Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.     

The amino-terminal domain of G-protein-coupled receptor kinase 2 is a regulatory Gbeta gamma binding site

G-protein-coupled receptor kinase 2 (GRK2) is activated by free Gbetagamma subunits. A Gbetagamma binding site of GRK2 is localized in the carboxyl-terminal pleckstrin homology domain. This Gbetagamma binding site of GRK2 also regulates Gbetagamma-stimulated signaling by sequestering free Gbetagamma subunits. We report here that truncation of the carboxyl-terminal Gbetagamma binding site of GRK2 did not abolish the Gbetagamma regulatory activity of GRK2 as determined by the inhibition of a Gbetagamma-stimulated increase in inositol phosphates in cells. This finding suggested the presence of a second Gbetagamma binding site in GRK2. And indeed, the amino terminus of GRK2 (GRK2(1-185)) inhibited a Gbetagamma-stimulated inositol phosphate signal in cells, purified GRK2(1-185) suppressed the Gbetagamma-stimulated phosphorylation of rhodopsin, and GRK2(1-185) bound directly to purified Gbetagamma subunits. The amino-terminal Gbetagamma regulatory site does not overlap with the RGS domain of GRK-2 because GRK2(1-53) with truncated RGS domain inhibited Gbetagamma-mediated signaling with similar potency and efficacy as did GRK2(1-185). In addition to the Gbetagamma regulatory activity, the amino-terminal Gbetagamma binding site of GRK2 affects the kinase activity of GRK2 because antibodies specifically cross-reacting with the amino terminus of GRK2 suppressed the GRK2-dependent phosphorylation of rhodopsin. The antibody-mediated inhibition was released by purified Gbetagamma subunits, strongly suggesting that Gbetagamma binding to the amino terminus of GRK2 enhances the kinase activity toward rhodopsin. Thus, the amino-terminal domain of GRK2 is a previously unrecognized Gbetagamma binding site that regulates GRK2-mediated receptor phosphorylation and inhibits Gbetagamma-stimulated signaling.     

The role of G beta gamma and domain interfaces in the activation of G protein-coupled receptor kinase 2

In response to extracellular signals, G protein-coupled receptors (GPCRs) catalyze guanine nucleotide exchange on Galpha subunits, enabling both activated Galpha and Gbetagamma subunits to target downstream effector enzymes. One target of Gbetagamma is G protein-coupled receptor kinase 2 (GRK2), an enzyme that initiates homologous desensitization by phosphorylating activated GPCRs. GRK2 consists of three distinct domains: an RGS homology (RH) domain, a protein kinase domain, and a pleckstrin homology (PH) domain, through which it binds Gbetagamma. The crystal structure of the GRK2-Gbetagamma complex revealed that the domains of GRK2 are intimately associated and left open the possibility for allosteric regulation by Gbetagamma. In this paper, we report the 4.5 A structure of GRK2, which shows that the binding of Gbetagamma does not induce large domain rearrangements in GRK2, although small rotations of the RH and PH domains relative to the kinase domain are evident. Mutation of residues within the larger domain interfaces of GRK2 generally leads to diminished expression and activity, suggesting that these interfaces are important for stability and remain intact upon activation of GRK2. Geranylgeranylated Gbetagamma, but not a soluble mutant of Gbetagamma, protects GRK2 from clostripain digestion at a site within its kinase domain that is 80 A away from the Gbetagamma binding site. Equilibrium ultracentrifugation experiments indicate that neither abnormally large detergent micelles nor protein oligomerization can account for the observed protection. The Gbetagamma-mediated binding of GRK2 to CHAPS micelles or lipid bilayers therefore appears to rigidify the kinase domain, perhaps by encouraging stable contacts between the RH and kinase domains.     

Mutation of a single TMVI residue, Phe(282), in the beta(2)-adrenergic receptor results in structurally distinct activated receptor conformations

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor (alpha(1B)-AR), a residue conserved in many G protein-coupled receptors (GPCRs), is critically involved in coupling agonist binding with TM helical movement and G protein activation. Here the equivalent residue, Phe(282), in the beta(2)-AR was evaluated by mutation to glycine, asparagine, alanine, or leucine. Except for F282N, which exhibits attenuated basal and maximal isoproterenol stimulation, the Phe(282) mutants display varying degrees of constitutive activity (F282L > F282A > F282G), and as shown by the results of substituted cysteine accessibility method (SCAM) studies, induce movement of endogenous cysteine(s) into the water-accessible ligand-binding pocket. For F282A, movement is confined to Cys(285) in TMVI, whereas F282L induces movement of both Cys(285) in TMVI and Cys(327) in TMVII. Further, engineered cysteine-sensor studies indicate that F282L causes movement of TMVI, both above and below an apparent kink-inducing TMVI proline (Pro(288)), whereas that due to F282A is confined to the domain below Pro(288). A plausible interpretation of these data is that receptor activation involves rigid body movement of TMVI which, because of its Pro(288)-induced kink, acts as a pivot to transduce and amplify the agonist-induced conformational change in the upper domain, to a change in the lower domain required for productive receptor-G protein coupling.     

Regulation of membrane targeting of the G protein-coupled receptor kinase 2 by protein kinase A and its anchoring protein AKAP79

The beta2 adrenergic receptor (beta2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta2AR markedly inhibit phosphorylation of beta2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbetagamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta2AR and phosphorylation of agonist-occupied beta2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization.     

Roles of phosphorylation-dependent and -independent mechanisms in the regulation of M1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 in hippocampal neurons

When co-expressed with the inositol 1,4,5-trisphosphate biosensor eGFP-PH(PLC delta), G protein-coupled receptor kinase 2 (GRK2) can suppress M1 muscarinic acetylcholine (mACh) receptor-mediated phospholipase C signaling in hippocampal neurons through a phosphorylation-independent mechanism, most likely involving the direct binding of the RGS homology domain of GRK2 to G alpha(q/11). To define the importance of this mechanism in comparison with classical, phosphorylation-dependent receptor regulation by GRKs, we have examined M1 mACh receptor signaling in hippocampal neurons following depletion of GRK2 and also in the presence of non-G alpha(q/11)-binding GRK2 mutants. Depletion of neuronal GRK2 using an antisense strategy almost completely inhibited M1 mACh receptor desensitization without enhancing acute agonist-stimulated phospholipase C activity. By stimulating neurons with a submaximal agonist concentration before (R1) and after (R2) a period of exposure to a maximal agonist concentration, an index (R2/R1) of agonist-induced desensitization of signaling could be obtained. Co-transfection of neurons with either a non-G alpha(q/11)-binding (D110A) GRK2 mutant or the catalytically inactive (D110A,K220R)GRK2 did not suppress acute M1 mACh receptor-stimulated inositol 1,4,5-trisphosphate production. However, using the desensitization (R2/R1) protocol, it could be shown that expression of (D110A)GRK2 enhanced, whereas (D110A,K220R)GRK2 inhibited, agonist-induced M1 mACh receptor desensitization. In Chinese hamster ovary cells, the loss of G alpha(q/11) binding did not affect the ability of the (D110A)GRK2 mutant to phosphorylate M1 mACh receptors, whereas expression of (D110A,K220R)GRK2 had no effect on receptor phosphorylation. These data indicate that in hippocampal neurons endogenous GRK2 is a key regulator of M1 mACh receptor signaling and that the regulatory process involves both phosphorylation-dependent and -independent mechanisms.     

Multiple residues in the second extracellular loop are critical for M3 muscarinic acetylcholine receptor activation

Recent studies suggest that the second extracellular loop (o2 loop) of bovine rhodopsin and other class I G protein-coupled receptors (GPCRs) targeted by biogenic amine ligands folds deeply into the transmembrane receptor core where the binding of cis-retinal and biogenic amine ligands is known to occur. In the past, the potential role of the o2 loop in agonist-dependent activation of biogenic amine GPCRs has not been studied systematically. To address this issue, we used the M(3) muscarinic acetylcholine receptor (M3R), a prototypic class I GPCR, as a model system. Specifically, we subjected the o2 loop of the M3R to random mutagenesis and subsequently applied a novel yeast genetic screen to identity single amino acid substitutions that interfered with M3R function. This screen led to the recovery of about 20 mutant M3Rs containing single amino acid changes in the o2 loop that were inactive in yeast. In contrast, application of the same strategy to the extracellular N-terminal domain of the M3R did not yield any single point mutations that disrupted M3R function. Pharmacological characterization of many of the recovered mutant M3Rs in mammalian cells, complemented by site-directed mutagenesis studies, indicated that the presence of several o2 loop residues is important for efficient agonist-induced M3R activation. Besides the highly conserved Cys(220) residue, Gln(207), Gly(211), Arg(213), Gly(218), Ile(222), Phe(224), Leu(225), and Pro(228) were found to be of particular functional importance. In general, mutational modification of these residues had little effect on agonist binding affinities. Our findings are therefore consistent with a model in which multiple o2 loop residues are involved in stabilizing the active state of the M3R. Given the high degree of structural homology found among all biogenic amine GPCRs, our findings should be of considerable general relevance.     

Agonist-induced signaling, desensitization, and internalization of a phosphorylation-deficient AT1A angiotensin receptor

An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.     

Agonist-dependent desensitization of the kappa opioid receptor by G protein receptor kinase and beta-arrestin.

We used the Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). kappa agonists increased the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current was modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increased the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone had no effect on the KOR desensitization rate. The desensitization was homologous as co-expressed delta opioid receptor-evoked responses were not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization was reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] did not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuated desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations did not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produced homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR.     

Effect of G Protein-Coupled Receptor Kinase 2 on the Sensitivity of M4 Muscarinic Acetylcholine Receptors to Agonist-Induced Internalization and Desensitization in NG108-15 Cells

NG108-15 cells express predominantly the M4 subtype of the muscarinic receptor for acetylcholine. Stimulation of these receptors by the agonist carbachol causes an inhibition of cellular adenylyl cyclase and a consequent fall in the intracellular cyclic AMP concentration. Pretreatment of the cells with carbachol caused both internalization and desensitization of the M4 receptor. Overexpression of G protein-coupled receptor kinase (GRK) 2 caused an increase in the rate constant for receptor endocytosis (from 0.06 to 0.18 min(-1)) and a decrease in the EC50 for carbachol stimulation of internalization (from 15 to 3 microM). Overexpression of a dominant negative form of GRK2 had more modest effects, reducing the rate constant for endocytosis (from 0.11 to 0.07 min(-1)) and increasing the EC50 for carbachol stimulation of internalization (from 8 to 17 microM). Neither GRK2 nor dominant negative GRK2 overexpression had any effect on the rate constant for receptor recycling following agonist removal. The time course and extent of receptor desensitization in control cells were identical to the corresponding values for receptor internalization, and the rate and extent of desensitization were again increased by GRK2 overexpression. Exposure of the cells to hyperosmolar sucrose (0.6 M) almost completely blocked agonist-induced receptor internalization in both control and GRK2-overexpressing cells. Sucrose treatment also blocked agonist-induced desensitization. We conclude that the internalization and desensitization of the M4 muscarinic receptor in NG108-15 cells can be modulated in response to changes in GRK2 activity and also that internalization plays a key role in desensitization.     

Agonist-induced formation of opioid receptor-G protein-coupled receptor kinase (GRK)-G beta gamma complex on membrane is required for GRK2 function in vivo

G protein-coupled receptor kinases (GRKs) catalyze agonist-induced receptor phosphorylation on the membrane and initiate receptor desensitization. Previous in vitro studies have shown that the binding of GRK to membrane-associated G beta gamma subunits plays an important role in translocation of GRK2 from the cytoplasm to the plasma membrane. The current study investigated the role of the interaction of GRK2 with the activated delta-opioid receptor (DOR) and G beta gamma subunits in the membrane translocation and function of GRK2 using intact human embryonic kidney 293 cells. Our results showed that agonist treatment induced GRK2 binding to DOR, GRK2 translocation to the plasma membrane, and DOR phosphorylation in cells expressing the wild-type DOR but not the mutant DOR lacking the carboxyl terminus, which contains all three GRK2 phosphorylation sites. DORs with the GRK2 phosphorylation sites modified (M3) or with the acidic residues flanking phosphorylation sites mutated (E355Q/D364N) failed to be phosphorylated in response to agonist stimulation. Agonist-induced GRK2 membrane translocation and GRK-receptor association were observed in cells expressing M3 but not E355Q/D364N. Moreover, over-expression of G beta gamma subunits promoted GRK2 binding to DOR, whereas over-expression of transducin alpha or the carboxyl terminus of GRK2 blocked binding. Further study demonstrated that agonist stimulation induced the formation of a complex containing DOR, GRK2, and G beta gamma subunits in the cell and that agonist-stimulated formation of this complex is essential for the stable localization of GRK2 on the membrane and for its catalytic activity in vivo.     

Role of the transmembrane domain 4/extracellular loop 2 junction of the human gonadotropin-releasing hormone receptor in ligand binding and receptor conformational selection

Recent crystal structures of G protein-coupled receptors (GPCRs) show the remarkable structural diversity of extracellular loop 2 (ECL2), implying its potential role in ligand binding and ligand-induced receptor conformational selectivity. Here we have applied molecular modeling and mutagenesis studies to the TM4/ECL2 junction (residues Pro(174(4.59))-Met(180(4.66))) of the human gonadotropin-releasing hormone (GnRH) receptor, which uniquely has one functional type of receptor but two endogenous ligands in humans. We suggest that the above residues assume an α-helical extension of TM4 in which the side chains of Gln(174(4.60)) and Phe(178(4.64)) face toward the central ligand binding pocket to make H-bond and aromatic contacts with pGlu(1) and Trp(3) of both GnRH I and GnRH II, respectively. The interaction between the side chains of Phe(178(4.64)) of the receptor and Trp(3) of the GnRHs was supported by reciprocal mutations of the interacting residues. Interestingly, alanine mutations of Leu(175(4.61)), Ile(177(4.63)), and Met(180(4.66)) decreased mutant receptor affinity for GnRH I but, in contrast, increased affinity for GnRH II. This suggests that these residues make intramolecular or intermolecular contacts with residues of transmembrane (TM) domain 3, TM5, or the phospholipid bilayer, which couple the ligand structure to specific receptor conformational switches. The marked decrease in signaling efficacy of I177A and F178A also indicates that IIe(177(4.63)) and Phe(178(4.64)) are important in stabilizing receptor-active conformations. These findings suggest that the TM4/ECL2 junction is crucial for peptide ligand binding and, consequently, for ligand-induced receptor conformational selection.     

c-Src tyrosine kinase binds the beta 2-adrenergic receptor via phospho-Tyr-350, phosphorylates G-protein-linked receptor kinase 2, and mediates agonist-induced receptor desensitization

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.     

The membrane proximal disulfides of the EGF receptor extracellular domain are required for high affinity binding and signal transduction but do not play a role in the localization of the receptor to lipid rafts

The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys-->Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.     

The tethering arm of the EGF receptor is required for negative cooperativity and signal transduction

The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-to-back dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use (125)I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys(558)-Cys(567) disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys(571)-Cys(593) disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys(571)-Cys(593) disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunit-subunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.     

Role of G protein-coupled receptor kinases on the agonist-induced phosphorylation and internalization of the follitropin receptor.

The experiments presented herein were designed to identify members of the G protein-coupled receptor kinase (GRK) family that participate in the agonist-induced phosphorylation and internalization of the rat FSH receptor (rFSHR). Western blots of human kidney 293 cells (the cell line used in transfection experiments) and MSC-1 cells (a cell line derived from Sertoli cells that displays many of the differentiated functions of their normal counterparts) reveal the presence of GRK2 and GRK6 in both cell lines as well as GRK4 in MSC-1 cells. Cotransfection of 293 cells with the rFSHR and GRK2, GRK4alpha, or GRK6 resulted in an increase in the agonist-induced phosphorylation of the rFSHR. Cotransfections of the rFSHR with GRKs or arrestin-3 enhanced the agonist-induced internalization of the rFHSR, and combinations of GRKs and arrestin-3 were more effective than the individual components. To characterize the involvement of endogenous GRKs on phosphorylation and internalization, we inhibited endogenous GRK2 by overexpression of a kinase-deficient mutant of GRK2 or G alpha t, a scavenger of G betagamma. We also inhibited endogenous GRK6 by overexpression of a kinase-deficient mutant of GKR6. All three constructs were effective inhibitors of phosphorylation, but only the kinase-deficient mutant of GRK2 and G alpha t inhibited internalization. The inhibition of internalization induced by these two constructs was less pronounced than that induced by a dominant-negative mutant of the nonvisual arrrestins, however. The finding that inhibitors of GRK2 and GRK6 impair phosphorylation, but only the inhibitors of GRK2 impair internalization, suggests that different GRKs have differential effects on receptor internalization.     

Role of the G protein-coupled receptor kinase site serine cluster in beta2-adrenergic receptor internalization, desensitization, and beta-arrestin translocation

There is considerable evidence for the role of carboxyl-terminal serines 355, 356, and 364 in G protein-coupled receptor kinase (GRK)-mediated phosphorylation and desensitization of beta(2)-adrenergic receptors (beta(2)ARs). In this study we used receptors in which these serines were changed to alanines (SA3) or to aspartic acids (SD3) to determine the role of these sites in beta-arrestin-dependent beta(2)AR internalization and desensitization. Coupling efficiencies for epinephrine activation of adenylyl cyclase were similar in wild-type and mutant receptors, demonstrating that the SD3 mutant did not drive constitutive GRK desensitization. Treatment of wild-type and mutant receptors with 0.3 nm isoproterenol for 5 min induced approximately 2-fold increases in the EC(50) for agonist activation of adenylyl cyclase, consistent with protein kinase A (PKA) site-mediated desensitization. When exposed to 1 mum isoproterenol to trigger GRK site-mediated desensitization, only wild-type receptors showed significant further desensitization. Using a phospho site-specific antibody, we determined that there is no requirement for these GRK sites in PKA-mediated phosphorylation at high agonist concentration. The rates of agonist-induced internalization of the SD3 and SA3 mutants were 44 and 13%, respectively, relative to that of wild-type receptors, but the SD3 mutant recruited enhanced green fluorescent protein (EGFP)-beta-arrestin 2 to the plasma membrane, whereas the SA3 mutant did not. EGFP-beta-Arrestin2 overexpression triggered a significant increase in the extent of SD3 mutant desensitization but had no effect on the desensitization of wild-type receptors or the SA3 mutant. Expression of a phosphorylation-independent beta-arrestin 1 mutant (R169E) significantly rescued the internalization defect of the SA3 mutant but inhibited the phosphorylation of serines 355 and 356 in wild-type receptors. Our data demonstrate that (i) the lack of GRK sites does not impair PKA site phosphorylation, (ii) the SD3 mutation inhibits GRK-mediated desensitization although it supports some agonist-induced beta-arrestin binding and receptor internalization, and (iii) serines 355, 356, and 364 play a pivotal role in the GRK-mediated desensitization, beta-arrestin binding, and internalization of beta(2)ARs.     

Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.     

The unique extracellular disulfide loop of the glycine receptor is a principal ligand binding element.

A loop structure, formed by the putative disulfide bridging of Cys198 and Cys209, is a principal element of the ligand binding site in the glycine receptor (GlyR). Disruption of the loop's tertiary structure by Ser mutations of these Cys residues either prevented receptor assembly on the cell surface, or created receptors unable to be activated by agonists or to bind the competitive antagonist, strychnine. Mutation of residues Lys200, Tyr202 and Thr204 within this loop reduced agonist binding and channel activation sensitivities by up to 55-, 520- and 190-fold, respectively, without altering maximal current sizes, and mutations of Lys200 and Tyr202 abolished strychnine binding to the receptor. Removal of the hydroxyl moiety from Tyr202 by mutation to Phe profoundly reduced agonist sensitivity, whilst removal of the benzene ring abolished strychnine binding, thus demonstrating that Tyr202 is crucial for both agonist and antagonist binding to the GlyR. Tyr202 also influences receptor assembly on the cell surface, with only large chain substitutions (Phe, Leu and Arg, but not Thr, Ser and Ala) forming functional receptors. Our data demonstrate the presence of a second ligand binding site in the GlyR, consistent with the three-loop model of ligand binding to the ligand-gated ion channel superfamily.     

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.