Structural determinants for ligand-receptor conformational selection in a peptide G protein-coupled receptor

G protein coupled receptors (GPCRs) modulate the majority of physiological processes through specific intermolecular interactions with structurally diverse ligands and activation of differential intracellular signaling. A key issue yet to be resolved is how GPCRs developed selectivity and diversity of ligand binding and intracellular signaling during evolution. We have explored the structural basis of selectivity of naturally occurring gonadotropin-releasing hormones (GnRHs) from different species in the single functional human GnRH receptor. We found that the highly variable amino acids in position 8 of the naturally occurring isoforms of GnRH play a discriminating role in selecting receptor conformational states. The human GnRH receptor has a higher affinity for the cognate GnRH I but a lower affinity for GnRH II and GnRHs from other species possessing substitutions for Arg(8). The latter were partial agonists in the human GnRH receptor. Mutation of Asn(7.45) in transmembrane domain (TM) 7 had no effect on GnRH I affinity but specifically increased affinity for other GnRHs and converted them to full agonists. Using molecular modeling and site-directed mutagenesis, we demonstrated that the highly conserved Asn(7.45) makes intramolecular interactions with a highly conserved Cys(6.47) in TM 6, suggesting that disruption of this intramolecular interaction induces a receptor conformational change which allosterically alters ligand specific binding sites and changes ligand selectivity and signaling efficacy. These results reveal GnRH ligand and receptor structural elements for conformational selection, and support co-evolution of GnRH ligand and receptor conformations.     

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site

The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe110^3.24, Asn115^3.29, Asn173^4.60, Phe179^45.39, Asn194^5.40, Phe195^5.41, Leu201^5.47, His252^6.54, and Tyr268^7.32) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [³H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian G_i proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn194^5.40 and Leu201^5.47, were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that—in contrast to most other rhodopsin-like GPCRs—the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.     

The functional microdomain in transmembrane helices 2 and 7 regulates expression, activation, and coupling pathways of the gonadotropin-releasing hormone receptor.

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp(2.50) and Asn(7.49) residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn(1.50(53)) residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.     

Genetic analysis of the first and third extracellular loops of the C5a receptor reveals an essential WXFG motif in the first loop

The extracellular loops of G protein-coupled receptors (GPCRs) frequently contain binding sites for peptide ligands. However, the mechanism of receptor activation following ligand binding and the influence of the extracellular loops in other aspects of receptor function are poorly understood. Here we report a structure-function analysis of the first and third extracellular loops of the human C5a receptor, a GPCR that binds a 74-amino acid peptide ligand. Amino acid substitutions were randomly incorporated into each loop, and functional receptors were identified in yeast. The first extracellular loop contains a large number of positions that cannot tolerate amino acid substitutions, especially residues within the WXFG motif found in many rhodopsin-like GPCRs, yet disruption of these residues does not alter C5a binding affinity. These results demonstrate an unanticipated role for the first extracellular loop, and the WXFG motif in particular, in ligand-mediated activation of the C5a receptor. This motif likely serves a similar role in other GPCRs. The third extracellular loop, in contrast, contains far fewer preserved residues and appears to play a less essential role in receptor activation.     

Common structural requirements for heptahelical domain function in class A and class C G protein-coupled receptors

G protein-coupled receptors (GPCRs) are key players in cell communication. Several classes of such receptors have been identified. Although all GPCRs possess a heptahelical domain directly activating G proteins, important structural and sequence differences within receptors from different classes suggested distinct activation mechanisms. Here we show that highly conserved charged residues likely involved in an interaction network between transmembrane domains (TM) 3 and 6 at the cytoplasmic side of class C GPCRs are critical for activation of the gamma-aminobutyric acid type B receptor. Indeed, the loss of function resulting from the mutation of the conserved lysine residue into aspartate or glutamate in the TM3 of gamma-aminobutyric acid type B(2) can be partly rescued by mutating the conserved acidic residue of TM6 into either lysine or arginine. In addition, mutation of the conserved lysine into an acidic residue leads to a nonfunctional receptor that displays a high agonist affinity. This is reminiscent of a similar ionic network that constitutes a lock stabilizing the inactive state of many class A rhodopsin-like GPCRs. These data reveal that despite their original structure, class C GPCRs share with class A receptors at least some common structural feature controlling G protein activation.     

Prediction of the odorant binding site of olfactory receptor proteins by human-mouse comparisons

Olfactory receptors (ORs) are a large family of proteins involved in the recognition and discrimination of numerous odorants. These receptors belong to the G-protein coupled receptor (GPCR) hyperfamily, for which little structural data are available. In this study we predict the binding site residues of OR proteins by analyzing a set of 1441 OR protein sequences from mouse and human. The central insight utilized is that functional contact residues would be conserved among pairs of orthologous receptors, but considerably less conserved among paralogous pairs. Using judiciously selected subsets of 218 ortholog pairs and 518 paralog pairs, we have identified 22 sequence positions that are both highly conserved among the putative orthologs and variable among paralogs. These residues are disposed on transmembrane helices 2 to 7, and on the second extracellular loop of the receptor. Strikingly, although the prediction makes no assumption about the location of the binding site, these amino acid positions are clustered around a pocket in a structural homology model of ORs, mostly facing the inner lumen. We propose that the identified positions constitute the odorant binding site. This conclusion is supported by the observation that all but one of the predicted binding site residues correspond to ligand-contact positions in other rhodopsin-like GPCRs.     

Identifying G-protein coupled receptors using weighted Levenshtein distance and the nearest neighbor method

G-protein coupled receptors （GPCRs） are a class of seven-helix transmembrane proteins that have been used in bioinformatics as the targets to facilitate drug discovery for human diseases. Although thousands of GPCR sequences have been collected, the ligand specificity of many GPCRs is still unknown and only one crystal structure of the rhodopsin-like family has been solved. Therefore, identifying GPCR types only from sequence data has become an important research issue. In this study, a novel technique for identifying GPCR types based on the weighted Levenshtein distance between two receptor sequences and the nearest neighbor method （NNM） is introduced, which can deal with receptor sequences with different lengths directly. In our experiments for classifying four classes （acetylcholine, adrenoceptor, dopamine, and serotonin） of the rhodopsin-like family of GPCRs, the error rates from the leave-one-out procedure and the leave-half-out procedure were 0.62% and 1.24%, respectively. These results are prior to those of the covariant discriminant algorithm, the support vector machine method, and the NNM with Euclidean distance.     

Polymorphisms and genetic linkage of histamine receptors

Histamine is a biogenic amine that plays an essential role in controlling many physiological functions, both in the central nervous system (CNS) and the peripheral nervous system (PNS). Most of these physiological effects are mediated through interactions with four histamine receptor subtypes, all of which are members of the larger family of rhodopsin-like class A G-protein coupled receptors (GPCRs) (Leurs et al., 2011; Lim et al., 2009). Here, we focus on the genetic variations and polymorphisms localized on the genes encoding for human histamine receptors where it provides an up to date collection of all polymorphisms found on genes encoding the histamine receptor subtypes and their association to diseases.     

Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases

G protein-coupled receptors (GPCRs) are integral membrane proteins that, in response to activation by extracellular stimuli, regulate intracellular second messenger levels via their coupling to heterotrimeric G proteins. GPCR activation also initiates a series of molecular events that leads to G protein-coupled receptor kinase-mediated receptor phosphorylation and the binding of beta-arrestin proteins to the intracellular face of the receptor. beta-Arrestin binding not only contributes to the G protein-uncoupling of GPCRs, but also mediates the targeting of many GPCRs for endocytosis in clathrin-coated pits. Several GPCRs internalize as a stable complex with beta-arrestin and the stability of this complex appears to regulate, at least in part, whether the receptors are dephosphorylated in early endosomes and recycled back to the cell surface as fully functional receptors, retained in early endosomes or targeted for degradation in lysosomes. More recently, it has become appreciated that the movement of GPCRs through functionally distinct intracellular membrane compartments is regulated by a variety of Rab GTPases and that the activity of these Rab GTPases may influence GPCR function. Moreover, it appears that GPCRs are not simply passive cargo molecules, but that GPCR activation may directly influence Rab GTPase activity and as such, GPCRs may directly control their own targeting between intracellular compartments. This review provides a synopsis of the current knowledge regarding the role of beta-arrestins and Rab GTPases in regulating the intracellular trafficking and function of GPCRs.     

Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

Sequence,Structure and Ligand Binding Evolution of Rhodopsin-Like G Protein-Coupled Receptors: A Crystal Structure-Based Phylogenetic Analysis

G protein-coupled receptors (GPCRs) form the largest family of membrane receptors in the human genome. Advances in membrane protein crystallization so far resulted in the determination of 24 receptors available as high-resolution atomic structures. We performed the first phylogenetic analysis of GPCRs based on the available set of GPCR structures. We present a new phylogenetic tree of known human rhodopsin-like GPCR sequences based on this structure set. We can distinguish the three separate classes of small-ligand binding GPCRs, peptide binding GPCRs, and olfactory receptors. Analyzing different structural subdomains, we found that small molecule binding receptors most likely have evolved from peptide receptor precursors, with a rhodopsin/S1PR1 ancestor, most likely an ancestral opsin, forming the link between both classes. A light-activated receptor therefore seems to be the origin of the small molecule hormone receptors of the central nervous system. We find hints for a common evolutionary path of both ligand binding site and central sodium/water binding site. Surprisingly, opioid receptors exhibit both a binding cavity and a central sodium/water binding site similar to the one of biogenic amine receptors instead of peptide receptors, making them seemingly prone to bind small molecule ligands, e.g. opiates. Our results give new insights into the relationship and the pharmacological properties of rhodopsin-like GPCRs.     

Simulations of a G protein-coupled receptor homology model predict dynamic features and a ligand binding site

A computational approach to predict structures of rhodopsin-like G protein-coupled receptors (GPCRs) is presented and evaluated by comparison to the X-ray structural models. By combining sequence alignment, the rhodopsin crystal structure, and point mutation data on the beta2 adrenoreceptor (b2ar), we predict a (-)-epinephrine-bound computational model of the beta2 adrenoreceptor. The model is evaluated by molecular dynamics simulations and by comparison with the recent X-ray structures of b2ar. The overall correspondence between the predicted and the X-ray structural model is high. Especially the prediction of the ligand binding site is accurate. This shows that the proposed dynamic homology modelling approach can be used to create reasonable models for the understanding of structure and dynamics of other rhodopsin-like GPCRs.     

Allosteric Effects of Sodium Ion Binding on Activation of the M3 Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) are important membrane proteins that mediate cellular signaling and represent primary targets for about one-third of currently marketed drugs. Recent x-ray crystallographic studies identified distinct conformations of GPCRs in the active and inactive states. An allosteric sodium ion was found bound to a highly conserved D2.50 residue in inactive GPCRs, whereas the D2.50 allosteric pocket became collapsed in active GPCR structures. However, the dynamic mechanisms underlying these observations remain elusive. In this study, we aimed to understand the mechanistic effects of sodium ion binding on dynamic activation of the M3 muscarinic GPCR through long-timescale accelerated molecular dynamics (aMD) simulations. Results showed that with the D2.50 residue deprotonated, the M3 receptor is bound by an allosteric sodium ion and confined mostly in the inactive state with remarkably reduced flexibility. In contrast, the D2.50-protonated receptor does not exhibit sodium ion binding to the D2.50 allosteric site and samples a significantly larger conformational space. The receptor activation is captured and characterized by large-scale structural rearrangements of the transmembrane helices via dynamic hydrogen bond and salt bridge interactions. The residue motions are highly correlated during receptor activation. Further network analysis revealed that the allosteric signaling between residue D2.50 and key residues in the intracellular, extracellular, and orthosteric pockets is significantly weakened upon sodium ion binding.     

Beta2 adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch

In many rhodopsin-like G-protein-coupled receptors, agonist binding to a cluster of aromatic residues in TM6 may promote receptor activation by altering the configuration of the TM6 Pro-kink and by the subsequent movement of the cytoplasmic end of TM6 away from TM3. We hypothesized that the highly conserved Cys(6.47), in the vicinity of the conserved Pro(6.50), modulates the configuration of the aromatic cluster and the TM6 Pro-kink through specific interactions in its different rotamer configurations. In the beta(2) adrenergic receptor, mutation of Cys(6.47) to Thr, which in an alpha-helix has a different rotamer distribution from Cys and Ser, produced a constitutively active receptor, whereas the Ser mutant was similar to wild-type receptor. Use of the biased Monte Carlo technique of Conformational Memories showed that the rotamer changes among Cys/Ser/Thr(6.47), Trp(6.48), and Phe(6.52) are highly correlated, representing a rotamer "toggle switch" that may modulate the TM6 Pro-kink. Differential modulation of the accessibility of Cys(6.47) and an engineered Cys(6.52) in wild type and a constitutively active background provides experimental support for the association of this rotamer switch with receptor activation.     

Two cytoplasmic loops of the glucagon receptor are required to elevate cAMP or intracellular calcium.

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure–function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure–function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22–Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.     

A molecular dissection of the glycoprotein hormone receptors

In glycoprotein hormone receptors, a subfamily of rhodopsin-like G protein-coupled receptors, the recognition and activation steps are carried out by separate domains of the proteins. Specificity of recognition of the hormones thyrotropin (TSH), lutropin (LH), human chorionic gonadotropin (hCG) and follitropin (FSH) involves leucine-rich repeats (LRRs) present in an N-terminal ectodomain, and can be associated with a limited number of residues at key positions of the LRRs. The mechanism by which binding of the hormones results in activation is proposed to involve switching of the ectodomain from a tethered inverse agonist to a full agonist of the serpentine, rhodopsin-like region of the receptor. Unexpectedly, the picture is complicated by the observation that promiscuous activation of one of the receptors (FSHr) by hCG or TSH can result from activating mutations affecting the serpentine region of the receptors.     

Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure-function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure-function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22-Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.     

Calindol, a positive allosteric modulator of the human Ca(2+) receptor, activates an extracellular ligand-binding domain-deleted rhodopsin-like seven-transmembrane structure in the absence of Ca(2+)

The extracellular calcium-sensing human Ca(2+) receptor (hCaR),2 a member of the family-3 G-protein-coupled receptors (GPCR) possesses a large amino-terminal extracellular ligand-binding domain (ECD) in addition to a seven-transmembrane helical domain (7TMD) characteristic of all GPCRs. Two calcimimetic allosteric modulators, NPS R-568 and Calindol ((R)-2-{1-(1-naphthyl)ethyl-aminom-ethyl}indole), that bind the 7TMD of the hCaR have been reported to potentiate Ca(2+) activation without independently activating the wild type receptor. Because agonists activate rhodopsin-like family-1 GPCRs by binding within the 7TMD, we examined the ability of Calindol, a novel chemically distinct calcimimetic, to activate a Ca(2+) receptor construct (T903-Rhoc) in which the ECD and carboxyl-terminal tail have been deleted to produce a rhodopsin-like 7TMD. Here we report that although Calindol has little or no agonist activity in the absence of extracellular Ca(2+) for the ECD-containing wild type or carboxyl-terminal deleted receptors, it acts as a strong agonist of the T903-Rhoc. In addition, Ca(2+) alone displays little or no agonist activity for the hCaR 7TMD, but potentiates the activation by Calindol. We confirm that activation of Ca(2+) T903-Rhoc by Calindol truly the is independent using in vitro reconstitution with purified G(q). These findings demonstrate distinct allosteric linkages between Ca(2+) site(s) in the ECD and 7TMD and the 7TMD site(s) for calcimimetics.     

Feedback inhibition of G protein-coupled receptor kinase 2 (GRK2) activity by extracellular signal-regulated kinases

G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and beta-arrestin binding uncouple G protein-coupled receptors (GPCRs) from their respective G proteins and initiates the process of receptor internalization. In the case of the beta(2)-adrenergic receptor and lysophosphatidic acid receptor, these processes can lead to ERK activation. Here we identify a novel mechanism whereby the activity of GRK2 is regulated by feedback inhibition. GRK2 is demonstrated to be a phosphoprotein in cells. Mass spectrometry and mutational analysis localize the site of phosphorylation on GRK2 to a carboxyl-terminal serine residue (Ser(670)). Phosphorylation at Ser(670) impairs the ability of GRK2 to phosphorylate both soluble and membrane-incorporated receptor substrates and dramatically attenuates Gbetagamma-mediated activation of this enzyme. Ser(670) is located in a peptide sequence that conforms to an ERK consensus phosphorylation sequence, and in vitro, in the presence of heparin, ERK1 phosphorylates GRK2. Inhibition of ERK activity in HEK293 cells potentiates GRK2 activity, whereas, conversely, ERK activation inhibits GRK2 activity. The discovery that ERK phosphorylates and inactivates GRK2 suggests that ERK participates in a feedback regulatory loop. By negatively regulating GRK-mediated receptor phosphorylation, beta-arrestin-mediated processes such as Src recruitment and clathrin-mediated internalization, which are required for GPCR-mediated ERK activation, are inhibited, thus dampening further ERK activation.