Tyrosine phosphorylation of G-protein-coupled-receptor kinase 2 (GRK2) by c-Src modulates its interaction with Gαq

Mariggiò, Stefania; García-Hoz, Carlota; Sarnago, Susana; Blasi, Antonio De; Mayor, Federico; Ribas, Catalina

doi:10.1016/j.cellsig.2006.03.004

Cited by 30 publications

(23 citation statements)

References 41 publications

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“…The charged face of the helix contains two aspartic acids, one glutamic acid, three alanines and a tyrosine (Tyr) at position 13 that can be phosphorylated by c-Src (38) and would likely contribute to the polarity of the helix. Since Asp-3 and Asp-10 on the polar side of the amphipathic helix do not appear to be involved in receptor binding (Fig.…”

Section: Resultsmentioning

confidence: 99%

Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor Phosphorylation

2009

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G protein-coupled receptor kinases (GRKs) specifically phosphorylate activated G protein-coupled receptors. While the X-ray crystal structures of several GRKs have been solved, the mechanism of GRK interaction with GPCRs is currently unknown. To further characterize the role of the GRK2 amino terminus in receptor interaction and phosphorylation, we generated a series of point mutations within the first 10 amino acids of GRK2 and tested their ability to phosphorylate receptor and nonreceptor substrates. Although all mutants showed some impairment in receptor phosphorylation, three of the mutants, D3K, L4A and D10A, were the most severely affected. Using the β 2 -adrenergic receptor and rhodopsin as receptor substrates and tubulin as a non-receptor substrate, we demonstrated that the kinase activity towards the receptors was severely decreased in the mutants, while they fully retained their ability to phosphorylate tubulin. Moreover, the amino terminal mutants were able to bind to the receptor but, in contrast to wild-type GRK2, were not activated by receptor binding. A synthetic peptide containing residues 1-14 of GRK2 served as a non-competitive inhibitor of receptor phosphorylation by GRK2, while a comparable peptide from GRK5 had no effect on GRK2 activity. Secondary structure prediction and circular dichroism suggest that the GRK2 amino terminal peptide forms an amphipathic alpha helix. Taken together, we propose a mechanism whereby the extreme amino terminus of GRK2 forms an intramolecular interaction that selectively enhances the catalytic activity of the kinase towards receptor substrates.G protein-coupled receptors (GPCRs 2 ) are a large class of plasma membrane proteins that respond to a wide variety of stimuli including hormones, odorants, peptides and lipids (1). Agonist binding transduces the signal into the cell by promoting receptor interaction with heterotrimeric G proteins, which subsequently activate effectors such as adenylyl cyclase, phosphodiesterases, phosphatidylinositol 3-kinase and various ion channels. Cellular responsiveness to external stimuli is tightly regulated. In GPCR-mediated signaling, the waning of receptor responsiveness to agonist is a key regulatory mechanism known as desensitization (2). Additional regulatory processes such as receptor endocytosis and downregulation limit the cellular response to continued stimuli by reducing the number of receptors on the cell surface (3,4). Some of these events are regulated by a family of serine/threonine protein kinases called GPCR kinases (GRKs) that specifically phosphorylate agonist occupied receptors (5,6). Receptor phosphorylation by GRKs promotes the binding of arrestins, which effectively uncouple the receptor from G protein and terminate signaling (7). + This work was supported by National Institutes of Health grants R01GM44944 (to JLB) and T32DK07705 (to BLB).*To whom correspondence should be addressed: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10 th Street, 350 BLSB, Philad...

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Section: Resultsmentioning

confidence: 99%

Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor Phosphorylation

2009

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“…The action of physically arresting Src is most likely due to the stimulation of GPCR endocytosis — a process often involving pSrc [36–37] — due to antagonist binding. These results also provide another potential example of interactions between G proteins and focal adhesion proteins such as Src to alter cytoskeletal function similar to recent reports [38].…”

Section: Discussionmentioning

confidence: 99%

5-HT2B antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

Hutcheson

Ryzhova

Setola

et al. 2012

Journal of Molecular and Cellular Cardiology

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Transforming growth factor-β1 (TGF-β1) induces myofibroblast activation of quiescent aortic valve interstitial cells (AVICs), a differentiation process implicated in calcific aortic valve disease (CAVD). The ubiquity of TGF-β1 signaling makes it difficult to target in a tissue specific manner; however, the serotonin 2B receptor (5-HT2B) is highly localized to cardiopulmonary tissues and agonism of this receptor displays pro-fibrotic effects in a TGF-β1-dependent manner. Therefore, we hypothesized that antagonism of 5-HT2B opposes TGF-β1-induced pathologic differentiation of AVICs and may offer a druggable target to prevent CAVD. To test this hypothesis, we assessed the interaction of 5-HT2B antagonism with canonical and non-canonical TGF-β1 pathways to inhibit TGF-β1-induced activation of isolated porcine AVICs in vitro. Here we show that AVIC activation and subsequent calcific nodule formation is completely mitigated by 5-HT2B antagonism. Interestingly, 5-HT2B antagonism does not inhibit canonical TGF-β1 signaling as identified by Smad3 phosphorylation and activation of a partial plasminogen activator inhibitor-1 promoter (PAI-1, a transcriptional target of Smad3), but prevents non-canonical p38 MAPK phosphorylation. It was initially suspected that 5-HT2B antagonism prevents Src tyrosine kinase phosphorylation; however, we found that this is not the case and time-lapse microscopy indicates that 5-HT2B antagonism prevents non-canonical TGF-β1 signaling by physically arresting Src tyrosine kinase. This study demonstrates the necessity of non-canonical TGF-β1 signaling in leading to pathologic AVIC differentiation. Moreover, we believe that the results of this study suggest 5-HT2B antagonism as a novel therapeutic approach for CAVD that merits further investigation.

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“…PKC-mediated phosphorylation of GRK2 alleviates the inhibition induced by calmodulin binding (92). c-Src-mediated phosphorylation of GRK2 at select N-terminal Tyrosine residues (amino acids 13, 86, and 92) enhances its kinase activity (92, 93) and selectively increases binding of GRK2 to Gαq as demonstrated by the use of phospho-tyrosine defective and mimic GRK2 mutants (94). Furthermore, increased tyrosine phosphorylation on GRK2 occurs in parallel with both increased co-immunoprecipitation of GRK2 and Gαq post activation of the Gαq-coupled M1 muscarinic receptor and inhibition of Gαq-mediated activation of phospholipase Cβ signaling in vitro, supporting a physiological role for this GRK2 regulatory mechanism (94).…”

Section: Grk2mentioning

confidence: 99%

Noncanonical Roles of G Protein-coupled Receptor Kinases in Cardiovascular Signaling

Schumacher

Koch

2017

Journal of Cardiovascular Pharmacology

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G protein-coupled receptor kinases (GRKs) are classically known for their role in regulating the activity of the largest known class of membrane receptors, which influence diverse biological processes in every cell type in the human body. As researchers have tried to uncover how this family of kinases, containing only 7 members, achieves selective and coordinated control of receptors, they have uncovered a growing number of non-canonical activities for these kinases. These activities include phosphorylation of non-receptor targets and kinase-independent molecular interactions. In particular, GRK2, GRK3, and GRK5 are the predominant members expressed in the heart. Their canonical and non-canonical actions within cardiac and other tissues have significant implications for cardiovascular function in healthy animals and for the development and progression of disease. This review summarizes what is currently known regarding the activity of these kinases, and particularly the role of GRK2 and GRK5 in the molecular alterations that occur during heart failure. This review further highlights areas of GRK regulation that remain poorly understood and how they may represent novel targets for therapeutic development.

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Tyrosine phosphorylation of G-protein-coupled-receptor kinase 2 (GRK2) by c-Src modulates its interaction with Gαq

Cited by 30 publications

References 41 publications

Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor Phosphorylation

Role of the Amino Terminus of G Protein-Coupled Receptor Kinase 2 in Receptor Phosphorylation

5-HT2B antagonism arrests non-canonical TGF-β1-induced valvular myofibroblast differentiation

Noncanonical Roles of G Protein-coupled Receptor Kinases in Cardiovascular Signaling

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