Ligand-induced internalization and recycling of the human neuropeptide Y2 receptor is regulated by its carboxyl-terminal tail

Agonist-induced internalization of G protein-coupled receptors plays an important role in signal regulation. The underlying mechanisms of the internalization of the human neuropeptide Y(2) receptor (hY(2)R), as well as its desensitization, endocytosis, and resensitization are mainly unknown. In the present study we have investigated the role of carboxyl-terminal (C-terminal) Ser/Thr residues and acidic amino acids in regulating receptor internalization, arrestin interaction, and recycling by fluorescence microscopy, cell surface enzyme-linked immunosorbent assay, and bioluminescence resonance energy transfer in several cell lines. Strikingly, C-terminal truncation mutants revealed two different internalization motifs. Whereas a distal motif (373)DSXTEXT(379) was found to be the primary regulatory internalization sequence acting in concert with arrestin-3, the proximal motif (347)DXXXSEXSXT(356) promoted ligand-induced internalization in an arrestin-3-independent manner. Moreover, we identified a regulatory sequence located between these internalization motifs ((357)FKAKKNLEVRKN(368)), which serves as an inhibitory element. We found that hY(2)R recycling is also governed by structural determinants within the proximal internalization motif. In conclusion, these results indicate that the hY(2)R C terminus is involved in multiple molecular events that regulate internalization, interaction with arrestin-3, and receptor resensitization. Our findings provide novel insights into complex mechanisms of controlled internalization of hY(2)R, which is likely applicable to other GPCRs.     

Dynamic Na+-H+ exchanger regulatory factor-1 association and dissociation regulate parathyroid hormone receptor trafficking at membrane microdomains

Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.     

Adaptor protein complex-2 (AP-2) and epsin-1 mediate protease-activated receptor-1 internalization via phosphorylation- and ubiquitination-dependent sorting signals

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs.     

Gamma-aminobutyric acid type B (GABA(B)) receptor internalization is regulated by the R2 subunit

γ-Aminobutyric acid type B (GABA(B)) receptors are important for slow synaptic inhibition in the CNS. The efficacy of inhibition is directly related to the stability of cell surface receptors. For GABA(B) receptors, heterodimerization between R1 and R2 subunits is critical for cell surface expression and signaling, but how this determines the rate and extent of receptor internalization is unknown. Here, we insert a high affinity α-bungarotoxin binding site into the N terminus of the R2 subunit and reveal its dominant role in regulating the internalization of GABA(B) receptors in live cells. To simultaneously study R1a and R2 trafficking, a new α-bungarotoxin binding site-labeling technique was used, allowing α-bungarotoxin conjugated to different fluorophores to selectively label R1a and R2 subunits. This approach demonstrated that R1a and R2 are internalized as dimers. In heterologous expression systems and neurons, the rates and extents of internalization for R1aR2 heteromers and R2 homomers are similar, suggesting a regulatory role for R2 in determining cell surface receptor stability. The fast internalization rate of R1a, which has been engineered to exit the endoplasmic reticulum, was slowed to that of R2 by truncating the R1a C-terminal tail or by removing a dileucine motif in its coiled-coil domain. Slowing the rate of internalization by co-assembly with R2 represents a novel role for GPCR heterodimerization whereby R2 subunits, via their C terminus coiled-coil domain, mask a dileucine motif on R1a subunits to determine the surface stability of the GABA(B) receptor.     

Noncanonical Control of Vasopressin Receptor Type 2 Signaling by Retromer and Arrestin

The vasopressin type 2 receptor (V2R) is a critical G protein-coupled receptor (GPCR) for vertebrate physiology, including the balance of water and sodium ions. It is unclear how its two native hormones, vasopressin (VP) and oxytocin (OT), both stimulate the same cAMP/PKA pathway yet produce divergent antinatriuretic and antidiuretic effects that are either strong (VP) or weak (OT). Here, we present a new mechanism that differentiates the action of VP and OT on V2R signaling. We found that vasopressin, as opposed to OT, continued to generate cAMP and promote PKA activation for prolonged periods after ligand washout and receptor internalization in endosomes. Contrary to the classical model of arrestin-mediated GPCR desensitization, arrestins bind the VP-V2R complex yet extend rather than shorten the generation of cAMP. Signaling is instead turned off by the endosomal retromer complex. We propose that this mechanism explains how VP sustains water and Na⁺ transport in renal collecting duct cells. Together with recent work on the parathyroid hormone receptor, these data support the existence of a novel “noncanonical” regulatory pathway for GPCR activation and response termination, via the sequential action of β-arrestin and the retromer complex.     

Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.     

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of histamine H2 receptor by G protein-coupled receptor kinase 2

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by β-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a Gα(s)-coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrin-homology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domain-mediated regulation of GPCRs beyond Gα(q)-coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

Identification of the orphan G protein-coupled receptor GPR31 as a receptor for 12-(S)-hydroxyeicosatetraenoic acid

Hydroxy fatty acids are critical lipid mediators involved in various pathophysiologic functions. We cloned and identified GPR31, a plasma membrane orphan G protein-coupled receptor that displays high affinity for the human 12-lipoxygenase-derived product 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE). Thus, GPR31 is named 12-(S)-HETE receptor (12-HETER) in this study. The cloned 12-HETER demonstrated high affinity binding for 12-(S)-[(3)H]HETE (K(d) = 4.8 ± 0.12 nm). Also, 12-(S)-HETE efficiently and selectively stimulated GTPγS coupling in the membranes of 12-HETER-transfected cells (EC(50) = 0.28 ± 1.26 nm). Activating GTPγS coupling with 12-(S)-HETE proved to be both regio- and stereospecific. Also, 12-(S)-HETE/12-HETER interactions lead to activation of ERK1/2, MEK, and NFκB. Moreover, knocking down 12-HRTER specifically inhibited 12-(S)-HETE-stimulated cell invasion. Thus, 12-HETER represents the first identified high affinity receptor for the 12-(S)-HETE hydroxyl fatty acids.     

阿片肽与P2Y12 受体信号通路的研究进展

糖尿病可增加心血管疾病危险性,因此糖尿病和心血管疾病的密切关系也日益被人们所重视。糖尿病引发的血小板功能亢进以及抗血小板药物抵抗的机制尚不明确。阿片肽类物质能够抑制血小板细胞活性以及凝集作用,本文对2型糖尿病血小板P2Y12信号通路高反应性、阿片肽及阿片受体对抗P2Y12信号通路高反应性的可能机制进行了归纳总结。     

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.     

Agonist-directed interactions with specific beta-arrestins determine mu-opioid receptor trafficking, ubiquitination, and dephosphorylation

Morphine and other opiates mediate their effects through activation of the μ-opioid receptor (MOR), and regulation of the MOR has been shown to critically affect receptor responsiveness. Activation of the MOR results in receptor phosphorylation, β-arrestin recruitment, and internalization. This classical regulatory process can differ, depending on the ligand occupying the receptor. There are two forms of β-arrestin, β-arrestin1 and β-arrestin2 (also known as arrestin2 and arrestin3, respectively); however, most studies have focused on the consequences of recruiting β-arrestin2 specifically. In this study, we examine the different contributions of β-arrestin1- and β-arrestin2-mediated regulation of the MOR by comparing MOR agonists in cells that lack expression of individual or both β-arrestins. Here we show that morphine only recruits β-arrestin2, whereas the MOR-selective enkephalin [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), recruits either β-arrestin. We show that β-arrestins are required for receptor internalization and that only β-arrestin2 can rescue morphine-induced MOR internalization, whereas either β-arrestin can rescue DAMGO-induced MOR internalization. DAMGO activation of the receptor promotes MOR ubiquitination over time. Interestingly, β-arrestin1 proves to be critical for MOR ubiquitination as modification does not occur in the absence of β-arrestin1 nor when morphine occupies the receptor. Moreover, the selective interactions between the MOR and β-arrestin1 facilitate receptor dephosphorylation, which may play a role in the resensitization of the MOR and thereby contribute to overall development of opioid tolerance.     

The Bile Acid Receptor TGR5 Does Not Interact with β-Arrestins or Traffic to Endosomes but Transmits Sustained Signals from Plasma Membrane Rafts

TGR5 is a G protein-coupled receptor that mediates bile acid (BA) effects on energy balance, inflammation, digestion, and sensation. The mechanisms and spatiotemporal control of TGR5 signaling are poorly understood. We investigated TGR5 signaling and trafficking in transfected HEK293 cells and colonocytes (NCM460) that endogenously express TGR5. BAs (deoxycholic acid (DCA), taurolithocholic acid) and the selective agonists oleanolic acid and 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated cAMP formation but did not induce TGR5 endocytosis or recruitment of β-arrestins, as assessed by confocal microscopy. DCA, taurolithocholic acid, and oleanolic acid did not stimulate TGR5 association with β-arrestin 1/2 or G protein-coupled receptor kinase (GRK) 2/5/6, as determined by bioluminescence resonance energy transfer. 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N, 5-dimethylisoxazole-4-carboxamide stimulated a low level of TGR5 interaction with β-arrestin 2 and GRK2. DCA induced cAMP formation at the plasma membrane and cytosol, as determined using exchange factor directly regulated by cAMP (Epac2)-based reporters, but cAMP signals did not desensitize. AG1478, an inhibitor of epidermal growth factor receptor tyrosine kinase, the metalloprotease inhibitor batimastat, and methyl-β-cyclodextrin and filipin, which block lipid raft formation, prevented DCA stimulation of ERK1/2. Bioluminescence resonance energy transfer analysis revealed TGR5 and EGFR interactions that were blocked by disruption of lipid rafts. DCA stimulated TGR5 redistribution to plasma membrane microdomains, as localized by immunogold electron microscopy. Thus, TGR5 does not interact with β-arrestins, desensitize, or traffic to endosomes. TGR5 signals from plasma membrane rafts that facilitate EGFR interaction and transactivation. An understanding of the spatiotemporal control of TGR5 signaling provides insights into the actions of BAs and therapeutic TGR5 agonists/antagonists.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

N-formyl peptide receptor 3 (FPR3) departs from the homologous FPR2/ALX receptor with regard to the major processes governing chemoattractant receptor regulation, expression at the cell surface, and phosphorylation

Among human N-formyl peptide chemoattractant receptors, FPR2/ALX and FPR3 share the highest degree of amino acid identity (83%), and trigger similar cell responses upon ligand binding. Although FPR2/ALX is a promiscuous receptor, FPR3 has only one specific high affinity ligand, F2L, and a more restricted tissue/cell distribution. In this study, we showed that FPR2/ALX behaved as the prototypical receptor FPR1. The agonist-dependent phosphorylation used a hierarchical mechanism with a prominent role of Ser(329), Thr(332), and Thr(335). Phosphorylation of FPR2/ALX was essential for its desensitization but the lack of phosphorylation did not result in enhanced or sustained responses. In contrast, resting FPR3 displayed a marked level of phosphorylation, which was only slightly increased upon agonist stimulation. Another noticeable difference between the two receptors was their subcellular distribution in unstimulated cells. Although FPR2/ALX was evenly distributed at the plasma membrane FPR3 was localized in small intracellular vesicles. By swapping domains between FPR2/ALX and FPR3, we uncovered the determinants involved in the basal phosphorylation of FPR3. Experiments aimed at monitoring receptor-bound antibody uptake showed that the intracellular distribution of FPR3 resulted from a constitutive internalization that was independent of C terminus phosphorylation. Unexpectedly, exchanging residues 1 to 53, which encompass the N-terminal extracellular region and the first transmembrane domain, between FPR2/ALX and FPR3 switched localization of the receptors from the plasma membrane to intracellular vesicles and vice versa. A clathrin-independent, possibly caveolae-dependent, mechanism was involved in FPR3 constitutive internalization. The peculiar behavior of FPR3 most probably serves distinct physiological functions that remain largely unknown.     

Helix 8 plays a crucial role in bradykinin B(2) receptor trafficking and signaling

Upon activation the human bradykinin B(2) receptor (B(2)R) acts as guanine nucleotide exchange factor for the G proteins G(q/11) and G(i). Thereafter, it gets phosphorylated by G protein-coupled receptor kinases (GRKs) and recruits β-arrestins, which block further G protein activation and promote B(2)R internalization via clathrin-coated pits. As for most G protein-coupled receptors of family A, an intracellular helix 8 after transmembrane domain 7 is also predicted for the B(2)R. We show here that disruption of helix 8 in the B(2)R by either C-terminal truncation or just by mutation of a central amino acid (Lys-315) to a helix-breaking proline resulted in strong reduction of surface expression. Interestingly, this malfunction could be overcome by the addition of the membrane-permeable B(2)R antagonist JSM10292, suggesting that helix 8 has a general role for conformational stabilization that can be accounted for by an appropriate antagonist. Intriguingly, an intact helix 8, but not the C terminus with its phosphorylation sites, was indispensable for receptor sequestration and for interaction of the B(2)R with GRK2/3 and β-arrestin2 as shown by co-immunoprecipitation. Recruitment of β-arrestin1, however, required the presence of the C terminus. Taken together, our results demonstrate that helix 8 of the B(2)R plays a crucial role not only in efficient trafficking to the plasma membrane or the activation of G proteins but also for the interaction of the B(2)R with GRK2/3 and β-arrestins. Additional data obtained with chimera of B(2)R with other G protein-coupled receptors of family A suggest that helix 8 might have similar functions in other GPCRs as well.     

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.     

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric G_i proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate G_i signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the G_i inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive G_i proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.