Agonist-dependent dissociation of human somatostatin receptor 2 dimers: a role in receptor trafficking

G-protein-coupled receptors (GPCRs) represent the largest and most diverse family of cell surface receptors. Several GPCRs have been documented to dimerize with resulting changes in pharmacology and signaling. We have previously reported, by means of photobleaching fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence correlation spectroscopic analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in the presence of the agonist SST-14. By contrast, hSSTR1 remained monomeric when expressed alone regardless of agonist exposure in live cells. However, the effect of the agonist on other hSSTR members remains unknown. Using pbFRET microscopy and Western blot, we provide evidence for agonist-dependent dissociation of self-associated hSSTR2 stably expressed in CHO-K1 and HEK-293 cells. Furthermore, the dissociation of the hSSTR2 dimer occurred in a concentration-dependent manner. Moreover, blocking receptor dissociation using a cross-linker agent perturbed receptor trafficking. Taking these data together, we suggest that the process of GPCR dimerization may operate differently, even among members of the same family, and that receptor dissociation as well as dimerization may be important steps for receptor dynamics.     

C-tail mediated modulation of somatostatin receptor type-4 homo- and heterodimerizations and signaling

Somatostatin receptors show great diversity in response to agonist mediated receptor-specific homo- and heterodimerizations. Here, using photobleaching-fluorescence resonance energy transfer, immunocytochemistry, western blot and co-immunoprecipitation, we investigated dimerization, trafficking, coupling to adenylyl cyclase and signaling of human somatostatin receptor-4 (hSSTR4) in HEK-293 cells. We also determined the role of the C-tail of hSSTR4 on physiological responses of the cells. wt-hSSTR4 exogenously expressed in HEK-293 cells exhibits constitutive dimerization, inhibits forskolin-stimulated cAMP, and displays agonist dependent changes in pERK1/2 and pERK5 expressions. Upon C-tail deletion, the receptor loses membrane expression and ability to dimerize and inhibition of cAMP and pERK5 however, displays several-fold increases in the expression of pERK1/2. Chimeric hSSTR4 with the C-tail of hSSTR5 functions like wt-hSSTR4, in contrast, with the C-tail of hSSTR1 functions like C-tail deleted hSSTR4. hSSTR4 dimerization and signaling are associated with increased cyclin-dependent-kinase p27^kip1 expression and inhibition of the cell proliferation. We also report heterodimerization between hSSTR4/hSSTR5, but not between hSSTR4/hSSTR1, with significant changes in receptor functions. Taken together, these data define a novel mechanism for the role of hSSTR4 in cell proliferation and modulation of signaling pathways.     

Agonist-dependent up-regulation of human somatostatin receptor type 1 requires molecular signals in the cytoplasmic C-tail.

We have previously reported that the human somatostatin receptor type 1 (hSSTR1) stably expressed in Chinese hamster ovary-K1 cells does not internalize but instead up-regulates at the membrane during continued agonist treatment (1 microM somatostatin (SST)-14 x 22 h). Here we have investigated the molecular basis of hSSTR1 up-regulation. hSSTR1 was up-regulated by SST in a time-, temperature-, and dose-dependent manner to saturable levels, in intact cells but not in membrane preparations. Although hSSTR1 was acutely desensitized to adenylyl cyclase coupling after 1 h SST-14 treatment, continued agonist exposure (22 h) restored functional effector coupling. Up-regulation was unaffected by cycloheximide but blocked by okadaic acid. Confocal fluorescence immunocytochemistry of intact and permeabilized cells showed progressive, time-dependent increase in surface hSSTR1 labeling, associated with depletion of intracellular SSTR1 immunofluorescent vesicles. To investigate the structural domains of hSSTR1 responsible for up-regulation, we constructed C-tail deletion (Delta) mutants and chimeric hSSTR1-hSSTR5 receptors. Human SSTR5 was chosen because it internalizes readily, displays potent C-tail internalization signals, and does not up-regulate. Like wild type hSSTR1, Delta C-tail hSSTR1 did not internalize and additionally lost the ability to up-regulate. Swapping the C-tail of hSSTR1 with that of hSSTR5 induced internalization (27%) but not up-regulation. Substitution of hSSTR5 C-tail with that of hSSTR1 converted the chimeric receptor to one resembling wild type hSSTR1 (poor internalization, 71% up-regulation). These results show that ligand-induced up-regulation of hSSTR1 occurs by a temperature-dependent active process of receptor recruitment from a pre-existing cytoplasmic pool to the plasma membrane. It does not require new protein synthesis or signal transduction, is sensitive to dephosphorylation events, and critically dependent on molecular signals in the receptor C-tail.     

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by beta-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys359-Ser360-Arg361. Moreover, point mutation of Ser360 to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of beta-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser360 being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with beta-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.     

Heterooligomerization of human dopamine receptor 2 and somatostatin receptor 2 Co-immunoprecipitation and fluorescence resonance energy transfer analysis

Somatostatin and dopamine receptors are well expressed and co-localized in several brain regions, suggesting the possibility of functional interactions. In the present study we used a combination of pharmacological, biochemical and photobleaching fluorescence resonance energy transfer (pbFRET) to determine the functional interactions between human somatostatin receptor 2 (hSSTR2) and human dopamine receptor 2 (hD2R) in both co-transfected CHO-K1 or HEK-293 cells as well as in cultured neuronal cells which express both the receptors endogenously. In monotransfected CHO-K1 or HEK-293 cells, D2R exists as a preformed dimer which is insensitive to agonist or antagonist treatment. In control CHO-K1 cells stably co-transfected with hD2R and hSSTR2, relatively low FRET efficiency and weak expression in co-immunoprecipitate from HEK-293 cells suggest the absence of preformed heterooligomers. However, upon treatment with selective ligands, hD2R and hSSTR2 exhibit heterodimerization. Agonist-induced heterodimerization was accompanied by increased affinity for dopamine and augmented hD2R signalling as well as prolonged hSSTR2 internalization. In contrast, cultured striatal neurons display constitutive heterodimerization between D2R and SSTR2, which were agonist-independent. However, heterodimerization in neurons was completely abolished in the presence of the D2R antagonist eticlopride. These findings suggest that hD2R and hSSTR2 operate as functional heterodimers modulated by ligands in situ, which may prove to be a useful model in designing new therapeutic drugs.     

Agonist-induced up-regulation of human somatostatin receptor type 1 is regulated by β-arrestin-1 and requires an essential serine residue in the receptor C-tail

We have previously shown that the human somatostatin receptor type 1 (hSSTR1) does not undergo agonist-induced internalization, but is instead up-regulated at the membrane upon prolonged somatostatin (SST) exposure. The deletion of the carboxyterminal C-tail of the receptor completely abolishes up-regulation. To identify molecular signals that mediate hSSTR1 up-regulation, we created mutant receptors with progressive C-tail deletions. Up-regulation was found to be absent in mutants lacking residues Lys³⁵⁹-Ser³⁶⁰-Arg³⁶¹. Moreover, point mutation of Ser³⁶⁰ to Ala completely abolished up-regulation. The coexpression of wild type hSSTR1 with V53D, a dominant negative mutant of β-arrestin-1, completely blocked hSSTR1 up-regulation. Further analysis demonstrated that calcium-calmodulin (CaM) dependent kinases were essential for the SST-induced up-regulation response. Like wild type receptors, all mutants failed to internalize after agonist exposure and were able to inhibit forskolin-stimulated cAMP accumulation. Taking these data together, we suggest that SST-induced hSSTR1 up-regulation is critically dependent upon a specific Lys-Ser-Arg sequence in the C-tail of the receptor, with Ser³⁶⁰ being essential. Up-regulation also requires the participation of CaM protein kinases and interactions with β-arrestins. In contrast, coupling to adenyl cyclase (AC) and internalization occur independently of molecular signals in the receptor's C-tail.     

Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast

G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs.     

Receptor specific crosstalk and modulation of signaling upon heterodimerization between β1-adrenergic receptor and somatostatin receptor-5

In the present study we describe heterodimerization, trafficking, coupling to adenylyl cyclase and signaling in HEK-293 cells cotransfected with human-somatostatin receptor 5 (hSSTR5) and β₁-adrenergic receptor (β₁AR). hSSTR5/β₁AR exists as heterodimers in basal conditions which was further enhanced upon synergistic activation of both receptors. Activation of either β₁AR or hSSTR5 displayed dissociation of heterodimerization. In cotransfectants, β₁AR effect on cAMP was predominant; however, blocking β₁AR with antagonist resulted in 60% inhibition of forskolin-stimulated cAMP in the presence of hSSTR5 agonists. cAMP/PKA pathway in cotransfected cells was regulated in receptor-specific manner, in contrast, the status of pERK1/2 and pPI3K/AKT was predominantly regulated by hSSTR5. The expression levels of phosphorylated NFAT remained unchanged indicating blockade of calcineurin-mediated dephosphorylation and nuclear translocation of NFAT, the process predominantly regulated by pJNK in SSTR5 dependent manner. Taken together, the functional consequences of results described here might have relevance in the cardiovascular system where SSTR and AR subtypes play important roles.     

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.     

The role of G-proteins in the dimerisation of human somatostatin receptor types 2 and 5

Somatostatin (SST) is a peptide hormone that acts through a family of heptahelical receptors belonging to the G-protein coupled receptor (GPCR) superfamily. There are five known SST receptor subtypes termed SSTR1–5 and all couple to Gα_i/o G-proteins. It has been previously demonstrated that these receptors can form both homo- and heterodimers within their family or with other GPCR family members. Although agonist was demonstrated as a factor in modulating certain dimeric pairs, the molecular mechanism(s) underlying this regulation remains undetermined. Here, we demonstrate the coupling of G-protein as a contributing factor in the homo- and heterodimerisation of human (h) SSTR2 and SSTR5. When cells stably expressing hSSTR2 are pretreated with pertussis toxin (PTX), dissociation of hSSTR2 dimers occurs. Interestingly, although dimerisation of hSSTR5 was unaffected following PTX treatment, heterodimerisation between hSSTR2 and hSSTR5 is potentiated in the absence of receptor-stimulation. These results demonstrate the importance of G-protein in the maintenance and regulation of hSSTR dimers.     

Prediction of heterodimerization interfaces of G-protein coupled receptors with a new subtractive correlated mutation method

Recent studies employing differential epitope tagging, selective immunoprecipitation of receptor complexes and fluorescence or bioluminescence resonance energy transfer techniques provide direct evidence for heterodimerization between both closely and distantly related members of the G-protein coupled receptor (GPCR) family. Since heterodimerization appears to play a role in modulating agonist affinity, efficacy and/or trafficking properties, the molecular models of GPCRs required to understand receptor function must consider these oligomerization hypotheses. To advance knowledge in this field, we present here a computational approach based on correlated mutation analysis and the structural information contained in three-dimensional molecular models of the transmembrane regions of GPCRs built using the rhodopsin crystal structure as a template. The new subtractive correlated mutation method reveals likely heterodimerization interfaces amongst the different alternatives for the positioning of two tightly packed bundles of seven transmembrane domains next to each other in contact heterodimers of GPCRs. Predictions are applied to GPCRs in the class of opioid receptors. However, in the absence of a known structure of any GPCR dimer, the features of the method and predictions are also illustrated and analyzed for a dimeric complex of known structure.     

Oligomerization, biogenesis, and signaling is promoted by a glycophorin A-like dimerization motif in transmembrane domain 1 of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form dimeric or oligomeric complexes in vivo. However, the functions and mechanisms of oligomerization remain poorly understood for most GPCRs, including the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Here we provide evidence indicating that alpha-factor receptor oligomerization involves a GXXXG motif in the first transmembrane domain (TM1), similar to the transmembrane dimerization domain of glycophorin A. Results of fluorescence resonance energy transfer, fluorescence microscopy, endocytosis assays of receptor oligomerization in living cells, and agonist binding assays indicated that amino acid substitutions affecting the glycine residues of the GXXXG motif impaired alpha-factor receptor oligomerization and biogenesis in vivo but did not significantly impair agonist binding affinity. Mutant receptors exhibited signaling defects that were not due to impaired cell surface expression, indicating that oligomerization promotes alpha-factor receptor signal transduction. Structure-function studies suggested that the GXXXG motif in TM1 of the alpha-factor receptor promotes oligomerization by a mechanism similar to that used by the GXXXG dimerization motif of glycophorin A. In many mammalian GPCRs, motifs related to the GXXXG sequence are present in TM1 or other TM domains, suggesting that similar mechanisms are used by many GPCRs to form dimers or oligomeric arrays.     

Subtypes of the somatostatin receptor assemble as functional homo- and heterodimers

The existence of receptor dimers has been proposed for several G protein-coupled receptors. However, the question of whether G protein-coupled receptor dimers are necessary for activating or modulating normal receptor function is unclear. We address this question with somatostatin receptors (SSTRs) of which there are five distinct subtypes. By using transfected mutant and wild type receptors, as well as endogenous receptors, we provide pharmacological, biochemical, and physical evidence, based on fluorescence resonance energy transfer analysis, that activation by ligand induces SSTR dimerization, both homo- and heterodimerization with other members of the SSTR family, and that dimerization alters the functional properties of the receptor such as ligand binding affinity and agonist-induced receptor internalization and up-regulation. Double label confocal fluorescence microscopy showed that when SSTR1 and SSTR5 subtypes were coexpressed in Chinese hamster ovary-K1 cells and treated with agonist they underwent internalization and were colocalized in cytoplasmic vesicles. SSTR5 formed heterodimers with SSTR1 but not with SSTR4 suggesting that heterodimerization is a specific process that is restricted to some but not all receptor subtype combinations. Direct protein interaction between different members of the SSTR subfamily defines a new level of molecular cross-talk between subtypes of the SSTR and possibly related receptor families.     

Agonist-dependent immobilization of chimeric bombesin/GRP receptors: dependence on c-Src activity and dissociation from internalization.

G-protein-coupled receptors (GPCRs) are membrane proteins that exhibit a decreased mobile fraction compared to a freely mobile plasma membrane protein. Recently, interest has focused on proteins other than heterotrimeric G-proteins that interact with GPCRs as scaffolding structures that affect receptor signal transduction. In order to investigate the physical state of receptors before and after agonist, we used fluorescence recovery after photobleaching of the bombesin/gastrin-releasing peptide (GRP) receptor fused to the intrinsically fluorescent green fluorescent protein (GFP-GRP receptor) expressed in KNRK cells to measure both the fraction of mobile receptors and their diffusion rate before and after agonist stimulation. In live cells at 37 degrees C, addition of GRP (100 nM) caused a rapid decrease in GFP-GRP receptor mobile fraction from 0.8 +/- 0.1 to 0.49 +/- 0.05, which was independent of endocytosis. Concurrently, the remaining mobile GFP-GRPreceptors showed an increase in the diffusion rate with the half-time of fluorescent recovery, tau(1/2) = 46 +/- 7 s for untreated cells, decreasing to tau(1/2) = 30 +/- 6 s for cells treated with GRP. Prior treatment with the Src-specific inhibitor PP-2 (10 microM) blocked GFP-GRP receptor immobilization while treatment with the inactive analog PP-3 (10 microM) did not affect receptor immobilization. These data suggest that agonist-bound GPCR have increased plasma membrane diffusion rates but an increased affinity for immobilization into a multiprotein complex that is mediated by Src activity.     

Somatostatin receptor-3 mediated intracellular signaling and apoptosis is regulated by its cytoplasmic terminal

In the present study, we describe the role of cytoplasmic terminal (C-tail) domain in regulating coupling to adenylyl cyclase, signaling, and apoptosis in human embryonic kidney (HEK-293) cells transfected with wild type (wt)-hSSTR3 and C-tail deleted mutants. Cells transfected with wt-hSSTR3 and C-tail mutants show comparable membrane expression; however, display decreased expression in presence of agonist. wt-hSSTR3 exists as preformed homodimer at cell surface in basal conditions and decreases in response to agonist. Cells expressing C-tail mutants also show evidence of homodimerization with the same intensity as wt-hSSTR3. The agonist-dependent inhibition of cyclic adenosine monophosphate (cAMP) was lost in cells expressing C-tail mutants. Agonist treatment in cells expressing wt-hSSTR3 resulted in inhibition of cell proliferation, increased expression of PARP-1, and TUNEL positivity in proliferating cell nuclear antigen (PCNA)-positive cells. The agonist mediated increase in membrane expression of protein tyrosine phosphatase (PTP) seen with wt-hSSTR3 was diminished in C-tail mutants, which was accompanied with the loss of receptor's ability to induce apoptosis. Taken together, our data provide new insights into C-tail-dependent regulation of cell signaling and apoptosis by hSSTR3.     

The orphan GPR50 receptor specifically inhibits MT1 melatonin receptor function through heterodimerization

One-third of the approximately 400 nonodorant G protein-coupled receptors (GPCRs) are still orphans. Although a considerable number of these receptors are likely to transduce cellular signals in response to ligands that remain to be identified, they may also have ligand-independent functions. Several members of the GPCR family have been shown to modulate the function of other receptors through heterodimerization. We show that GPR50, an orphan GPCR, heterodimerizes constitutively and specifically with MT(1) and MT(2) melatonin receptors, using biochemical and biophysical approaches in intact cells. Whereas the association between GPR50 and MT(2) did not modify MT(2) function, GPR50 abolished high-affinity agonist binding and G protein coupling to the MT(1) protomer engaged in the heterodimer. Deletion of the large C-terminal tail of GPR50 suppressed the inhibitory effect of GPR50 on MT(1) without affecting heterodimerization, indicating that this domain regulates the interaction of regulatory proteins to MT(1). Pairing orphan GPCRs to potential heterodimerization partners might be of clinical importance and may become a general strategy to better understand the function of orphan GPCRs.     

Multiplex Detection of Homo- and Heterodimerization of G Protein-Coupled Receptors by Proximity Biotinylation

Dimerization of G protein-coupled receptors (GPCRs) represents a potential mechanism by which GPCR functions are regulated. Several resonance energy transfer (RET)-based methods have revealed GPCR homo- and heterodimerization. However, interpretation of an increase in FRET efficiency could be attributed to either dimerization/oligomerization events or conformational changes within an already dimerized/oligomerized receptor complex. Furthermore, RET-based methods can only measure pairwise dimerization, and cannot easily achieve multiplex detection. In this study, we applied proximity-based biotinylation for detecting receptor dimerization by utilizing a specific enzyme-substrate pair that are fused to GPCRs. The biotin ligase BirA is fused to CXCR4 and site-specifically biotinylates an acceptor peptide (AP) in the presence of biotin. As a test case for our newly developed assay, we have characterized the homo-dimerization of chemokine receptor CXCR4 and heterodimerization of CXCR4 with CCR2 or CCR5. The degree of biotinylation varies with the amount of GPCR-AP as well as biotinylation time. Using enzyme/substrate receptor pairs and measuring receptor biotinylation, we demonstrate that CXCR4 can homo-dimerize and hetero-dimerize with CCR2 and CCR5. The effect of CXCL12, agonist for CXCR4, was found to decrease surface biotinylation of CXCR4-AP. This effect is due to a combination of CXCR4 endocytosis and stabilization of CXCR4 homodimers. Finally, when CXCR4-AP, CCR2-AP, and CCR5-AP were expressed together, we observed CXCR4-CXCR4 homodimers and CXCR4-CCR2 and CXCR4-CCR5 heterodimers. The newly developed assay opens new opportunity for multiplex detection for GPCR homo- and heterodimerization within the same cellular context.     

Resistance of the human beta 1-adrenergic receptor to agonist-mediated down-regulation. Role of the C terminus in determining beta-subtype degradation

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human beta1-adrenergic receptor (beta 1AR) to agonist over a 24-h period, we instead observed an increase of approximately 30% in both beta 1AR binding activity and immune-detected receptors. In contrast, beta 2AR expressed in these cells exhibited a decrease of > or =50%. We determined that the basal turnover rates of the two subtypes were similar (t(1/2) approximately 7 h) and that agonist stimulation increased beta 2AR but not beta 1AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A1 had no effect on basal turnover of either subtype but blocked agonist-stimulated beta 2AR turnover. As beta 1AR mRNA levels increased in agonist-stimulated cells, beta 1AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; beta 1AR with a beta 2AR C-tail underwent down-regulation, and beta 2AR with a beta 1AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being beta 2AR > beta 1AR with beta 2AR C-tail > beta 2AR with a beta 1AR C-tail > beta 1AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although beta 1AR internalization was increased to that of beta 2AR, down-regulation still did not occur. Instead, beta 1AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and beta 2AR but not beta 1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.     

A pH-sensitive fluor,CypHer 5, used to monitor agonist-induced G protein-coupled receptor internalization in live cells

G protein-coupled receptors (GPCRs) are the largest family of proteins involved in transmembrane signal transduction and are actively studied because of their suitability as therapeutic small-molecule drug targets. Agonist activation of GPCRs almost invariably results in the receptor being desensitized. One of the key events in receptor desensitization is the sequestration of the receptor from the cell surface into acidic intracellular endosomes. Therefore, a convenient, generic, and noninvasive monitor of this process is desirable. A novel, pH-sensitive, red-excited fluorescent dye, CypHer 5, was synthesized. This dye is non-fluorescent at neutral pH and is fluorescent at acidic pH. Anti-epitope antibodies labeled with this dye were internalized in an agonist concentration- and time-dependent manner, following binding on live cells to a range of GPCRs that had been modified to incorporate the epitope tags in their extracellular N-terminal domain. This resulted in a large signal increase over background. When protonated, the red fluorescence of CypHer 5 provides a generic reagent suitable for monitoring the internalization of GPCRs into acidic vesicles. This approach should be amenable to the study of many other classes of cell surface receptors that also internalize following stimulation.     

Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors

Visual arrestin, betaarrestin1, and betaarrestin2 comprise a family of intracellular proteins that desensitize G protein-coupled receptors (GPCRs). In addition, betaarrestin1 and betaarrestin2 target desensitized receptors to clathrin-coated pits for endocytosis. Whether arrestins differ in their ability to interact with GPCRs in cells is not known. In this study, we visualize the interaction of arrestin family members with GPCRs in real time and in live cells using green fluorescent protein-tagged arrestins. In the absence of agonist, visual arrestin and betaarrestin1 were found in both the cytoplasm and nucleus of HEK-293 cells, whereas betaarrestin2 was found only in the cytoplasm. Analysis of agonist-mediated arrestin translocation to multiple GPCRs identified two major classes of receptors. Class A receptors (beta2 adrenergic receptor, mu opioid receptor, endothelin type A receptor, dopamine D1A receptor, and alpha1b adrenergic receptor) bound betaarrestin2 with higher affinity than betaarrestin1 and did not interact with visual arrestin. In contrast, class B receptors (angiotensin II type 1A receptor, neurotensin receptor 1, vasopressin V2 receptor, thyrotropin-releasing hormone receptor, and substance P receptor) bound both betaarrestin isoforms with similar high affinities and also interacted with visual arrestin. Switching the carboxyl-terminal tails of class A and class B receptors completely reversed the affinity of each receptor for the visual and non-visual arrestins. In addition, exchanging the betaarrestin1 and betaarrestin2 carboxyl termini reversed their extent of binding to class A receptors as well as their subcellular distribution. These results reveal for the first time marked differences in the ability of arrestin family members to bind GPCRs at the plasma membrane. Moreover, they show that visual arrestin can interact in cells with GPCRs other than rhodopsin. These findings suggest that GPCR signaling may be differentially regulated depending on the cellular complement of arrestin isoforms and the ability of arrestins to interact with other cellular proteins.