Studies with chimeras of the gonadotropin receptors reveal the importance of third intracellular loop threonines on the formation of the receptor/nonvisual arrestin complex

Using chimeras and more discrete exchange mutations of the rat (r) and human (h) gonadotropin receptors, we had previously identified multiple noncontiguous residues of the lutropin (LHR) and follitropin (FSHR) receptors that dictate their rates of internalization. Since the internalization of the LHR and the FSHR is driven by their abilities to associate with the nonvisual arrestins, we hypothesized that one or more of the residues previously identified by the internalization assays are involved in the formation of the receptor/nonvisual arrestin complex. In the studies reported herein, we tested this hypothesis by measuring the association of arrestin-3 with a large number of rLHR/hLHR and rFSHR/hFSHR exchange mutants that affect internalization. The results presented show that the same residues that dictate the rate of internalization of these two receptor pairs affect their ability to associate with arrestin-3. Although these residues are located in distinct topological domains, our analyses show that threonine residues in the third intracellular loop of both receptor pairs are particularly important for the formation of the receptor/arrestin-3 complexes and internalization. We conclude that the different rates of internalization of the gonadotropin receptors are dictated by their different abilities to associate with the nonvisual arrestins and that this association is, in turn, largely dictated by the presence of threonine residues in their third intracellular loops.     

Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.     

Mutations of the second extracellular loop of the human lutropin receptor emphasize the importance of receptor activation and de-emphasize the importance of receptor phosphorylation in agonist-induced internalization

Alanine scanning mutagenesis of the second extracellular loop of the human lutropin receptor (hLHR) showed that mutation of most of the residues present in this region either enhance or impair the internalization of agonist. A more complete analysis of four mutants, two that enhanced internalization (F515A and T521A) and two that impaired internalization (S512A and V519A), showed that the two mutants that impaired internalization also show a decrease in the sensitivity for agonist-induced cAMP accumulation, whereas the two mutants that enhanced internalization show an increase in the sensitivity for agonist-induced cAMP accumulation. None of these mutants had an effect on the agonist-induced phosphorylation of the hLHR, however. We conclude that, in contrast to the prevailing view of the relative importance of receptor phosphorylation in the internalization of G protein-coupled receptors, the phosphorylation of the hLHR is less important than the agonist-induced activation of the hLHR in the process of internalization.     

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.     

Small ubiquitin-like modifier modification of arrestin-3 regulates receptor trafficking

Nonvisual arrestins are regulated by direct post-translational modifications, such as phosphorylation, ubiquitination, and nitrosylation. However, whether arrestins are regulated by other post-translational modifications remains unknown. Here we show that nonvisual arrestins are modified by small ubiquitin-like modifier 1 (SUMO-1) upon activation of β(2)-adrenergic receptor (β(2)AR). Lysine residues 295 and 400 in arrestin-3 fall within canonical SUMO consensus sites, and mutagenic analysis reveals that Lys-400 represents the main SUMOylation site. Depletion of the SUMO E2 modifying enzyme Ubc9 blocks arrestin-3 SUMOylation and attenuates β(2)AR internalization, suggesting that arrestin SUMOylation mediates G protein-coupled receptor endocytosis. Consistent with this, expression of a SUMO-deficient arrestin mutant failed to promote β(2)AR internalization as compared with wild-type arrestin-3. Our data reveal an unprecedented role for SUMOylation in mediating GPCR endocytosis and provide novel mechanistic insight into arrestin function and regulation.     

The role of phosphorylation in D1 dopamine receptor desensitization: evidence for a novel mechanism of arrestin association

Homologous desensitization of D(1) dopamine receptors is thought to occur through their phosphorylation leading to arrestin association which interdicts G protein coupling. In order to identify the relevant domains of receptor phosphorylation, and to determine how this leads to arrestin association, we created a series of mutated D(1) receptor constructs. In one mutant, all of the serine/threonine residues within the 3rd cytoplasmic domain were altered (3rdTOT). A second construct was created in which only three of these serines (serines 256, 258, and 259) were mutated (3rd234). We also created four truncation mutants of the carboxyl terminus (T347, T369, T394, and T404). All of these constructs were comparable with the wild-type receptor with respect to expression and adenylyl cyclase activation. In contrast, both of the 3rd loop mutants exhibited attenuated agonist-induced receptor phosphorylation that was correlated with an impaired desensitization response. Sequential truncation of the carboxyl terminus of the receptor resulted in a sequential loss of agonist-induced phosphorylation. No phosphorylation was observed with the most severely truncated T347 mutant. Surprisingly, all of the truncated receptors exhibited normal desensitization. The ability of the receptor constructs to promote arrestin association was evaluated using arrestin-green fluorescent protein translocation assays and confocal fluorescence microscopy. The 3rd234 mutant receptor was impaired in its ability to induce arrrestin translocation, whereas the T347 mutant was comparable with wild type. Our data suggest a model in which arrestin directly associates with the activated 3rd cytoplasmic domain in an agonist-dependent fashion; however, under basal conditions, this is sterically prevented by the carboxyl terminus of the receptor. Receptor activation promotes the sequential phosphorylation of residues, first within the carboxyl terminus and then the 3rd cytoplasmic loop, thereby dissociating these domains and allowing arrestin to bind to the activated 3rd loop. Thus, the role of receptor phosphorylation is to allow access of arrestin to its receptor binding domain rather than to create an arrestin binding site per se.     

Characterization of G protein-coupled receptor regulation in antisense mRNA-expressing cells with reduced arrestin levels.

Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins have established a role for these proteins in beta2-adrenergic receptor (beta2AR) internalization, desensitization, and resensitization. To validate and extend such findings, we employed an antisense strategy to target the nonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of G protein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targeting arrestin-2 exhibited a selective reduction (approximately 50%) in arrestin-2 levels, while arrestin-3 antisense constructs resulted in reductions (>/=50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cells demonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent of agonist-induced internalization of exogenously expressed beta2ARs, but had no effect on internalization of either m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing the role of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantly decreased the rate of endogenous beta2AR internalization, desensitization, and resensitization. Further analysis demonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels. The effects on the beta2-adrenergic, A2b adenosine, and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction in arrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility of antisense approaches in the examination of arrestin-mediated GPCR regulation.     

The nature of the arrestin x receptor complex determines the ultimate fate of the internalized receptor

The vast majority of G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation of an active receptor followed by arrestin binding. The arrestin x receptor complex is then internalized. Internalized receptor can be recycled back to the plasma membrane (resensitization) or targeted to lysosomes for degradation (down-regulation). The intracellular compartment where this choice is made and the molecular mechanisms involved are largely unknown. Here we used two arrestin2 mutants that bind with high affinity to phosphorylated and unphosphorylated agonist-activated beta 2-adrenergic receptor to manipulate the receptor-arrestin interface. We found that mutants support rapid internalization of beta 2-adrenergic receptor similar to wild type arrestin2. At the same time, phosphorylation-independent arrestin2 mutants facilitate receptor recycling and sharply reduce the rate of receptor loss, effectively protecting beta 2-adrenergic receptor from down-regulation even after very long (up to 24 h) agonist exposure. Phosphorylation-independent arrestin2 mutants dramatically reduce receptor phosphorylation in response to an agonist both in vitro and in cells. Interestingly, co-expression of high levels of beta-adrenergic receptor kinase restores receptor down-regulation in the presence of mutants to the levels observed with wild type arrestin2. Our data suggest that unphosphorylated receptor internalized in complex with mutant arrestins recycles faster than phosphoreceptor and is less likely to get degraded. Thus, targeted manipulation of the characteristics of an arrestin protein that binds to a G protein-coupled receptors can dramatically change receptor trafficking and its ultimate fate in a cell.     

Inhibition of chemoattractant N-formyl peptide receptor trafficking by active arrestins

Recent studies have highlighted the emergence of a class of G protein-coupled receptors that are internalized in an arrestin-independent manner. In addition to demonstrating that the N-formyl peptide receptor belongs in this family, we have recently shown that recycling of the receptor requires the presence of arrestins. To further elucidate mechanisms of arrestin-dependent regulation of G protein-coupled receptor processing, we examined the effects of altering the receptor-arrestin complex on ternary complex formation and cellular trafficking of the N-formyl peptide receptor by studying two active arrestin-2 mutants (truncated arrestin-2 [1-382], and arrestin-2 I386A, V387A, F388A). Complexes between the N-formyl peptide receptor and active arrestins exhibited higher affinity in vitro than the complex between the N-formyl peptide receptor and wild-type arrestin and furthermore were observed in vivo by colocalization studies using confocal microscopy. To assess the effects of these altered interactions on receptor trafficking, we demonstrated that active, but not wild-type, arrestin expression retards N-formyl peptide receptor internalization. Furthermore, expression of arrestin-2 I386A/V387A/F388A but not arrestin-2 [1-382] inhibited recycling of the N-formyl peptide receptor, reflecting an expanded role for arrestins in G protein-coupled receptor processing and trafficking. Whereas the extent of N-formyl peptide receptor phosphorylation had no effect on the inhibition of internalization, N-formyl peptide receptor recycling was restored when the receptor was only partially phosphorylated. These results indicate not only that a functional interaction between receptor and arrestin is required for recycling of certain G protein-coupled receptors, such as the N-formyl peptide receptor, but that the pattern of receptor phosphorylation further regulates this process.     

Agonist-induced internalization of the platelet-activating factor receptor is dependent on arrestins but independent of G-protein activation. Role of the C terminus and the (D/N)PXXY motif.

As with most G-protein-coupled receptors, repeated agonist stimulation of the platelet-activating factor receptor (PAFR) results in its desensitization, sequestration, and internalization. In this report, we show that agonist-induced PAFR internalization is independent of G-protein activation but is dependent on arrestins and involves the interaction of arrestins with a limited region of the PAFR C terminus. In cotransfected COS-7 cells, both arrestin-2 and arrestin-3 could be coimmunoprecipitated with PAFR, and agonist stimulation of PAFR induced the translocation of both arrestin-2 and arrestin-3. Furthermore, coexpression of arrestin-2 with PAFR potentiated receptor internalization, whereas agonist-induced PAFR internalization was inhibited by a dominant negative mutant of arrestin-2. The coexpression of a minigene encoding the C-terminal segment of the receptor abolished PAF-induced arrestin translocation and inhibited PAFR internalization. Using C terminus deletion mutants, we determined that the association of arrestin-2 with the receptor was dependent on the region between threonine 305 and valine 330 because arrestin-2 could be immunoprecipitated with the mutant PAFRstop330 but not PAFRstop305. Consistently, stop330 could mediate agonist-induced arrestin-2 translocation, whereas stop305 could not. Two other deletion mutants with slightly longer regions of the C terminus, PAFRstop311 and PAFRstop317, also failed to induce arrestin-2 translocation. Finally, the PAFR mutant Y293A, containing a single substitution in the putative internalization motif DPXXY in the seventh transmembrane domain (which we had shown to be able to internalize but not to couple to G-proteins) could efficiently induce arrestin translocation. Taken together, our results indicate that ligand-induced PAFR internalization is dependent on arrestins, that PAFR can associate with both arrestin-2 and -3, and that their translocation involves interaction with the region of residues 318-330 in the PAFR C terminus but is independent of G-protein activation.     

N-formyl peptide receptor phosphorylation domains differentially regulate arrestin and agonist affinity

Arrestins regulate the signaling and endocytosis of many G protein-coupled receptors (GPCRs). It has been suggested that the functions of arrestins are dependent upon both the number and pattern of phosphorylation sites present in an activated GPCR. However, little is currently known about the relationships between the sites of receptor phosphorylation, the resulting affinities of arrestin binding, and the ensuing mechanisms of receptor regulation for any given GPCR. To investigate these interactions, we used an active truncated mutant of arrestin (amino acids 1-382) and phosphorylation-deficient mutants of the N-formyl peptide receptor (FPR). In contrast to results with wild type arrestins, the truncated arrestin-2 protein bound to the unphosphorylated wild type FPR, although with lower affinity and a low affinity for the agonist as revealed by competition studies with heterotrimeric G proteins. Using FPR mutants, we further demonstrated that the phosphorylation status of serines and threonines between residues 328-332 is a key determinant that regulates the affinity of the FPR for arrestins. Furthermore, we found that the phosphorylation status of serine and threonine residues between amino acids 334 and 339 regulates the affinity of the receptor for agonist when arrestin is bound. These results suggest that the agonist affinity state of the receptor is principally regulated by phosphorylation at specific sites and is not simply a consequence of arrestin binding as has previously been proposed. Furthermore, this is the first demonstration that agonist affinity of a GPCR and the affinity of arrestin binding to the phosphorylated receptor are regulated by distinct receptor phosphodomains.     

Arrestin binding to the M(2) muscarinic acetylcholine receptor is precluded by an inhibitory element in the third intracellular loop of the receptor

Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation-independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation.     

Arrestin isoforms dictate differential kinetics of A2B adenosine receptor trafficking

Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A(2A) and A(2B) adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study, we investigate the role of arrestins in A(2B)AR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A(2B)AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (<1 min) from the cytosol to the plasma membrane following A(2B)AR activation. However, longer incubations with agonist (>10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR in rab-5 and transferrin receptor containing early endosomes. At later times, the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensitization.     

Association of beta-Arrestin 1 with the type 1A angiotensin II receptor involves phosphorylation of the receptor carboxyl terminus and correlates with receptor internalization

Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT(1A)) angiotensin II (AngII) receptors, the contribution of arrestins to AT(1A) receptor internalization is controversial, and the physical association of arrestins with the AT(1A) receptor has not been established. In this study, by coimmunoprecipitating AT(1A) receptors and beta-arrestin 1, we provide direct evidence for an association between arrestins and the AT(1A) receptor that was agonist- and time-dependent and contingent upon the level of beta-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of beta-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT(1A) receptor to lysine(325) prevented AngII-induced phosphorylation and beta-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT(1A) receptor carboxyl terminus (Thr(332), Ser(335), Thr(336), Ser(338)) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser(346), Ser(347), Ser(348)) and at three PKC phosphorylation sites (Ser(331), Ser(338), Ser(348)) had no effect on AngII-induced beta-arrestin 1 association or receptor internalization. While AT(1A) receptor internalization could be inhibited by a dominant-negative beta-arrestin 1 mutant (beta arr1(319-418)), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII, [Sar(1)Ile(4)Ile(8)]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong beta-arrestin 1 association with the full-length AT(1A) receptor. These results identify the central portion of the AT(1A) receptor carboxyl terminus as the important determinant for beta-arrestin 1 binding and internalization and indicate that AT(1A) receptor phosphorylation is crucial for beta-arrestin docking.     

Phosphorylation uncouples the gastrin-releasing peptide receptor from G(q)

Previous work on the desensitization of G protein-coupled receptors has focused on the role of arrestin binding following receptor phosphorylation. We have examined the hypothesis that phosphorylation alone contributes to desensitization. In this study we demonstrate that for the G(q)-coupled gastrin-releasing peptide receptor (GRP-R), phosphorylation by GRK2 to a stoichiometry of approximately 1 mol PO(4)/mol GRP-R is sufficient in the absence of arrestin to reduce the rate of receptor catalyzed G protein activation by approximately 80%. Furthermore, GRP-Rs exposed in vivo to agonist are rapidly phosphorylated to a similar stoichiometry and are desensitized to a similar degree. Finally, the molecular mechanism for both in vitro GRK2-induced and in vivo agonist-induced desensitization is primarily a decrease in the maximum velocity (V(max)) for the catalysis of guanine nucleotide exchange by the GRP-R rather than a change in the affinity of the receptor for the alpha(q) or betagamma subunits. Based on these results, we suggest that, for some G protein-coupled receptors, phosphorylation has a role in desensitization that is independent of arrestin.     

Arrestin variants display differential binding characteristics for the phosphorylated N-formyl peptide receptor carboxyl terminus.

The phosphorylation-dependent binding of arrestins to cytoplasmic domains of G protein-coupled receptors (GPCRs) is thought to be a crucial step in receptor desensitization. In some GPCR systems, arrestins have also been demonstrated to be involved in receptor internalization, resensitization, and the activation of signaling cascades. The objective of the current study was to examine binding interactions of members of the arrestin family with the formyl peptide receptor (FPR), a member of the GPCR family of receptors. Peptides representing the unphosphorylated and phosphorylated carboxyl terminus of the FPR were synthesized and bound to polystyrene beads via a biotin/streptavidin interaction. Using fluorescein-conjugated arrestins, binding interactions between arrestins and the bead-bound FPR carboxyl terminus were analyzed by flow cytometry. Arrestin-2 and arrestin-3 bound to the FPR carboxyl-terminal peptide in a phosphorylation-dependent manner, with K(d) values in the micromolar range. Binding of visual arrestin, which binds rhodopsin with high selectivity, was not observed. Arrestin-2-(1--382) and arrestin-3-(1--393), truncated mutant forms of arrestin that display phosphorylation-independent binding to intact receptors, were also observed to bind the bead-bound FPR terminus in a phosphorylation-dependent manner, but with much greater affinity than the full-length arrestins, yielding K(d) values in the 5--50 nm range. Two additional arrestin mutants, which are full-length but display phosphorylation-independent binding to intact GPCRs, were evaluated for their binding affinity to the FPR carboxyl terminus. Whereas the single point mutant, arrestin-2 R169E, displayed an affinity similar to that of the full-length arrestins, the triple point mutant, arrestin-2 I386A/V387A/F388A, displayed an affinity more similar to that of the truncated forms of arrestin. The results suggest that the carboxyl terminus of arrestin is a critical determinant in regulating the binding affinity of arrestin for the phosphorylated domains of GPCRs.     

Role of the rate of internalization of the agonist-receptor complex on the agonist-induced down-regulation of the lutropin/choriogonadotropin receptor.

The extent of agonist-induced down-regulation of the LH/CG receptor (LHR) in human kidney 293 cells transfected with the rat LHR (rLHR) is much lower than in two Leydig tumor cell lines (MA-10 and R2C) that express the rodent LHR endogenously. This difference can not be attributed to differences in the recycling of internalized receptors, or in the replenishment of new receptors at the cell surface. It can be correlated, however, with the half-life of internalization of the bound agonist, which is approximately 60 min in Leydig tumor cells and about 100 min in transfected 293 cells. To determine whether the rate of internalization of the bound agonist affects down-regulation, we compared these two parameters in 293 cells expressing four rLHR mutants that enhance internalization and three mutants that impair internalization. We show that all four mutations of the rLHR that enhanced internalization enhanced down-regulation, while only one of the three mutations that impaired internalization impaired down-regulation. In addition, cotransfections of 293 cells with the rLHR-wt and three constructs that enhanced internalization (G protein-coupled receptor kinase 2, beta-arrestin, and arrestin-3) increased down-regulation, while a related construct (visual arrestin) that had no effect on internalization also had no effect on down-regulation. We conclude that the rate of internalization of the agonist-LHR complex is the main determinant of the extent of down-regulation of the LHR.     

mu-Opioid receptors desensitize less rapidly than delta-opioid receptors due to less efficient activation of arrestin

Receptor desensitization by G-protein receptor kinases (GRK) and arrestins is likely to be an important component underlying the development of tolerance to opioid drugs. Reconstitution of this process in Xenopus oocytes revealed distinct differences in the kinetics of GRK and arrestin regulation of the closely related opioid receptors mu (MOR), delta (DOR), and kappa (KOR). We demonstrated that under identical conditions, GRK and arrestin-dependent desensitization of MOR proceeds dramatically slower than that of DOR. Furthermore, GRK3 phosphorylation sites required for opioid receptor desensitization also greatly differ. The determinants for DOR and KOR desensitization reside in the carboxyl-terminal tail, whereas MOR depends on Thr-180 in the second intracellular loop. Although this later finding might indicate an inefficient phosphorylation of MOR Thr-180, increasing the amount of arrestin expressed greatly increased the rate of MOR desensitization to a rate comparable with that of DOR. Similarly, coexpression of a constitutively active arrestin 2(R169E) with MOR and DOR desensitized both receptors in an agonist-dependent, GRK-independent manner at rates that were indistinguishable. Together, these data suggest that it is the activation of arrestin, rather than its binding, that is the rate-limiting step in MOR desensitization. In addition, mutation of Thr-161 in DOR, homologous to MOR Thr-180, significantly inhibited the faster desensitization of DOR. These results suggest that DOR desensitization involves phosphorylation of both the carboxyl-terminal tail and the second intracellular loop that together leads to a more efficient activation of arrestin and thus faster desensitization.     

Conservation of the phosphate-sensitive elements in the arrestin family of proteins.

Arrestins play a key role in the homologous desensitization of G protein-coupled receptors (GPCRs). These cytosolic proteins selectively bind to the agonist-activated and GPCR kinase-phosphorylated forms of the GPCR, precluding its further interaction with the G protein. Certain mutations in visual arrestin yield "constitutively active" proteins that bind with high affinity to the light-activated form of rhodopsin without requiring phosphorylation. The crystal structure of visual arrestin shows that these activating mutations perturb two groups of intramolecular interactions that keep arrestin in its basal (inactive) state. Here we introduced homologous mutations into arrestin2 and arrestin3 and found that the resulting mutants bind to the beta(2)-adrenoreceptor in vitro in a phosphorylation-independent fashion. The same mutants effectively desensitize both the beta(2)-adrenergic and delta-opioid receptors in the absence of receptor phosphorylation in Xenopus oocytes. Moreover, the arrestin mutants also desensitize the truncated delta-opioid receptor from which the C terminus, containing critical phosphorylation sites, has been removed. Conservation of the phosphate-sensitive hot spots in non-visual arrestins suggests that the overall fold is similar to that of visual arrestin and that the mechanisms whereby receptor-attached phosphates drive arrestin transition into the active binding competent state are conserved throughout the arrestin family of proteins.     

Partial phosphorylation of the N-formyl peptide receptor inhibits G protein association independent of arrestin binding

It is now well accepted that G protein-coupled receptors activated by agonist binding become targets for phosphorylation, leading to desensitization of the receptor. Using a series of phosphorylation deficient mutants of the N-formyl peptide receptor (FPR), we have explored the role of phosphorylation on the ability of the receptor to interact with G proteins and arrestins. Using a fluorometric assay in conjunction with solubilized receptors, we demonstrate that phosphorylation of the wild type FPR lowers its affinity for G protein, whereas mutant receptors lacking four potential phosphorylation sites retain their ability to couple to G protein. Phosphorylated mutant receptors lacking only two potential phosphorylation sites are again unable to couple to G protein. Furthermore, whereas stimulated wild type FPR in whole cells colocalizes with arrestin-2, and the solubilized, phosphorylated FPR binds arrestin-2, the stimulated receptors lacking four potential phosphorylation sites display no interaction with arrestin-2. However, the mutant receptors lacking only two potential phosphorylation sites are restored in their ability to bind and colocalize with arrestin-2. Thus, there is a submaximal threshold of FPR phosphorylation that simultaneously results in an inhibition of G protein binding and an induction of arrestin binding. These results are the first to demonstrate that less than maximal levels of receptor phosphorylation can block G protein binding, independent of arrestin binding. We therefore propose that phosphorylation alone may be sufficient to desensitize the FPR in vivo, raising the possibility that for certain G protein-coupled receptors, desensitization may not be the primary function of arrestin.