GPCR activation and GRK2 assembly by a biased intracellular agonist.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1-6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR-GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR-GRK interactions and GRK2-mediated biased signalling.

The short third intracellular loop and cytoplasmic tail of bitter taste receptors provide functionally relevant GRK phosphorylation sites in TAS2R14.

For most G protein-coupled receptors, the third intracellular loop (IL3) and carboxy-terminal tail (CT) are sites for G protein-coupled receptor kinase (GRK)-mediated phosphorylation, leading to ß-arrestin binding and agonist-specific desensitization. These regions of bitter taste receptors (TAS2Rs) are extremely short compared with the superfamily, and their function in desensitization is unknown. TAS2R14 expressed on human airway smooth muscle cells relax the cell, suggesting a novel target for bronchodilators. To assess IL3 and CT in agonist-promoted TAS2R14 desensitization (tachyphylaxis), we generated fusion proteins of both the WT sequence and Ala substituted for Ser/Thr in the IL3 and CT sequences. In vitro, activated GRK2 phosphorylated WT IL3 and WT CT proteins but not Ala-substituted forms. TAS2R14s with mutations in IL3 (IL-5A), CT (CT-5A), and in both regions (IL/CT-10A) were expressed in human embryonic kidney 293T cells. IL/CT-10A and CT-5A failed to undergo desensitization of the intracellular calcium response compared with WT, indicating that functional desensitization by GRK phosphorylation is at residues in the CT. Desensitization of TAS2R14 was blocked by GRK2 knockdown in human airway smooth muscle cells. Receptor:ß-arrestin binding was absent in IL/CT-10A and CT-5A and reduced in IL-5A, indicating a role for IL3 phosphorylation in the ß-arrestin interaction for this function. Agonist-promoted internalization of IL-5A and CT-5A receptors was impaired, and they failed to colocalize with early endosomes. Thus, agonist-promoted functional desensitization of TAS2R14 occurs by GRK phosphorylation of CT residues and ß-arrestin binding. However, ß-arrestin function in the internalization and trafficking of the receptor also requires GRK phosphorylation of IL3 residues.

A peptide of the amino-terminus of GRK2 induces hypertrophy and yet elicits cardioprotection after pressure overload.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression and activity are elevated early on in response to several forms of cardiovascular stress and are a hallmark of heart failure. Interestingly, though, in addition to its well-characterized role in regulating GPCRs, mounting evidence suggests a GRK2 "interactome" that underlies a great diversity in its functional roles. Several such GRK2 interacting partners are important for adaptive and maladaptive myocyte growth; therefore, an understanding of domain-specific interactions with signaling and regulatory molecules could lead to novel targets for heart failure therapy. Herein, we subjected transgenic mice with cardiac restricted expression of a short, amino terminal fragment of GRK2 (ßARKnt) to pressure overload and found that unlike their littermate controls or previous GRK2 fragments, they exhibited an increased left ventricular wall thickness and mass prior to cardiac stress that underwent proportional hypertrophic growth to controls after acute pressure overload. Importantly, despite this enlarged heart, ßARKnt mice did not undergo the expected transition to heart failure observed in controls. Further, ßARKnt expression limited adverse left ventricular remodeling and increased cell survival signaling. Proteomic analysis to identify ßARKnt binding partners that may underlie the improved cardiovascular phenotype uncovered a selective functional interaction of both endogenous GRK2 and ßARKnt with AKT substrate of 160 kDa (AS160). AS160 has emerged as a key downstream regulator of insulin signaling, integrating physiological and metabolic cues to couple energy demand to membrane recruitment of Glut4. Our preliminary data indicate that in ßARKnt mice, cardiomyocyte insulin signaling is improved during stress, with a coordinate increase in spare respiratory activity and ATP production without metabolite switching. Surprisingly, these studies also revealed a significant decrease in gonadal fat weight, equivalent to human abdominal fat, in male ßARKnt mice at baseline and following cardiac stress. These data suggest that the enhanced AS160-mediated signaling in the ßARKnt mice may ameliorate pathological cardiac remodeling through direct modulation of insulin signaling within cardiomyocytes, and translate these to beneficial effects on systemic metabolism.

TRH receptor mobility in the plasma membrane is strongly affected by agonist binding and by interaction with some cognate signaling proteins.

OBJECTIVES: Extensive research has been dedicated to elucidating the mechanisms of signal transduction through different G protein-coupled receptors (GPCRs). However, relatively little is known about the regulation of receptor movement within the cell membrane upon ligand binding. In this study we focused our attention on the thyrotropin-releasing hormone (TRH) receptor that typically couples to Gq/11 proteins. METHODS: We monitored receptor diffusion in the plasma membrane of HEK293 cells stably expressing yellow fluorescent protein (YFP)-tagged TRH receptor (TRHR-YFP) by fluorescence recovery after photobleaching (FRAP). RESULTS: FRAP analysis indicated that the lateral movement of the TRH receptor was markedly reduced upon TRH binding as the value of its diffusion coefficient fell down by 55%. This effect was prevented by the addition of the TRH receptor antagonist midazolam. We also found that siRNA-mediated knockdown of Gq/11α, Gß, ß-arrestin2 and phospholipase Cß1, but not of Giα1, ß-arrestin1 or G protein-coupled receptor kinase 2, resulted in a significant decrease in the rate of TRHR-YFP diffusion, indicating the involvement of the former proteins in the regulation of TRH receptor behavior. The observed partial reduction of the TRHR-YFP mobile fraction caused by down-regulation of Giα1 and ß-arrestin1 suggests that these proteins may also play distinct roles in THR receptor-mediated signaling. CONCLUSION: These results demonstrate for the first time that not only agonist binding but also abundance of some signaling proteins may strongly affect TRH receptor dynamics in the plasma membrane.

An essential role for Grk2 in Hedgehog signalling downstream of Smoothened.

The G-protein-coupled receptor kinase 2 (adrbk2/GRK2) has been implicated in vertebrate Hedgehog (Hh) signalling based on the effects of its transient knock-down in mammalian cells and zebrafish embryos. Here, we show that the response to Hh signalling is effectively abolished in the absence of Grk2 activity. Zebrafish embryos lacking all Grk2 activity are refractory to both Sonic hedgehog (Shh) and oncogenic Smoothened (Smo) activity, but remain responsive to inhibition of cAMP-dependent protein kinase (PKA) activity. Mutation of the kinase domain abrogates the rescuing activity of grk2 mRNA, suggesting that Grk2 acts in a kinase-dependent manner to regulate the response to Hh. Previous studies have suggested that Grk2 potentiates Smo activity by phosphorylating its C-terminal tail (CTT). In the zebrafish embryo, however, phosphomimetic Smo does not display constitutive activity, whereas phospho-null mutants retain activity, implying phosphorylation is neither sufficient nor necessary for Smo function. Since Grk2 rescuing activity requires the integrity of domains essential for its interaction with GPCRs, we speculate that Grk2 may regulate Hh pathway activity by downregulation of a GPCR.

The EGF receptor inhibits the signaling of dopamine D3 receptor through the phosphorylation of GRK2 on tyrosine residues.

Receptor transactivation or crosstalk are terms referring to instances in which the signaling of a given receptor is regulated by a different class of receptor. The epidermal growth factor (EGF) and the dopaminergic systems in the brain are closely related to schizophrenia with respect to both etiology and treatment. Thus, we investigated the functional interactions between the EGF receptor (EGFR), which belongs to the receptor tyrosine kinase family, and the dopamine D2-like receptors (D2R, D3R, and D4R), which are members of the G protein-coupled receptor (GPCR) family. Among D2-like receptors, the signaling of D3R was selectively inhibited by EGFR stimulation. Moreover loss-of-function assays showed that tyrosine-phosphorylated GRK2 mediates this inhibition by acting on the second intracellular loop of D3R. Considering that both EGFR and D3R are closely related to schizophrenia, this study could provide new molecular insight into the etiology of the disorder.

Effect of Lipid Composition on the Membrane Orientation of the G Protein-Coupled Receptor Kinase 2-Gß1γ2 Complex.

Interactions between proteins and cell membranes are critical for biological processes such as transmembrane signaling, and specific components of the membrane may play roles in helping to organize or mandate particular conformations of both integral and peripheral membrane proteins. One example of a signaling enzyme whose function is dependent on membrane binding and whose activity is affected by specific lipid components is G protein-coupled receptor (GPCR) kinase 2 (GRK2). Efficient GRK2-mediated phosphorylation of activated GPCRs is dependent not only on its recruitment to the membrane by heterotrimeric Gßγ subunits but also on the presence of highly negatively charged lipids, in particular phosphatidylinositol 4',5'-bisphosphate (PIP2). We hypothesized that PIP2 may favor a distinct orientation of the GRK2-Gßγ complex on the membrane that is more optimal for function. In this study, we compared the possible orientations of the GRK2-Gßγ complex and Gßγ alone on model cell membranes prepared with various anionic phospholipids as deduced from sum frequency generation vibrational and attenuated total reflectance Fourier transform infrared spectroscopic methods. Our results indicate that PIP2 affects the membrane orientation of the GRK2-Gß1γ2 complex but not that of complexes formed with anionic phospholipid binding deficient mutations in the GRK2 pleckstrin homology (PH) domain. Gß1γ2 exhibits a similar orientation on the lipid bilayer regardless of its lipid composition. The PIP2-induced orientation of the GRK2-Gß1γ2 complex is therefore most likely caused by specific interactions between PIP2 and the GRK2 PH domain. Thus, PIP2 not only helps recruit GRK2 to the membrane but also "fine tunes" the orientation of the GRK2-Gßγ complex so that it is better positioned to phosphorylate activated GPCRs.

Role of amino acid residues surrounding the phosphorylation site in peptide substrates of G protein-coupled receptor kinase 2 (GRK2).

A series of amino acid substitutions was made in a previously identified ß-tubulin-derived GRK2 substrate peptide (404DEMEFTEAESNMN416) to examine the role of amino acid residues surrounding the phosphorylation site. Anionic amino acid residues surrounding the phosphorylation site played an important role in the affinity for GRK2. Compared to the original peptide, a modified peptide (Ac-EEMEFSEAEANMN-NH2) exhibited markedly higher affinity for GRK2, but very low affinity for GRK5, suggesting that it can be a sensitive and selective peptide for GRK2.

Mapping the putative G protein-coupled receptor (GPCR) docking site on GPCR kinase 2: insights from intact cell phosphorylation and recruitment assays.

G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and ß-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α(2A)-adrenergic receptors (α(2A)ARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gßγ and α(2A)AR with EC50 values of 15 nM and 8 µM, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu(4), Val(7), Leu(8), Val(11), and Ser(12), directly interacts with receptors, whereas residues such as Asp(10), Tyr(13), Ala(16), Met(17), Gly(475), Val(477), and Ile(485) are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity.

Exploiting functional domains of GRK2/3 to alter the competitive balance of pro- and anticontractile signaling in airway smooth muscle.

To clarify the potential utility of targeting GRK2/3-mediated desensitization as a means of manipulating airway smooth muscle (ASM) contractile state, we assessed the specificity of GRK2/3 regulation of procontractile and relaxant G-protein-coupled receptors in ASM. Functional domains of GRK2/3 were stably expressed, or siRNA-mediated GRK2/3 knockdown was performed, in human ASM cultures, and agonist-induced signaling was assessed. Regulation of contraction of murine tracheal rings expressing GRK2 C terminus was also assessed. GRK2/3 knockdown or expression of the GRK2 C terminus caused a significant (∼ 30-90%) increase in maximal ß-agonist and histamine [phosphoinositide (PI) hydrolysis] signaling, without affecting the calculated EC50. GRK2 C-terminal expression did not affect signaling by methacholine, thrombin, or LTD4. Expression of the GRK2 N terminus or kinase-dead holo-GRK2 diminished (∼ 30-70%) both PI hydrolysis and Ca(2+) mobilization by every Gq-coupled receptor examined. Under conditions of GRK2 C-terminal expression, ß-agonist inhibition of methacholine-stimulated PI hydrolysis was greater. Finally, transgenic expression of the GRK2 C terminus in murine ASM enabled ∼ 30-50% greater ß-agonist-mediated relaxation of methacholine-induced contraction. Collectively these data demonstrate the relative selectivity of GRKs for the ß2AR in ASM and the ability to exploit GRK2/3 functional domains to render ASM hyporesponsive to contractile agents while increasing responsiveness to bronchodilating ß-agonist.

Monitoring of phosphorylated peptides by radioactive assay and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is frequently used to monitor phosphorylated peptides or protein kinase activities. However, few reports have compared a radioactivity assay with MALDI-TOF-MS analysis. We analyzed the phosphorylation ratios of 23 peptide substrates for G protein-coupled receptor kinase 2 (GRK2) with different lengths and numbers of negatively charged amino acids by MALDI-TOF-MS. We then examined the correlations between the phosphorylation ratios determined by MALDI-TOF-MS and the radioactivity levels (counts per minute, CPM) determined using a radioactive assay. Using MALDI-TOF-MS, the phosphorylation ratios were greater in the negative mode than in the positive mode. The phosphorylation ratio measured in the negative mode was strongly correlated with the CPM (r = 0.86). The number of acidic amino acids was related to the phosphorylation of peptide substrates by GRK2 (r = 0.53 and 0.46 for the phosphorylation ratio and CPM, respectively). These results suggest that MALDI-TOF-MS is an alternative to radioactive assays for monitoring phosphorylated peptides.

Cardiac progenitor cells engineered with ßARKct have enhanced ß-adrenergic tolerance.

Stem cell survival and retention in myocardium after injury following adoptive transfer is low. Elevated catecholamine levels coinciding with myocardial injury adversely affect cardiac progenitor cell (CPC) survival. The G protein-coupled receptor kinase 2 (GRK2)-derived inhibitory peptide, ßARKct, enhance myocyte contractility, survival, and normalize the neurohormonal axis in failing heart, however salutary effects of ßARKct on CPC survival and proliferation are unknown. Herein, we investigated whether the protective effects of ßARKct expression seen in the failing heart relate to CPCs. Modified CPCs expressing ßARKct enhanced AKT/eNOS signaling through protective ß2-adrenergic receptors (ß2-ARs). In addition, to the actions of ßARKct expression on ß2- AR signaling, pharmacologic inhibition of GRK2 also increased ß2-AR signaling in nonengineered CPCs (lacking ßARKct) but had limited effects in ßARKct engineered CPCs providing evidence for the strength of the ßARKct in inhibiting GRK2 in these cells. Increased proliferation and metabolic activity were observed in ßARKct-engineered CPCs following catecholamine stimulation indicating improved adrenergic tolerance. ßARKct modification of CPCs increased survival and proliferation following adoptive transfer in an acute myocardial infarction model concomitant with increased expression of ß-AR. Thus, ßARKct engineering of CPCs promotes survival and proliferation of injected cells following myocardial infarction, which includes improved ß-adrenergic tolerance essential for stem cell survival.

Structural and functional analysis of g protein-coupled receptor kinase inhibition by paroxetine and a rationally designed analog.

Recently we identified the serotonin reuptake inhibitor paroxetine as an inhibitor of G protein-coupled receptor kinase 2 (GRK2) that improves cardiac performance in live animals. Paroxetine exhibits up to 50-fold selectivity for GRK2 versus other GRKs. A better understanding of the molecular basis of this selectivity is important for the development of even more selective and potent small molecule therapeutics and chemical genetic probes. We first sought to understand the molecular mechanisms underlying paroxetine selectivity among GRKs. We directly measured the K(D) for paroxetine and assessed its mechanism of inhibition for each of the GRK subfamilies and then determined the atomic structure of its complex with GRK1, the most weakly inhibited GRK tested. Our results suggest that the selectivity of paroxetine for GRK2 largely reflects its lower affinity for adenine nucleotides. Thus, stabilization of off-pathway conformational states unique to GRK2 will likely be key for the development of even more selective inhibitors. Next, we designed a benzolactam derivative of paroxetine that has optimized interactions with the hinge of the GRK2 kinase domain. The crystal structure of this compound in complex with GRK2 confirmed the predicted interactions. Although the benzolactam derivative did not significantly alter potency of inhibition among GRKs, it exhibited 20-fold lower inhibition of serotonin reuptake. However, there was an associated increase in the potency for inhibition of other AGC kinases, suggesting that the unconventional hydrogen bond formed by the benzodioxole ring of paroxetine is better accommodated by GRKs.

Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane.

Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein ß(1)γ(2) subunits (Gßγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gßγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gßγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

Structural domains required for Caenorhabditis elegans G protein-coupled receptor kinase 2 (GRK-2) function in vivo.

G protein-coupled receptor kinases (GRKs) are key regulators of signal transduction that specifically phosphorylate activated G protein-coupled receptors (GPCRs) to terminate signaling. Biochemical and crystallographic studies have provided great insight into mammalian GRK2/3 interactions and structure. However, despite extensive in vitro characterization, little is known about the in vivo contribution of these described GRK structural domains and interactions to proper GRK function in signal regulation. We took advantage of the disrupted chemosensory behavior characteristic of Caenorhabditis elegans grk-2 mutants to discern the interactions required for proper in vivo Ce-GRK-2 function. Informed by mammalian crystallographic and biochemical data, we introduced amino acid substitutions into the Ce-grk-2 coding sequence that are predicted to selectively disrupt GPCR phosphorylation, Gα(q/11) binding, Gßγ binding, or phospholipid binding. Changing the most amino-terminal residues, which have been shown in mammalian systems to be required specifically for GPCR phosphorylation but not phosphorylation of alternative substrates or recruitment to activated GPCRs, eliminated the ability of Ce-GRK-2 to restore chemosensory signaling. Disrupting interaction between the predicted Ce-GRK-2 amino-terminal α-helix and kinase domain, posited to stabilize GRKs in their active ATP- and GPCR-bound conformation, also eliminated Ce-GRK-2 chemosensory function. Finally, although changing residues within the RH domain, predicted to disrupt interaction with Gα(q/11), did not affect Ce-GRK-2 chemosensory function, disruption of the predicted PH domain-mediated interactions with Gßγ and phospholipids revealed that both contribute to Ce-GRK-2 function in vivo. Combined, we have demonstrated functional roles for broadly conserved GRK2/3 structural domains in the in vivo regulation of organismal behavior.

Selectivity mechanism of GRK2/5 inhibition through in silico investigation.

As two representative isoforms of G protein-coupled receptor kinases family, the largest known membrane receptor family, GRK2 and GRK5 are ubiquitously distributed in human heart, brain, lung, kidney, skeletal muscle and other tissues. GRK2 and GRK5 have common functions implicated in the regulation of heart failure, though GRK5 has also been involved in diseases like hypertension, cancer, diabetes and Alzheimer's disease. Therefore, to clarify the selectivity mechanism towards GRK2 and GRK5 will be of great significance for the discovery of effective and selective inhibitors. To this end, the structures and chemical properties of key residues were analyzed among GRK2 and GRK5 derived from their respective protein crystal structures. Furthermore, a combination of multiple computational strategies, including sequence superposition, receptor-ligand docking, molecular dynamics, MM-GBSA calculation, QM/MM approach and pharmacological modeling, were integrated to validated and elucidate their unique binding modes towards highly selective inhibitors. In addition, the specific amino acid distribution within the GRK2/5 target site is also analyzed in this paper, which can guide future research and development of selective inhibitors in a more targeted manner. Overall, our study comprehensively clarifies the selectivity mechanism of GRK2/5 inhibition, thereby providing guidance for further rational design of selective inhibitors targeting GRK2/5.

Molecular mechanism of selectivity among G protein-coupled receptor kinase 2 inhibitors.

G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-Gßγ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-Gßγ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

Rational design of a selective covalent modifier of G protein ßγ subunits.

G protein-coupled receptors transduce signals through heterotrimeric G protein Gα and Gßγ subunits, both of which interact with downstream effectors to regulate cell function. Gßγ signaling has been implicated in the pathophysiology of several diseases, suggesting that Gßγ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a protein-protein interaction "hot spot" on Gßγ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with Gßγ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies GßCys204, but also a second undefined site. The high potency of selenocystamine in Gßγ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on Gß. Using structural information about the "hot spot," we developed a strategy to selectively target redox reversible compounds to a specific site on Gßγ using peptide carriers such as SIGCAFKILGY(-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on Gß, likely GßCys204, and inhibits Gßγ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the Gßγ "hot spot" through rational incorporation into molecules that noncovalently bind to Gßγ.

The Open Question of How GPCRs Interact with GPCR Kinases (GRKs).

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR-GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.

G Protein-Coupled Receptor Kinase 2 as Novel Therapeutic Target in Fibrotic Diseases.

G protein-coupled receptor kinase 2 (GRK2), an important subtype of GRKs, specifically phosphorylates agonist-activated G protein-coupled receptors (GPCRs). Besides, current research confirms that it participates in multiple regulation of diverse cells via a non-phosphorylated pathway, including interacting with various non-receptor substrates and binding partners. Fibrosis is a common pathophysiological phenomenon in the repair process of many tissues due to various pathogenic factors such as inflammation, injury, drugs, etc. The characteristics of fibrosis are the activation of fibroblasts leading to myofibroblast proliferation and differentiation, subsequent aggerate excessive deposition of extracellular matrix (ECM). Then, a positive feedback loop is occurred between tissue stiffness caused by ECM and fibroblasts, ultimately resulting in distortion of organ architecture and function. At present, GRK2, which has been described as a multifunctional protein, regulates copious signaling pathways under pathophysiological conditions correlated with fibrotic diseases. Along with GRK2-mediated regulation, there are diverse effects on the growth and apoptosis of different cells, inflammatory response and deposition of ECM, which are essential in organ fibrosis progression. This review is to highlight the relationship between GRK2 and fibrotic diseases based on recent research. It is becoming more convincing that GRK2 could be considered as a potential therapeutic target in many fibrotic diseases.