Gßγ subunits inhibit Epac-induced melanoma cell migration.

BACKGROUND: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein ßγ subunits (Gßγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gßγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gßγ and Epac in melanoma, which plays a role in regulation of cell migration. METHODS: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. RESULTS: The effect of Gßγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gßγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-ß-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gßγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of ß1 and γ2, which is the major combination of Gßγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of ß adrenergic receptor kinase (ßARK-CT), an endogenous inhibitor for Gßγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gßγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of ß1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gßγ with ßARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPßS), a GDP analogue that inactivates Gßγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gßγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gßγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration. CONCLUSION: We found the cross talk of Ca 2+ signaling between Gßγ and Epac, which plays a major role in melanoma cell migration.

Taking the heart failure battle inside the cell: small molecule targeting of Gßγ subunits.

Heart failure (HF) is devastating disease with poor prognosis. Elevated sympathetic nervous system activity and outflow, leading to pathologic attenuation and desensitization of ß-adrenergic receptors (ß-ARs) signaling and responsiveness, are salient characteristic of HF progression. These pathologic effects on ß-AR signaling and HF progression occur in part due to Gßγ-mediated signaling, including recruitment of receptor desensitizing kinases such as G-protein coupled receptor (GPCR) kinase 2 (GRK2) and phosphoinositide 3-kinase (PI3K), which subsequently phosphorylate agonist occupied GPCRs. Additionally, chronic GPCR signaling signals chronically dissociated Gßγ subunits to interact with multiple effector molecules that activate various signaling cascades involved in HF pathophysiology. Importantly, targeting Gßγ signaling with large peptide inhibitors has proven a promising therapeutic paradigm in the treatment of HF. We recently described an approach to identify small molecule Gßγ inhibitors that selectively block particular Gßγ functions by specifically targeting a Gßγ protein-protein interaction "hot spot." Here we describe their effects on Gßγ downstream signaling pathways, including their role in HF pathophysiology. We suggest a promising therapeutic role for small molecule inhibition of pathologic Gßγ signaling in the treatment of HF. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

Pasireotide and octreotide stimulate distinct patterns of sst2A somatostatin receptor phosphorylation.

Pasireotide (SOM230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, Cushing's disease, and carcinoid tumors. Whereas octreotide acts primarily via the sst(2A) somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst(2A) activity combined with enhanced binding to other somatostatin receptor subtypes. In the present study, we used phophosite-specific antibodies to examine agonist-induced phosphorylation of the rat sst(2A) receptor. We show that somatostatin and octreotide stimulate the complete phosphorylation of a cluster of four threonine residues within the cytoplasmic (353)TTETQRT(359) motif in a variety of cultured cell lines in vitro as well as in intact animals in vivo. This phosphorylation was mediated by G protein-coupled receptor kinases (GRK) 2 and 3 and followed by rapid cointernalization of the receptor and ss-arrestin into the same endocytic vesicles. In contrast, pasireotide failed to promote substantial phosphorylation and internalization of the rat sst(2A) receptor. In the presence of octreotide or SS-14, SOM230 showed partial agonist behavior, inhibiting phosphorylation, and internalization of sst(2A). Upon overexpression of GRK2 or GRK3, pasireotide stimulated selective phosphorylation of Thr356 and Thr359 but not of Thr353 or Thr354 within the (353)TTETQRT(359) motif. Pasireotide-mediated phosphorylation led to the formation of relatively unstable beta-arrestin-sst(2A) complexes that dissociated at or near the plasma membrane. Thus, octreotide and pasireotide are equally active in inducing classical G protein-dependent signaling via the sst(2A) somatostatin receptor. Yet, we find that they promote strikingly different patterns of sst(2A) receptor phosphorylation and, hence, stimulate functionally distinct pools of beta-arrestin.

GRK2 negatively regulates glycogen synthesis in mouse liver FL83B cells.

G-protein-coupled receptor (GPCR) kinases (GRKs) are serine/threonine kinases that desensitize agonist-occupied classical GPCRs. Although the insulin receptor (IR) is a tyrosine kinase receptor, the IR also couples to G-proteins and utilizes G-protein signaling components. The present study was designed to test the hypothesis that GRK2 negatively regulates IR signaling. FL83B cells, derived from mouse liver, were treated with insulin and membrane translocation of GRK2 was determined using immunofluoresecence and Western blotting. Insulin caused an increase in the translocation of GRK-2 from cytosol to the plasma membrane. To determine the role of GRK2 in IR signaling, GRK2 was selectively down-regulated ( approximately by 90%) in FL83B cells using a small interfering RNA technique. Basal as well as insulin-induced glycogen synthesis (measured by d-[U-(14)C]glucose incorporation) was increased in GRK2-deficient cells compared with control cells. Similarly, GRK2 deficiency increased the basal and insulin-stimulated phosphorylation of Ser(21) in glycogen synthase kinase-3alpha. Insulin-induced tyrosine phosphorylation of the IR was similar in control and GRK2-deficient cells. Basal and insulin-stimulated phosphorylation of Tyr(612) in insulin receptor subunit 1 was significantly increased while phosphorylation of Ser(307) was decreased in GRK2-deficient FL83B cells compared with control cells. Chronic insulin treatment (24 h) in control cells caused an increase in GRK2 (56%) and a decrease in IR (50%) expression associated with the absence of an increase in glycogen synthesis, suggesting impairment of IR function. However, chronic insulin treatment (24 h) did not decrease IR expression or impair IR effects on glycogen synthesis in GRK2-deficient cells. We conclude that (i) GRK2 negatively regulates basal and insulin-stimulated glycogen synthesis via a post-IR signaling mechanism, and (ii) GRK2 may contribute to reduced IR expression and function during chronic insulin exposure.

Regulatory mechanisms underlying GKR2 levels in U937 cells: evidence for GRK3 involvement.

G protein-coupled receptors represent the most diverse group of proteins involved in transmembrane signalling, that participate in the regulation of a wide range of physicochemical messengers through the interaction with heterotrimeric G proteins. In addition, GPCRs stimulation also triggers a negative feedback mechanism, known as desensitization that prevents the potentially harmful effects caused by persistent receptor stimulation. In this adaptative response, G protein-coupled receptor kinases (GRKs) play a key role and alterations in their function are related to diverse pathophysiological situations. Based on the scarce knowledge about the regulation of GRK2 by other kinases of the same family, the aim of the present work was to investigate the regulation of GRK2 levels in systems where other GRKs are diminished by antisense technique. Present findings show that in U937 cells GRK2 levels are regulated by GRK3 and not by GRK6 through a mechanism involving InsP upregulation. This work reports a novel GRK3-mediated GRK2 regulatory mechanism and further suggests that GRK2 may also act as a compensatory kinase tending to counterbalance the reduction in GRK3 levels. This study provides the first evidence for the existence of GRKs cross-regulation.

Phosphorylation of the endogenous thyrotropin-releasing hormone receptor in pituitary GH3 cells and pituitary tissue revealed by phosphosite-specific antibodies.

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr(365) in the receptor tail; distal sites were more phosphorylated in some heterologous models. beta-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nm TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t((1/2)) approximately 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t((1/2)) approximately 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin- and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.

Adrenal GRK2 upregulation mediates sympathetic overdrive in heart failure.

Cardiac overstimulation by the sympathetic nervous system (SNS) is a salient characteristic of heart failure, reflected by elevated circulating levels of catecholamines. The success of beta-adrenergic receptor (betaAR) antagonists in heart failure argues for SNS hyperactivity being pathogenic; however, sympatholytic agents targeting alpha2AR-mediated catecholamine inhibition have been unsuccessful. By investigating adrenal adrenergic receptor signaling in heart failure models, we found molecular mechanisms to explain the failure of sympatholytic agents and discovered a new strategy to lower SNS activity. During heart failure, there is substantial alpha2AR dysregulation in the adrenal gland, triggered by increased expression and activity of G protein-coupled receptor kinase 2 (GRK2). Adrenal gland-specific GRK2 inhibition reversed alpha2AR dysregulation in heart failure, resulting in lowered plasma catecholamine levels, improved cardiac betaAR signaling and function, and increased sympatholytic efficacy of a alpha2AR agonist. This is the first demonstration, to our knowledge, of a molecular mechanism for SNS hyperactivity in heart failure, and our study identifies adrenal GRK2 activity as a new sympatholytic target.

Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2.

Dopamine, via activation of D1-like receptors, inhibits Na,K-ATPase and Na,H-exchanger in renal proximal tubules and promotes sodium excretion. This effect of dopamine is not seen in conditions associated with oxidative stress such as hypertension, diabetes, and aging due to uncoupling of D1-like receptors from G proteins. To identify the role of oxidative stress in uncoupling of the D1-like receptors, we utilized primary cultures from rat renal proximal tubules. Hydrogen peroxide (H2O2), an oxidant, treatment to the cell cultures increased the level of malondialdehyde, a marker of oxidative damage. Further, H2O2 decreased membranous D1-like receptor numbers and proteins, D1-like agonist (SKF 38393)-mediated [35S]GTPgammaS binding and SKF 38393-mediated inhibition of Na,K-ATPase. Moreover, H2O2 treatment to the cultures caused membranous translocation of G-protein-coupled receptor kinase 2 (GRK 2) and increased serine phosphorylation of D1A receptors accompanied by an increase in protein kinase C (PKC) activity. Interestingly, PKC inhibitors blocked the H2O2-mediated stimulation of GRK 2 and serine phosphorylation of D1A receptors. Further, GRK 2 antisense but not scrambled oligonucleotides attenuated the effect of H2O2 on membranous expression of GRK 2. Moreover, direct activation of PKC with phorbol ester (PMA) resulted in reduction of SKF 38393-mediated [35S]GTPgammaS binding. We conclude that H2O2 stimulates PKC leading to the activation of GRK 2, which causes serine phopshorylation of D1A receptors and receptor G-protein uncoupling in these cells, resulting in impairment in D1-like receptor function.

Defects in cardiomyocyte function: role of beta-adrenergic receptor dysfunction.

Heart failure is a common clinical syndrome characterized by increased levels of circulating catecholamines and extensive abnormalities in the beta-adrenergic receptor (betaAR) system. Interestingly, whether dampening of betaAR signals is beneficial or detrimental for the failing cardiomyocyte is still controversial. In this review we will discuss a number of studies addressing the role of betaAR dysfunction in the development and progression of cardiomyocyte failure, and novel possible strategies to ameliorate cardiomyocyte contractility in heart failure through the normalization of betaAR signaling.