Regulation of melanin-concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) belongs to the class A G protein-coupled receptors (GPCRs). The MCH-MCH1R system plays a central role in energy metabolism, and thus the regulation of signaling pathways activated by this receptor is of particular interest. Regulator of G protein signaling (RGS) proteins work by increasing the GTPase activity of G protein alpha subunits and attenuate cellular responses coupled with G proteins. Recent evidence has shown that RGS proteins are not simple G protein regulators but equally inhibit the signaling from various GPCRs. Here, we demonstrate that RGS8, which is highly expressed in the brain, functions as a negative modulator of MCH1R signaling. By using biochemical approaches, RGS8 was found to selectively and directly bind to the third intracellular (i3) loop of MCH1R in vitro. When expressed in HEK293T cells, RGS8 and MCH1R colocalized to the plasma membrane and RGS8 potently inhibited the calcium mobilization induced by MCH. The N-terminal 9 amino acids of RGS8 were required for the optimal capacity to downregulate the receptor signaling. Furthermore, Arg(253) and Arg(256) at the distal end of the i3 loop were found to comprise a structurally important site for the functional interaction with RGS8, since coexpression of RGS8 with R253Q/R256Q mutant receptors resulted in a loss of induction of MCH-stimulated calcium mobilization. This functional association suggests that RGS8 may represent a new therapeutic target for the development of novel pharmaceutical agents.     

Role for the third intracellular loop in cell surface stabilization of the alpha2A-adrenergic receptor.

Previous studies have shown that alpha2A-adrenergic receptor (alpha2A-AR) retention at the basolateral surface of polarized MDCKII cells involves its third intracellular (3i loop). The present studies examining mutant alpha2A-ARs possessing short deletions of the 3i loop indicate that no single region can completely account for the accelerated surface turnover of the Delta3ialpha2A-AR, suggesting that the entire 3i loop is involved in basolateral retention. Both wild-type and Delta3i loop alpha2A-ARs are extracted from polarized Madin-Darby canine kidney (MDCK) cells with 0.2% Triton X-100 and with a similar concentration/response profile, suggesting that Triton X-100-resistant interactions of the alpha2A-AR with cytoskeletal proteins are not involved in receptor retention on the basolateral surface. The indistinguishable basolateral t(1)/(2) for either the wild-type or nonsense 3i loop alpha2A-AR suggests that the stabilizing properties of the alpha2A-AR 3i loop are not uniquely dependent on a specific sequence of amino acids. The accelerated turnover of Delta3i alpha2A-AR cannot be attributed to alteration in agonist-elicited alpha2A-AR redistribution, because alpha2A-ARs are not down-regulated in response to agonist. Taken together, the present studies show that stabilization of the alpha2A-AR on the basolateral surface of MDCKII cells involves multiple mechanisms, with the third intracellular loop playing a central role in regulating these processes.     

RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling

RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate Galpha signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G(q/11)-coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G(i/o)-coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G(q)alpha and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G(11)alpha in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.     

An asn to lys polymorphism in the third intracellular loop of the human alpha 2A-adrenergic receptor imparts enhanced agonist-promoted Gi coupling

alpha(2A)-Adrenergic receptors (alpha(2A)AR) are presynaptic autoinhibitory receptors of noradrenergic neurons in the central and peripheral sympathetic nervous systems, which act to dynamically regulate neurotransmitter release. Signaling through the G(i)/G(o) family of G-proteins, the receptor subserves numerous homeostatic and central nervous system functions. A single nucleotide polymorphism of this receptor, which results in an Asn to Lys substitution at amino acid 251 of the third intracellular loop, was identified in the human population. The frequency of Lys-251 was 10-fold greater in African-Americans than in Caucasians, but was not associated with essential hypertension. To determine the consequences of this substitution, wild-type and Lys-251 receptors were expressed in CHO and COS-7 cells. Expression, ligand binding, and basal receptor function were unaffected by the substitution. However, agonist-promoted [(35)S]GTPgammaS binding was approximately 40% greater with the Lys-251 receptor. This enhanced agonist function was observed with catecholamines, azepines, and imidazolines albeit to different degrees. In studies of agonist-promoted functional coupling to G(i), the polymorphic receptor displayed enhanced inhibition of adenylyl cyclase (60 +/- 4. 4 versus 46 +/- 4.1% inhibition) and markedly enhanced stimulation of MAP kinase (57 +/- 9 versus 15- +/- 2-fold increase over basal) compared with wild-type alpha(2A)AR. The potency of epinephrine in stimulating inositol phosphate accumulation was increased approximately 4 fold with the Lys-251 receptor. Unlike previously described variants of G-protein-coupled receptors, where the minor species causes either a loss of function or increased non-agonist function, Lys-251 alpha(2A)AR represents a new class of polymorphism whose phenotype is a gain of agonist-promoted function.     

Sites in the third intracellular loop of the alpha 2A-adrenergic receptor confer short term agonist-promoted desensitization. Evidence for a receptor kinase-mediated mechanism.

To investigate the mechanisms of agonist-promoted desensitization of the alpha 2-adrenergic receptor (alpha 2AR), the human alpha 2AAR and a mutated form of the receptor were expressed in CHW cells. After cells were exposed to epinephrine for 30 min, the ability of the wild type alpha 2AAR to mediate inhibition of forskolin-stimulated adenylyl cyclase was depressed by approximately 78%. To assess the role of receptor phosphorylation during desensitization, cells were incubated with 32Pi, exposed to agonist, and alpha 2AAR purified by immunoprecipitation with a fusion protein antibody. Agonist-promoted desensitization was found to be accompanied by phosphorylation of the alpha 2AAR in vivo. The beta-adrenergic receptor kinase (beta ARK) is known to phosphorylate purified alpha 2AAR in vitro. We found that heparin, a beta ARK inhibitor, ablated short term agonist-induced desensitization of alpha 2AAR, while such desensitization was unaffected by inhibition of protein kinase A. To further assess the role of beta ARK, we constructed a mutated alpha 2AAR which has a portion of the third intracellular loop containing 9 serines and threonines (potential phosphorylation sites) deleted. This mutated alpha 2AAR failed to undergo short term agonist-induced desensitization. Agonist promoted in vivo phosphorylation of this mutated receptor was reduced by 90%, consistent with the notion that receptor phosphorylation at sites in the third intracellular loop plays a critical role in alpha 2AAR desensitization. After 24 h of agonist exposure, an even more profound desensitization of alpha 2AAR occurred, which was not accompanied by a decrease in receptor expression. Rather, long term agonist-induced desensitization was found to be due in part to a decrease in the amount of cellular Gi, which was not dependent on receptor third loop phosphorylation sites.     

Potent inhibition of angiotensin AT1 receptor signaling by RGS8: importance of the C-terminal third exon part of its RGS domain

R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in regulation of many GPCR-mediated responses. Multiple RGS proteins are usually expressed in a cell, and it is difficult to point out which RGS protein species are functionally important in the cell. To evaluate intrinsic potency of these RGS proteins, we compared inhibitory effects of RGS1, RGS2, RGS3, RGS4, RGS5, RGS8 and RGS16 on AT₁ receptor signaling. Intracellular Ca²⁺ responses to angiotensin II were markedly attenuated by transiently expressed RGS2, RGS3 and RGS8, compared to weak inhibition by RGS1, RGS4, RGS5 and RGS16. N-terminally deleted RGS2 (RGS2 domain) lost this potent inhibitory effect, whereas RGS domains of RGS3 and RGS8 showed strong inhibition similar to those of the full-length proteins. To investigate key determinants that specify the differences in potency, we constructed chimeric domains by replacing one or two of three exon parts of RGS8 domain with the corresponding part of RGS5. The chimeric RGS8 domains containing the first or the second exon part of RGS5 showed strong inhibitory effects similar to that of wild type RGS8, but the chimeric domain with the third exon part of RGS5 lost its activity. On the contrary, replacement of the third exon part of RGS5 with the corresponding residues of RGS8 increased the inhibitory effect. The role of the third exon part of RGS8 domain was further confirmed with the chimeric RGS8/RGS4 domains. These results indicate the potent inhibitory activity of RGS8 among R4/B subfamily proteins and importance of the third exon.     

Structure of the third intracellular loop of the human cannabinoid 1 receptor

The third cytoplasmic loop (IC3) is a determinant in the dynamic life cycle of G protein-coupled receptors, including the activation, internalization, desensitization, and resensitization processes. Here, we characterize the structural features of the IC3 of the cannabinoid 1 receptor (CB1) in micelle solution using heteronuclear, (1)H,(15)N-high-resolution NMR methods. The IC3 construct was designed to contain one-third of each of the transmembrane helices (TMs 5 and 6) to tether the protein to the hydrophobic portion of the micelle. Indeed, the NMR analysis illustrates prominent alpha-helices at the N-terminus (G1-R10) and C-terminus (Q37-T47) of the IC3 receptor domain, corresponding to the cytoplasmic termini of TM5 and TM6. The structural features of the central portion of the IC3 consist of a small alpha-helix, adjacent to the terminus of TM5. The remainder is mostly unstructured as indicated by the NMR-based observables (NOEs and chemical shifts). Despite the lack of secondary structure, the hydrophobic triplet of isoleucine residues in the center of the IC3 is found in molecular dynamics simulations to associate with the lipid environment, producing two smaller loops out of the IC3. Previous studies examining mastoparan and related peptides and their ability to activate G proteins have concluded an alpha-helix is required for efficient binding and activation. Our structural results for the IC3 of CB1 would then suggest that in the intact receptor the G protein is activated by the alpha-helices of the cytoplasmic ends of TM5 or TM6 and not the unstructured central region of the IC3.     

Mapping structural determinants within third intracellular loop that direct signaling specificity of type 1 corticotropin-releasing hormone receptor

The type 1 corticotropin-releasing hormone receptor (CRH-R1) influences biological responses important for adaptation to stressful stimuli, through activation of multiple downstream effectors. The structural motifs within CRH-R1 that mediate G protein activation and signaling selectivity are unknown. The aim of this study was to gain insights about important structural determinants within the third intracellular loop (IC3) of the human CRH-R1α important for cAMP and ERK1/2 pathways activation and selectivity. We investigated the role of the juxtamembrane regions of IC3 by mutating amino acid cassettes or specific residues to alanine. Although simultaneous tandem alanine mutations of both juxtamembrane regions Arg(292)-Met(295) and Lys(311)-Lys(314) reduced ligand binding and impaired signaling, all other mutant receptors retained high affinity binding, indistinguishable from wild-type receptor. Agonist-activated receptors with tandem mutations at the proximal or distal terminal segments enhanced activation of adenylyl cyclase by 50-75% and diminished activation of inositol trisphosphate and ERK1/2 by 60-80%. Single Ala mutations identified Arg(292), Lys(297), Arg(310), Lys(311), and Lys(314) as important residues for the enhanced activation of adenylyl cyclase, partly due to reduced inhibition of adenylyl cyclase activity by pertussis toxin-sensitive G proteins. In contrast, mutation of Arg(299) reduced receptor signaling activity and cAMP response. Basic as well as aliphatic amino acids within both juxtamembrane regions were identified as important for ERK1/2 phosphorylation through activation of pertussis toxin-sensitive G proteins as well as G(q) proteins. These data uncovered unexpected roles for key amino acids within the highly conserved hydrophobic N- and C-terminal microdomains of IC3 in the coordination of CRH-R1 signaling activity.     

Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.     

The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by Gi/Go-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a Gi/Gq chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while mu-opioid receptors (MOR) and P2Y12 purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T6.30A or N6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.     

Potent and selective inhibition of angiotensin AT1 receptor signaling by RGS2: roles of its N-terminal domain

Emerging evidence indicates that R4/B subfamily RGS (regulator of G protein signaling) proteins play roles in functional regulation in the cardiovascular system. In this study, we compared effects of three R4/B subfamily proteins, RGS2, RGS4 and RGS5 on angiotensin AT1 receptor signaling, and investigated roles of the N-terminus of RGS2. In HEK293T cells expressing AT1 receptor stably, intracellular Ca²⁺ responses induced by angiotensin II were much more strongly attenuated by RGS2 than by RGS4 and RGS5. N-terminally deleted RGS2 proteins lost this potent inhibitory effect. Replacement of the N-terminal residues 1-71 of RGS2 with the corresponding residues (1-51) of RGS5 decreased significantly the inhibitory effect. On the other hand, replacement of the residues 1-51 of RGS5 with the residues 1-71 of RGS2 increased the inhibitory effect dramatically. Furthermore, we investigated functional contribution of N-terminal subdomains of RGS2, namely, an N-terminal region (residues 16-55) with an amphipathic α helix domain (the subdomain N1), a probable non-specific membrane-targeting subdomain, and another region (residues 56-71) between the α helix and the RGS box (the subdomain N2), a probable GPCR-recognizing subdomain. RGS2 chimera proteins with the residues 1-33 or 34-52 of RGS5 showed weak inhibitory activity, and either of RGS5 chimera proteins with residues 1-55 or 56-71 of RGS2 showed strong inhibitory effects on AT1 receptor signaling. The present study indicates the essential roles of both N-terminal subdomains for the potent inhibitory activity of RGS2 on AT1 receptor signaling.     

A cyclic peptide mimicking the third intracellular loop of the V2 vasopressin receptor inhibits signaling through its interaction with receptor dimer and G protein

In this study, we investigated the mechanism by which a peptide mimicking the third cytoplasmic loop of the vasopressin V2 receptor inhibits signaling. This loop was synthesized as a cyclic peptide (i3 cyc) that adopted defined secondary structure in solution. We found that i3 cyc inhibited the adenylyl cyclase activity induced by vasopressin or a nonhydrolyzable analog of GTP, guanosine 5'-O-(3-thio)triphosphate. This peptide also affected the specific binding of [3H]AVP by converting vasopressin binding sites from a high to a low affinity state without any effect on the global maximal binding capacity. The inhibitory actions of i3 cyc could also be observed in the presence of maximally uncoupling concentration of guanosine 5'-O-(3-thio)triphosphate, indicating a direct effect on the receptor itself and not exclusively on the interaction between the Gs protein and the V2 receptor (V2-R). Bioluminescence resonance energy-transfer experiments confirmed this assumption, because i3 cyc induced a significant inhibition of the bioluminescence resonance energy-transfer signal between the Renilla reniformis luciferase and the enhanced yellow fluorescent protein fused V2-R. This suggests that the proper arrangement of the dimer could be an important prerequisite for triggering Gs protein activation. In addition to its effect on the receptor itself, the peptide exerted some of its actions at the G protein level, because it could also inhibit guanosine 5'-O-(3-thio)triphosphate-stimulated AC activity. Taken together, the data demonstrate that a peptide mimicking V2-R third intracellular loop affects both the dimeric structural organization of the receptor and has direct inhibitory action on Gs.     

Structure and function of the third intracellular loop of the 5-hydroxytryptamine2A receptor: the third intracellular loop is alpha-helical and binds purified arrestins

Understanding the precise structure and function of the intracellular domains of G protein-coupled receptors is essential for understanding how receptors are regulated, and how they transduce their signals from the extracellular milieu to intracellular sites. To understand better the structure and function of the intracellular domain of the 5-hydroxytryptamine2A (5-HT2A) receptor, a model G(alpha)q-coupled receptor, we overexpressed and purified to homogeneity the entire third intracellular loop (i3) of the 5-HT2A receptor, a region previously implicated in G-protein coupling. Circular dichroism spectroscopy of the purified i3 protein was consistent with alpha-helical and beta-loop, -turn, and -sheet structure. Using random peptide phage libraries, we identified several arrestin-like sequences as i3-interacting peptides. We subsequently found that all three known arrestins (beta-arrestin, arrestin-3, and visual arrestin) bound specifically to fusion proteins encoding the i3 loop of the 5-HT(2A) receptor. Competition binding studies with synthetic and recombinant peptides showed that the middle portion of the i3 loop, and not the extreme N and C termini, was likely to be involved in i3-arrestin interactions. Dual-label immunofluorescence confocal microscopic studies of rat cortex indicated that many cortical pyramidal neurons coexpressed arrestins (beta-arrestin or arrestin-3) and 5-HT2A receptors, particularly in intracellular vesicles. Our results demonstrate (a) that the i3 loop of the 5-HT2A receptor represents a structurally ordered domain composed of alpha-helical and beta-loop, -turn, and -sheet regions, (b) that this loop interacts with arrestins in vitro, and is hence active, and (c) that arrestins are colocalized with 5-HT2A receptors in vivo.     

The zeta isoform of 14-3-3 proteins interacts with the third intracellular loop of different alpha2-adrenergic receptor subtypes

The alpha2-adrenergic receptors (alpha2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the alpha2AAR (Val217-Ala377), alpha2BAR (Lys210-Trp354), and alpha2CAR (Arg248-Val363) were used as ligands in gel overlay assays. A protein doublet of approximately 30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (alpha2B >/= alpha2C> alpha2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the zeta isoform of 14-3-3 proteins. [35S]Met-14-3-3zeta binds to all three native alpha2AR subtypes, assessed using a solid phase binding assay (alpha2A>/=alpha2B> alpha2C), and this binding depends on the presence of the 3i loops. Attenuation of the alpha2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251-266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the alpha2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.     

The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype

We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.     

Structure/function analysis of alpha2A-adrenergic receptor interaction with G protein-coupled receptor kinase 2

G protein-coupled receptors (GPCRs) mediate the ability of a diverse array of extracellular stimuli to control intracellular signaling. Many GPCRs are phosphorylated by G protein-coupled receptor kinases (GRKs), a process that mediates agonist-specific desensitization in many cells. Although GRK binding to activated GPCRs results in kinase activation and receptor phosphorylation, relatively little is known about the mechanism of GRK/GPCR interaction or how this interaction results in kinase activation. Here, we used the alpha2A-adrenergic receptor (alpha(2A)AR) as a model to study GRK/receptor interaction because GRK2 phosphorylation of four adjacent serines within the large third intracellular loop of this receptor is known to mediate desensitization. Various domains of the alpha(2A)AR were expressed as glutathione S-transferase fusion proteins and tested for the ability to bind purified GRK2. The second and third intracellular loops of the alpha(2A)AR directly interacted with GRK2, whereas the first intracellular loop and C-terminal domain did not. Truncation mutagenesis identified three discrete regions within the third loop that contributed to GRK2 binding, the membrane proximal N- and C-terminal regions as well as a central region adjacent to the phosphorylation sites. Site-directed mutagenesis revealed a critical role for specific basic residues within these regions in mediating GRK2 interaction with the alpha(2A)AR. Mutation of these residues within the holo-alpha(2A)AR diminished GRK2-promoted phosphorylation of the receptor as well as the ability of the kinase to be activated by receptor binding. These studies provide new insight into the mechanism of interaction and activation of GRK2 by GPCRs and suggest that GRK2 binding is critical not only for receptor phosphorylation but also for full activity of the kinase.     

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.     

Phosphorylation-independent regulation of metabotropic glutamate receptor 1 signaling requires g protein-coupled receptor kinase 2 binding to the second intracellular loop

Metabotropic glutamate receptors (mGluRs) are members of a unique class of G protein-coupled receptors (class III) that include the calcium-sensing and gamma-aminobutyric acid type B receptors. The activity of mGluRs is regulated by second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). The attenuation of both mGluR1a and mGluR1b signaling by GRK2 is phosphorylation- and beta-arrestin-independent and requires the concomitant association of GRK2 with both the receptor and Galpha(q/11). G protein interactions are mediated, in part, by the mGluR1 intracellular second loop, but the domains required for GRK2 binding are unknown. In the present study, we showed that GRK2 binds to the second intracellular loop of mGluR1a and mGluR1b and also to the mGluR1a carboxyl-terminal tail. Alanine scanning mutagenesis revealed a discrete domain within loop 2 that contributes to GRK2 binding, and the mutation of either lysine 691 or 692 to an alanine within this domain resulted in a loss of GRK2 binding to both mGluR1a and mGluR1b. Mutation of either Lys(691) or Lys(692) prevented GRK2-mediated attenuation of mGluR1b signaling, whereas the mutation of only Lys(692) prevented GRK2-mediated inhibition of mGluR1a signaling. Thus, the mGluR1a carboxyl-terminal tail may also be involved in regulating the signaling of the mGluR1a splice variant. Taken together, our findings indicated that kinase binding to an mGluR1 domain involved in G protein-coupling is essential for the phosphorylation-independent attenuation of signaling by GRK2.     

RGS4 and RGS2 bind coatomer and inhibit COPI association with Golgi membranes and intracellular transport

COPI, a protein complex consisting of coatomer and the small GTPase ARF1, is an integral component of some intracellular transport carriers. The association of COPI with secretory membranes has been implicated in the maintenance of Golgi integrity and the normal functioning of intracellular transport in eukaryotes. The regulator of G protein signaling, RGS4, interacted with the COPI subunit beta'-COP in a yeast two-hybrid screen. Both recombinant RGS4 and RGS2 bound purified recombinant beta'-COP in vitro. Endogenous cytosolic RGS4 from NG108 cells and RGS2 from HEK293T cells cofractionated with the COPI complex by gel filtration. Binding of beta'-COP to RGS4 occurred through two dilysine motifs in RGS4, similar to those contained in some aminoglycoside antibiotics that are known to bind coatomer. RGS4 inhibited COPI binding to Golgi membranes independently of its GTPase-accelerating activity on G(ialpha). In RGS4-transfected LLC-PK1 cells, the amount of COPI in the Golgi region was considerably reduced compared with that in wild-type cells, but there was no detectable difference in the amount of either Golgi-associated ARF1 or the integral Golgi membrane protein giantin, indicating that Golgi integrity was preserved. In addition, RGS4 expression inhibited trafficking of aquaporin 1 to the plasma membrane in LLC-PK1 cells and impaired secretion of placental alkaline phosphatase from HEK293T cells. The inhibitory effect of RGS4 in these assays was independent of GTPase-accelerating activity but correlated with its ability to bind COPI. Thus, these data support the hypothesis that these RGS proteins sequester coatomer in the cytoplasm and inhibit its recruitment onto Golgi membranes, which may in turn modulate Golgi-plasma membrane or intra-Golgi transport.     

alpha 2A/alpha 2C-adrenergic receptor third loop chimera show that agonist interaction with receptor subtype backbone establishes G protein-coupled receptor kinase phosphorylation

The alpha(2A)-adrenergic receptor (AR) undergoes rapid agonist-promoted desensitization due to phosphorylation by G protein-coupled receptor kinases (GRKs) 2 and 3 at serines in the third intracellular loop of the receptor. In contrast, the alpha(2C)AR fails to display such desensitization or phosphorylation, which has been presumed to be due to this receptor lacking GRK phosphorylation sites. However, the alpha(2C)AR has multiple serines and threonines in putative favorable motifs within its third intracellular loop. We considered that the conformation of the third intracellular loop imposed by agonists binding to the transmembrane-spanning domains could be the basis of this subtype-specific property, rather than the presence or absence of phosphoacceptors per se. To address this, alpha(2A)/alpha(2C) third loop chimeric receptors were constructed. In whole cell phosphorylation studies, the alpha(2A) with the alpha(2C) third loop receptor underwent agonist-promoted phosphorylation while the alpha(2C) with the alpha(2A) third loop receptor did not, indicating that the agonist interaction with the parent receptor backbone establishes the phosphorylation phenotype. We postulated then that agonists with diverse structures that distinctly interact with alpha(2)AR should display different degrees of phosphorylation independent of receptor activation. Indeed, several full and partial agonists were identified, which evoked phosphorylation that was not related to intrinsic activity as established by [(35)S]guanosine 5'-3-O-(thio)triphosphate binding. Taken together, it appears that phosphorylation of the alpha(2)AR evoked by agonist is highly sensitive to the conformation of the third intracellular loop induced/stabilized by agonist to such an extent that these properties dictate the extent of phosphorylation of the loop when phosphoacceptors are present, and are the basis for subtype-specific phosphorylation.