Human substance P receptor undergoes agonist-dependent phosphorylation by G protein-coupled receptor kinase 5 in vitro

G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes activated G protein-coupled receptors (GPCRs). Here, we identify ezrin as a novel non-GPCR substrate of GRK2. GRK2 phosphorylates glutathione S-transferase (GST)-ezrin, but not an ezrin fusion protein lacking threonine 567 (T567), in vitro. These results suggest that T567, the regulatory phosphorylation site responsible for maintaining ezrin in its active conformation, represents the principle site of GRK2-mediated phosphorylation. Two lines of evidence indicate that GRK2-mediated ezrin-radixinmoesin (ERM) phosphorylation serves to link GPCR activation to cytoskeletal reorganization. First, in Hep2 cells muscarinic M1 receptor (M1MR) activation causes membrane ruffling. This ruffling response is ERM dependent and is accompanied by ERM phosphorylation. Inhibition of GRK2, but not rho kinase or protein kinase C, prevents ERM phosphorylation and membrane ruffling. Second, agonist-induced internalization of the β2-adrenergic receptor (β2AR) and M1MR is accompanied by ERM phosphorylation and localization of phosphorylated ERM to receptor-containing endocytic vesicles. The colocalization of internalized β2AR and phosphorylated ERM is not dependent on Na+/H+ exchanger regulatory factor binding to the β2AR. Inhibition of ezrin function impedes β2AR internalization, further linking GPCR activation, GRK activity, and ezrin function. Overall, our results suggest that GRK2 serves not only to attenuate but also to transduce GPCR-mediated signals.

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An RNA molecule that specifically inhibits G-protein-coupled receptor kinase 2 in vitro

RNA ◽

10.1261/rna.821908 ◽

2008 ◽

Vol 14 (3) ◽

pp. 524-534 ◽

Cited By ~ 42

Author(s):

G. Mayer ◽

B. Wulffen ◽

C. Huber ◽

J. Brockmann ◽

B. Flicke ◽

...

Keyword(s):

G Protein ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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Hypothesis

Journal of Cardiovascular Pharmacology and Therapeutics ◽

10.1177/1074248416644350 ◽

2016 ◽

Vol 22 (1) ◽

pp. 51-53 ◽

Cited By ~ 4

Author(s):

Jonathan M. Powell ◽

Emanuel Ebin ◽

Steven Borzak ◽

Anastasios Lymperopoulos ◽

Charles H. Hennekens

Keyword(s):

G Protein ◽

Wall Thickness ◽

Basic Research ◽

Left Ventricular ◽

Conditional Knockout ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

G Protein Coupled

The hypothesis that paroxetine decreases morbidity and mortality in patients with heart failure (HF) is plausible but unproven. Basic research demonstrates that inhibition of G protein-coupled receptor kinase 2 (GRK2) both in vitro and in vivo in the myocardium may be beneficial. G protein-coupled receptor kinase 2 antagonism is purported to exert cardioprotective effects immediately following myocardial injury by blunting toxic overstimulation on a recently injured heart. In addition, chronic overexpression of GRK2 inhibits catecholamine induction of vital positive chronotropic and ionotropic effects required to preserve cardiac output leading to worsening of congestive HF. In cardiac-specific GRK2 conditional knockout mice, there is significant improvement in left ventricular wall thickness, left ventricular end-diastolic diameter (LVEDD), and ejection fraction (EF) compared to controls. Paroxetine is a selective serotonin reuptake inhibitor which was recently shown to have the ability to directly inhibit GRK2 both in vitro and in vivo. At physiologic temperatures, paroxetine inhibits GRK2-dependent phosphorylation of an activated G-protein-coupled receptor with a half maximal inhibitory concentration of 35 micromoles, a substantially greater affinity than for other G protein-coupled receptor kinases. In a randomized trial in mice with systolic HF and depressed EF postmyocardial infarction, those treated with paroxetine had a 30% increase in EF, improved contractility, and LVEDD and wall thickness compared to those treated with medical therapy alone. While further basic research may continue to elucidate plausible mechanisms of benefit and observational studies will contribute important relevant information, large scale randomized trials designed a priori to do so are necessary to test the hypothesis.

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G-protein-coupled receptor kinase-2 is a critical regulator of TNFα signaling in colon epithelial cells

Biochemical Journal ◽

10.1042/bcj20170093 ◽

2017 ◽

Vol 474 (14) ◽

pp. 2301-2313 ◽

Cited By ~ 3

Author(s):

Michael D. Steury ◽

Peter C. Lucas ◽

Laura R. McCabe ◽

Narayanan Parameswaran

Keyword(s):

Wound Healing ◽

Epithelial Cell ◽

Epithelial Cells ◽

G Protein ◽

Critical Role ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

G Protein Coupled

G-protein-coupled receptor kinase-2 (GRK2) belongs to the GRK family of serine/threonine protein kinases critical in the regulation of G-protein-coupled receptors. Apart from this canonical role, GRK2 is also involved in several signaling pathways via distinct intracellular interactomes. In the present study, we examined the role of GRK2 in TNFα signaling in colon epithelial cell–biological processes including wound healing, proliferation, apoptosis, and gene expression. Knockdown of GRK2 in the SW480 human colonic cells significantly enhanced TNFα-induced epithelial cell wound healing without any effect on apoptosis/proliferation. Consistent with wound-healing effects, GRK2 knockdown augmented TNFα-induced matrix metalloproteinases (MMPs) 7 and 9, as well as urokinase plasminogen activator (uPA; factors involved in cell migration and wound healing). To assess the mechanism by which GRK2 affects these physiological processes, we examined the role of GRK2 in TNFα-induced MAPK and NF-κB pathways. Our results demonstrate that while GRK2 knockdown inhibited TNFα-induced IκBα phosphorylation, activation of ERK was significantly enhanced in GRK2 knockdown cells. Our results further demonstrate that GRK2 inhibits TNFα-induced ERK activation by inhibiting generation of reactive oxygen species (ROS). Together, these data suggest that GRK2 plays a critical role in TNFα-induced wound healing by modulating MMP7 and 9 and uPA levels via the ROS–ERK pathway. Consistent with in vitro findings, GRK2 heterozygous mice exhibited enhanced intestinal wound healing. Together, our results identify a novel role for GRK2 in TNFα signaling in intestinal epithelial cells.

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Abstract 311: In vitro Regulation of G-protein Coupled Receptor Kinase 5 (GRK5) Induced Cardiac Hypertrophy by a Novel Interaction With the Phosphatase PHLPP2

Circulation Research ◽

10.1161/res.123.suppl_1.311 ◽

2018 ◽

Vol 123 (Suppl_1) ◽

Author(s):

Szu-Tsen Yeh ◽

Cristina Zambrano ◽

Walter Koch ◽

Nicole Purcell

Keyword(s):

Cardiac Hypertrophy ◽

G Protein ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

G Protein Coupled

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β-Arrestin 1 and 2 and G Protein-Coupled Receptor Kinase 2 Expression in Pituitary Adenomas: Role in the Regulation of Response to Somatostatin Analogue Treatment in Patients With Acromegaly

Endocrinology ◽

10.1210/en.2013-1672 ◽

2013 ◽

Vol 154 (12) ◽

pp. 4715-4725 ◽

Cited By ~ 29

Author(s):

Federico Gatto ◽

Richard Feelders ◽

Rob van der Pas ◽

Johan M. Kros ◽

Fadime Dogan ◽

...

Keyword(s):

G Protein ◽

Pituitary Adenomas ◽

Somatostatin Analogue ◽

Receptor Kinase ◽

G Protein Coupled Receptor ◽

Sstr Subtype ◽

G Protein Coupled ◽

Grk2 Expression

Recent in vitro studies highlighted G protein-coupled receptor kinase (GRK)2 and β-arrestins as important players in driving somatostatin receptor (SSTR) desensitization and trafficking. Our aim was to characterize GRK2 and β-arrestins expression in different pituitary adenomas and to investigate their potential role in the response to somatostatin analog (SSA) treatment in GH-secreting adenomas (GHomas). We evaluated mRNA expression of multiple SSTRs, GRK2, β-arrestin 1, and β-arrestin 2 in 41 pituitary adenomas (31 GHomas, 6 nonfunctioning [NFPAs], and 4 prolactinomas [PRLomas]). Within the GHomas group, mRNA data were correlated with the in vivo response to an acute octreotide test and with the GH-lowering effect of SSA in cultured primary cells. β-Arrestin 1 expression was low in all 3 adenoma histotypes. However, its expression was significantly lower in GHomas and PRLomas, compared with NFPAs (P < .01). GRK2 expression was higher in PRLomas and NFPAs compared with GHomas (P < .05). In the GHoma group, GRK2 expression was inversely correlated to β-arrestin 1 (P < .05) and positively correlated to β-arrestin 2 (P < .0001). SSA treatment did not affect GRK2 and β-arrestin expression in GHomas or in cultured rat pituitary tumor GH3 cells. Noteworthy, β-arrestin 1 was significantly lower (P < .05) in tumors responsive to octreotide treatment in vitro, whereas GRK2 and SSTR subtype 2 were significantly higher (P < .05). Likewise, β-arrestin 1 levels were inversely correlated with the in vivo response to acute octreotide test (P = .001), whereas GRK2 and SSTR subtype 2 expression were positively correlated (P < .05). In conclusion, for the first time, we characterized GRK2, β-arrestin 1, and β-arrestin 2 expression in a representative number of pituitary adenomas. β-Arrestin 1 and GRK2 seem to have a role in modulating GH secretion during SSA treatment.

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