Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus

The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arrestin- and clathrin-independent pathway.     

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.     

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.     

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.     

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.     

Differential roles of arrestin-2 interaction with clathrin and adaptor protein 2 in G protein-coupled receptor trafficking

The non-visual arrestins, arrestin-2 and arrestin-3, play a critical role in regulating the signaling and trafficking of many G protein-coupled receptors (GPCRs). Molecular insight into the role of arrestins in GPCR trafficking has suggested that arrestin interaction with clathrin, beta(2)-adaptin (the beta-subunit of the adaptor protein AP2), and phosphoinositides contributes to this process. In the present study, we have attempted to better define the molecular basis and functional role of arrestin-2 interaction with clathrin and beta(2)-adaptin. Site-directed mutagenesis revealed that the C-terminal region of arrestin-2 mediated beta(2)-adaptin and clathrin interaction with Phe-391 and Arg-395 having an essential role in beta(2)-adaptin binding and LIELD (residues 376-380) having an essential role in clathrin binding. Interestingly, arrestin-2-R169E, an activated form of arrestin that binds to GPCRs in a phosphorylation-independent manner, has significantly enhanced binding to beta(2)-adaptin and clathrin. This suggests that receptor-induced conformational changes in the C-terminal tail of arrestin-2 will likely play a major role in mediating arrestin interaction with clathrin-coated pits. In an effort to clarify the role of these interactions in GPCR trafficking we generated arrestin mutants that were completely and selectively defective in either clathrin (arrestin-2-DeltaLIELD) or beta(2)-adaptin (arrestin-2-F391A) interaction. Analysis of these mutants in COS-1 cells revealed that arrestin/clathrin interaction was essential for agonist-promoted internalization of the beta(2)-adrenergic receptor, while arrestin/beta(2)-adaptin interaction appeared less critical. Arrestin-2 mutants defective in both clathrin and beta(2)-adaptin binding functioned as effective dominant negatives in HEK293 cells and significantly attenuated beta(2)-adrenergic receptor internalization. These mutants should prove useful in better defining the role of arrestins in mediating receptor trafficking.     

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.     

Identification of a short linear sequence present in the C-terminal tail of the rat follitropin receptor that modulates arrestin-3 binding in a phosphorylation-independent fashion

The rat follitropin receptor (rFSHR) is an unusual G protein-coupled receptor in that agonist-induced activation leads to the phosphorylation of the first and third intracellular loops instead of the C-terminal tail. To determine regions of G protein-coupled receptors that affect internalization independently of phosphorylation we examined the effects of truncations of the C-terminal tail of the rFSHR on agonist-induced internalization. Our studies show that progressive truncations of a region flanked by residues 642 and 651 enhance the internalization of human follicle-stimulating hormone (hFSH). Further characterization of a mutant truncated at residue 649 (designated rFSHR-t649) and another mutant in which the 642-651 region was deleted in the context of the full-length rFSHR, designated rFSHR(Delta642-651), showed that both of them internalized hFSH at rates that were 2-3 times faster than rFSHR-wild type (wt). Like rFSHR-wt, however, the internalization of hFSH mediated by rFSHR-t649 and rFSHR(Delta642-651) can be inhibited with dominant-negative mutants of the non-visual arrestins or dynamin. Alanine-scanning mutagenesis of the 642-651 region suggests that the effects on internalization are not mediated by a single residue, however. In an attempt to understand the molecular basis of the enhanced internalization of hFSH mediated by these mutants we used an assay that can be readily used to assess the association of the rFSHR with the arrestin-3 in co-transfected cells. Using this assay we were able to show that, when compared with rFSHR-wt, rFSHR(Delta642-651) displays an approximately 4-fold enhancement in binding affinity for arrestin-3 and an approximately 1.7-fold reduction in maximal arrestin-3 binding capacity. We conclude that a short linear sequence present in the C-terminal tail of the rFSHR (642SATHNFHARK651) that is not phosphorylated limits internalization by lowering the affinity of the rFSHR for the endogenous non-visual arrestins.     

Role of arrestins in endocytosis and signaling of alpha2-adrenergic receptor subtypes

We investigated the role of arrestins in the trafficking of human alpha2-adrenergic receptors (alpha2-ARs) and the effect of receptor trafficking on p42/p44 MAP kinase activation. alpha2-ARs expressed in COS-1 cells demonstrated a modest level of agonist-mediated internalization, with alpha2c > alpha2b > alpha2a. However, upon coexpression of arrestin-2 (beta-arrestin-1) or arrestin-3 (beta-arrestin-2), internalization of the alpha2b AR was dramatically enhanced and redistribution of receptors to clathrin coated vesicles and endosomes was observed. Internalization of the alpha2c AR was selectively promoted by coexpression of arrestin-3, while alpha2a AR internalization was only slightly stimulated by coexpression of either arrestin. Coexpression of GRK2 had no effect on the internalization of any alpha2-AR subtype, either in the presence or absence of arrestins. Internalization of the alpha2b and alpha2c ARs was inhibited by coexpression of dominant negative dynamin-K44A. However, alpha2-AR-mediated activation of either endogenous or cotransfected p42/p44 mitogen-activated protein (MAP) kinase was not affected by either dynamin-K44A or arrestin-3. Moreover, activation of p42/p44 MAP kinase by endogenous epidermal growth factor, lysophosphatidic acid, and beta2-adrenergic receptors was also unaltered by dynamin-K44A. In summary, our data suggest that internalization of the alpha2b, alpha2c, and to a lesser extent alpha2a ARs, is both arrestin- and dynamin-dependent. However, endocytosis does not appear to be required for alpha2-adrenergic, epidermal growth factor, lysophosphatidic acid, or beta2-adrenergic receptor-mediated p42/p44 MAP kinase activation in COS-1 cells.     

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.     

The third intracellular loop of alpha 2-adrenergic receptors determines subtype specificity of arrestin interaction

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the alpha(2b)- and alpha(2c)-adrenergic receptors (ARs) while having no effect on the alpha(2a)AR, here we used alpha(2)ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the alpha(2a)AR, alpha(2b)AR, or alpha(2c)AR. These studies revealed that arrestin-3 directly binds to the alpha(2b)AR and alpha(2c)AR but not the alpha(2a)AR, whereas arrestin-2 only binds to the alpha(2b)AR. Truncation mutagenesis of the alpha(2b)AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the alpha(2b)AR third intracellular loop. Mutation of these residues in the holo-alpha(2b)AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant alpha(2b)AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the alpha(2b)AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.     

Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus

Platelet-activating factor (PAF) is a phospholipid with multiple physiological and pathological actions. The PAF receptor (PAFR) belongs to the G protein-coupled, heptahelical receptor superfamily. Recently, we have shown that PAF signals through the Janus kinase (Jak)/STAT pathway and that Tyk2 plays an essential role in PAF-induced PAFR promoter 1 activation. In the present study we found that PAF stimulated Jak2 tyrosine phosphorylation in the monocytic cell line MonoMac-1 as well as in COS-7 cells transfected with PAFR and Jak2 cDNAs. The use of a G protein-uncoupled PAFR (D289A) mutant indicated that Jak2 activation was G protein independent. Interestingly, following PAF stimulation, Jak2 coimmunoprecipitated with PAFR in the presence of active Tyk2, but not with a kinase-inactive Tyk2 mutant, K930I. Moreover, Tyk2-K930I completely blocked PAF-stimulated Jak2 phosphorylation. Gradual deletion of C-terminal residues of the PAFR resulted in progressively decreased Jak2 activation. Deletion of 12 C-terminal residues in mutant V330Stop diminished Jak2 tyrosine phosphorylation by 17%. Further deletions of 25-37 residues from the PAFR C-tail (C317Stop, M311Stop, and T305Stop) resulted in a 50% decrease in Jak2 phosphorylation compared with the wild-type receptor. Complete removal of the C tail resulted in a mutant (K298Stop) that failed to activate Jak2, suggesting that the receptor C-terminal region contains important domains for Jak2 activation. Finally, the coexpression of a minigene encoding the C terminus of PAFR partially inhibited PAF-induced kinase activation. Taken together, our results indicate that PAF activates Jak2 and that Tyk2 and the C-terminal tail of PAFR are of critical importance for PAF-induced Jak2 activation.     

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.     

Internalization and sequestration of the human prostacyclin receptor

Prostacyclin (PGI(2)), the major product of cyclooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of agonist-induced desensitization (Smyth, E. M., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C terminus to the green fluorescent protein (GFP), was coupled to increased cAMP (EC(50) = 0.39 +/- 0.09 nm) and inositol phosphate (EC(50) = 86. 6 +/- 18.3 nm) generation when overexpressed in HEK 293 cells. Iloprost-induced sequestration of HAhIP-GFP, followed in real time by confocal microscopy, was partially colocalized to clathrin-coated vesicles. Iloprost induced a time- and concentration-dependent loss of cell surface HA, indicating receptor internalization, which was prevented by inhibitors of clathrin-mediated trafficking and partially reduced by cotransfection of cells with a dynamin dominant negative mutant. Sequestration (EC(50) = 27.6 +/- 5.7 nm) was evident at those concentrations of iloprost that induce PKC-dependent desensitization. Neither the PKC inhibitor GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, altered receptor sequestration indicating that, unlike desensitization, internalization is PKC-independent. Deletion of the C terminus prevented iloprost-induced internalization, demonstrating the critical nature of this region for sequestration. Internalization was unaltered by cotransfection of cells with G protein-coupled receptor kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant negative mutant, indicating that GRKs and arrestins do not play a role in hIP trafficking. The hIP is sequestered in response to agonist activation via a PKC-independent pathway that is distinct from desensitization. Trafficking is dependent on determinants located in the C terminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-dependent clathrin-coated vesicular endocytotic pathway although other dynamin-independent pathways may also be involved.     

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.     

Arrestin binding to the G protein-coupled N-formyl peptide receptor is regulated by the conserved "DRY" sequence

Following activation by ligand, the N-formyl peptide receptor (FPR) undergoes processing events initiated by phosphorylation that lead to receptor desensitization and internalization. Our previous results have shown that FPR internalization can occur in the absence of receptor desensitization, suggesting that FPR desensitization and internalization are controlled by distinct mechanisms. More recently, we have provided evidence that internalization of the FPR occurs via a mechanism that is independent of the actions of arrestin, dynamin, and clathrin. In the present report, we demonstrate that stimulation of the FPR with agonist leads to a significant translocation of arrestin-2 from the cytosol to the membrane. Fluorescence microscopy revealed that the translocated arrestin-2 is highly colocalized with the ligand-bound FPR. A D71A mutant FPR, which does not undergo activation or phosphorylation in response to ligand, did not colocalize with arrestin-2. Surprisingly, an R123G mutant FPR, which does not bind G protein but does become phosphorylated and subsequently internalized, also did not bind arrestin. These results indicate that arrestin binding is not required for FPR internalization and demonstrate for the first time that a common motif, the conserved "DRY" domain of G protein-coupled receptors, is essential for phosphorylation-dependent arrestin binding, as well as G protein activation.     

Internalization of the TXA2 receptor alpha and beta isoforms. Role of the differentially spliced cooh terminus in agonist-promoted receptor internalization

Thromboxane A2 (TXA2) potently stimulates platelet aggregation and smooth muscle constriction and is thought to play a role in myocardial infarction, atherosclerosis, and bronchial asthma. The TXA2 receptor (TXA2R) is a member of the G protein-coupled receptor family and is found as two alternatively spliced isoforms, alpha (343 residues) and beta (407 residues), which share the first 328 residues. In the present report, we demonstrate by enzyme-linked immunosorbent assay and immunofluorescence microscopy that the TXA2Rbeta, but not the TXA2Ralpha, undergoes agonist-induced internalization when expressed in HEK293 cells as well as several other cell types. Various dominant negative mutants were used to demonstrate that the internalization of the TXA2Rbeta is dynamin-, GRK-, and arrestin-dependent in HEK293 cells, suggesting the involvement of receptor phosphorylation and clathrin-coated pits in this process. Interestingly, the agonist-stimulated internalization of both the alpha and beta isoforms, but not of a mutant truncated after residue 328, can be promoted by overexpression of arrestin-3, identifying the C-tails of both receptors as necessary in arrestin-3 interaction. Simultaneous mutation of two dileucine motifs in the C-tail of TXA2Rbeta did not affect agonist-promoted internalization. Analysis of various C-tail deletion mutants revealed that a region between residues 355 and 366 of the TXA2Rbeta is essential for agonist-promoted internalization. These data demonstrate that alternative splicing of the TXA2R plays a critical role in regulating arrestin binding and subsequent receptor internalization.     

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.     

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.     

The association of arrestin-3 with the human lutropin/choriogonadotropin receptor depends mostly on receptor activation rather than on receptor phosphorylation.

Although the involvement of the nonvisual arrestins in the agonist-induced internalization of the human lutropin receptor (hLHR) has been documented previously with the use of dominant-negative mutants, a physical association of the nonvisual arrestins with the hLHR in intact cells has not been established. In the studies presented herein, we used a cross-linking/coimmunoprecipitation/immunoblotting approach as well as confocal microscopy to document the association of the hLHR with the nonvisual arrestins in co-transfected 293 cells. We also used this approach to examine the relative importance of receptor activation and receptor phosphorylation in the formation of this complex. Using hLHR mutants that impair phosphorylation, activation, or both, we show that the formation of the hLHR-nonvisual arrestin complex depends mostly on the agonist-induced activation of the hLHR rather than on the phosphorylation of the hLHR. These results stand in contrast to those obtained with several other G protein-coupled receptors (i.e. the beta2-adrenergic receptor, the m2 muscarinic receptor, rhodopsin, and the type 1A angiotensin receptor) where arrestin binding depends mostly on receptor phosphorylation rather than on receptor activation. We have also examined the association of the nonvisual arrestins with naturally occurring gain-of-function mutations of the hLHR found in boys with Leydig cell hyperplasia or Leydig cell adenomas. Our results show that these mutants associate with the nonvisual arrestins in an agonist-independent fashion.