lead to robust and persistent activation of extracellular-signal regulated kinase 1/2 (ERK1/2) localized on endosomes. The co-trafficking of receptor-ß-arrestin complexes to endosomes requires stable ß-arrestin ubiquitination. Lysines at positions 11 and 12 in ß-arrestin2 are specific and required sites for AngII-mediated sustained ubiquitination of arrestin. Thus, upon AngII stimulation the mutant ß-arrestin2(K11,12R) is only transiently ubiquitinated, does not form stable endocytic complexes with the AT1aR, and is impaired in scaffolding-activated ERK1/2. Fusion of a ubiquitin moiety in-frame to ß-arrestin2(K11,12R) restores AngII-mediated trafficking and signaling. Wild type ß-arrestin2 and ß-arrestin2(K11R,K12R)-Ub, but not ß-arrestin2(K11R,K12R), prevent nuclear translocation of pERK. These findings imply that sustained ß-arrestin ubiquitination not only directs co-trafficking of receptor-ß-arrestin complexes but also orchestrates the targeting of '7TMR signalosomes' to microcompartments within the cell. Surprisingly, binding of ß-arrestin2(K11R,K12R) to V2R and NK1R is indistinguishable from that of wild type ß-arrestin2. Moreover, ubiquitination patterns and ERK scaffolding of ß-arrestin2(K11,12R) are unimpaired with respect to V2R stimulation. In contrast, a quintuple lysine mutant [ß-arrestin2(K18R,K107R,K108R,K207R,K296R)] is impaired in endosomal trafficking in response to V2R but not AT1aR stimulation. These findings delineate a novel regulatory mechanism for 7TMR signaling, dictated by the ubiquitination of ß-arrestin on specific lysines that become accessible for modification due to the specific receptor-bound conformational states of ß-arrestin2 (Shenoy, 2005).
Arrestins are important proteins that regulate the function of serpentine heptahelical receptors and contribute to multiple signaling pathways downstream of receptors. The ubiquitous mammalian ß-arrestins are believed to function exclusively as monomers, although self-association is assumed to control the activity of visual arrestin in the retina, where this isoform is particularly abundant. Oligomerization status of ß-arrestins was investigated using different approaches, including co-immunoprecipitation of epitope-tagged ß-arrestins and resonance energy transfer (BRET and FRET) in living cells. At steady state and at physiological concentrations, ß-arrestins constitutively form both homo- and hetero-oligomers. Co-expression of ß-arrestin2 and ß-arrestin1 prevents ß-arrestin1 accumulation into the nucleus, suggesting that hetero-oligomerization may have functional consequences. These data clearly indicate that ß-arrestins can exist as homo- and hetero-oligomers in living cells and raise the hypothesis that the oligomeric state may regulate their subcellular distribution and functions (Storez, 2005).
Wnt proteins, regulators of development in many organisms, bind to seven transmembrane-spanning (7TMS) receptors called frizzleds, thereby recruiting the cytoplasmic molecule dishevelled (Dvl) to the plasma membrane. Frizzled-mediated endocytosis of Wg (a Drosophila Wnt protein) and lysosomal degradation may regulate the formation of morphogen gradients. Endocytosis of Frizzled 4 (Fz4) in human embryonic kidney 293 cells is dependent on added Wnt5A protein and is accomplished by the multifunctional adaptor protein ß-arrestin 2 (ßarr2), which is recruited to Fz4 by binding to phosphorylated Dvl2. These findings provide a previously unrecognized mechanism for receptor recruitment of ß-arrestin and demonstrate that Dvl plays an important role in the endocytosis of frizzled, as well as in promoting signaling (Chen, 2003).
The otd/Otx gene family encodes paired-like homeodomain proteins that are involved in the regulation of anterior head structure and sensory organ development. Using the yeast one-hybrid screen with a bait containing the Ret 4 site from the bovine rhodopsin promoter, a new member of the otd family, Crx (Cone rod homeobox) has been isolated. Crx encodes a 299 amino acid residue protein with a paired-like homeodomain near its N terminus. In the adult, it is expressed predominantly in photoreceptors and pinealocytes. In the developing mouse retina, it is expressed by embryonic day 12.5 (E12.5). Recombinant Crx binds in vitro not only to the Ret 4 site but also to the Ret 1 and BAT-1 sites. In transient transfection studies, Crx transactivates rhodopsin promoter-reporter constructs. Its activity is synergistic with that of Nrl. Crx also binds to and transactivates the genes for several other photoreceptor cell-specific proteins (interphotoreceptor retinoid-binding protein, ß-phosphodiesterase, and arrestin). Human Crx maps to chromosome 19q13.3, the site of a cone rod dystrophy (CORDII). These studies implicate Crx as a potentially important regulator of photoreceptor cell development and gene expression and also identify it as a candidate gene for CORDII and other retinal diseases (Chen, 1997).
The homeobox gene CHX10 is required for retinal progenitor cell proliferation early in retinogenesis and subsequently for bipolar neuron differentiation. To clarify the molecular mechanisms employed by CHX10, attempts were made to identify its target genes. In a yeast one-hybrid assay Chx10 interacted with the Ret1 site of the photoreceptor-specific gene Rhodopsin. Gel shift assays using in vitro translated protein confirmed that CHX10 binds to Ret1, but not to the similar Rhodopsin sites Ret4 and BAT-1. Using retinal nuclear lysates, interactions were observed between Chx10 and additional photoreceptor-specific elements including the PCE-1 (Rod arrestin/S-antigen) and the Cone opsin locus control region (Red/green cone opsin). However, chromatin immunoprecipitation assays revealed that in vivo, Chx10 binds sites upstream of the Rod arrestin and Interphotoreceptor retinoid-binding protein genes but not Rhodopsin or Cone opsin. Thus, in a chromatin context, Chx10 associates with a specific subset of elements that it binds with comparable apparent affinity in vitro. The data suggest that CHX10 may target these motifs to inhibit rod photoreceptor gene expression in bipolar cells (Dorval, 2006).
Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. This study shows that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of ß-arrestin 1 (ßarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, ßarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. These results reveal a novel function of ßarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification (Kang, 2005)
Although regulation of G protein-coupled receptor signaling by receptor kinases and arrestins is a well established biochemical process, the physiological significance of such regulation remains poorly understood. To better understand the in vivo consequences of arrestin function, the function of the sole arrestin in Caenorhabditis elegans, ARR-1, were studied. ARR-1 is primarily expressed in the nervous system, including the HSN neuron and various chemosensory neurons involved in detecting soluble and volatile odorants. arr-1 null mutants exhibit normal chemotaxis but have significant defects in olfactory adaptation and recovery to volatile odorants. In contrast, adaptation is enhanced in animals overexpressing ARR-1. Both the adaptation and recovery defects of arr-1 mutants are rescued by transgenic expression of wild-type ARR-1, whereas expression of a C-terminally truncated ARR-1 effectively rescues only the adaptation defect. A potential mechanistic basis for these findings is revealed by in vitro studies demonstrating that wild-type ARR-1 binds proteins of the endocytic machinery and promotes receptor endocytosis, whereas C-terminally truncated ARR-1 does not. These results demonstrate that ARR-1 functions to regulate chemosensory signaling, enabling organisms to adapt to a variety of environmental cues, and provide an in vivo link between arrestin, receptor endocytosis, and temporal recovery from adaptation (Palmitessa, 2005).
A study was performed to determine whether constitutive signal flow arising from defective rhodopsin shut-off causes photoreceptor cell death in arrestin knockout mice. The retinas of cyclic-light-reared, pigmented arrestin knockout mice and wild-type littermate control mice were examined histologically for photoreceptor cell loss from 100 days to 1 year of age. In separate experiments, to determine whether constant light would accelerate the degeneration in arrestin knockout mice, these animals and wild-type control mice were exposed for 1, 2, or 3 weeks to fluorescent light at an intensity of 115 to 150 fc. The degree of photoreceptor cell loss was quantified histologically by obtaining a mean outer nuclear layer thickness for each animal. In arrestin knockout mice maintained in cyclic light, photoreceptor loss was evident at 100 days of age, and it became progressively more severe, with less than 50% of photoreceptors surviving at 1 year of age. The photoreceptor degeneration appeared to be caused by light, because when these mice were reared in the dark, the retinal structure was indistinguishable from normal. When exposed to constant light, the retinas of wild-type pigmented mice showed no light-induced damage, regardless of exposure duration. By contrast, the retinas of arrestin knockout mice showed rapid degeneration in constant light, with a loss of 30% of photoreceptors after 1 week of exposure and greater than 60% after 3 weeks of exposure. The results indicate that constitutive signal flow due to arrestin knockout leads to photoreceptor degeneration. Excessive light accelerates the cell death process in pigmented arrestin knockout mice. Human patients with naturally occurring mutations that lead to nonfunctional arrestin and rhodopsin kinase have Oguchi disease, a form of stationary night blindness. The present findings suggest that such patients may be at greater risk of the damaging effects of light than those with other forms of retinal degeneration, and they provide an impetus to restrict excessive light exposure as a protective measure in patients with constitutive signal flow in phototransduction (Chen, 1999).
Retinitis pigmentosa (RP) is a clinically and genetically heterogeneous degenerative eye disease. Mutations at Arg135 of rhodopsin are associated with a severe form of autosomal dominant RP. This report presents evidence that Arg135 mutant rhodopsins (e.g., R135L, R135G, and R135W) are hyperphosphorylated and bind with high affinity to visual arrestin. Mutant rhodopsin recruits the cytosolic arrestin to the plasma membrane, and the rhodopsin-arrestin complex is internalized into the endocytic pathway. Furthermore, the rhodopsin-arrestin complexes alter the morphology of endosomal compartments and severely damage receptor-mediated endocytic functions. The biochemical and cellular defects of Arg135 mutant rhodopsins are distinct from those previously described for class I and class II RP mutations, and, hence, it is proposed that they be named class III. Impaired endocytic activity may underlie the pathogenesis of RP caused by class III rhodopsin mutations (Chuang, 2004).
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