Up-regulation of Endogenous RGS2 Mediates Cross-desensitization between Gs and Gq Signaling in Osteoblasts

Regulator of G protein signaling (RGS) proteins limit G protein signals. In this study, we investigated the role of RGS2 in the control of G protein signaling cascades in osteoblasts, the cells responsible for bone formation. Expression of RGS2 was up-regulated in primary cultures of mouse calvarial osteoblasts by parathyroid hormone-related peptide (PTHrP)-(1-34), which stimulates Gs signaling. RGS2 was also up-regulated by extracellular ATP, which selectively activates Gq, as well as by forskolin and phorbol myristate acetate, which activate targets downstream of Gs and Gq, respectively. To assess the role of endogenous RGS2, we characterized Gs and Gq signaling in osteoblasts derived from wild type and rgs2-/- mice. Under control conditions, nucleotide-stimulated calcium release, endothelin-stimulated accumulation of inositol phosphates, and PTHrP-stimulated cAMP accumulation were equivalent in osteoblasts isolated from wild type and rgs2-/- mice. Thus, basal levels of endogenous RGS2 do not appear to regulate Gs or Gq signaling in osteoblasts. Interestingly, forskolin treatment of wild type but not rgs2-/- osteoblasts suppressed both endothelin-stimulated accumulation of inositol phosphates and nucleotide-stimulated calcium release, indicating that up-regulation of RGS2 by Gs signaling desensitizes Gq signals. Furthermore, pretreatment with ATP suppressed PTHrP-dependent cAMP accumulation in wild type but not rgs2-/- osteoblasts, implying that up-regulation of RGS2 by Gq signaling desensitizes Gs signals. Our findings demonstrate that endogenously expressed RGS2 can limit Gs signaling. Moreover, up-regulation of RGS2 contributes to cross-desensitization of Gs- and Gq-coupled signals.

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Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

Endocrinology ◽

10.1210/en.2011-1662 ◽

2012 ◽

Vol 153 (6) ◽

pp. 2747-2755 ◽

Cited By ~ 5

Author(s):

Jesse J. Parry ◽

Ronald Chen ◽

Rebecca Andrews ◽

Kimberly A. Lears ◽

Buck E. Rogers

Keyword(s):

Ligand Binding ◽

G Protein ◽

Calcium Release ◽

Expression Profiles ◽

Somatostatin Receptor ◽

Receptor Subtype ◽

G Protein Signaling ◽

Protein Signaling ◽

Wild Type ◽

Somatostatin Receptor Subtype 2

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling.

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GPR158/179 regulate G protein signaling by controlling localization and activity of the RGS7 complexes

The Journal of Cell Biology ◽

10.1083/jcb.201202123 ◽

2012 ◽

Vol 197 (6) ◽

pp. 711-719 ◽

Cited By ~ 50

Author(s):

Cesare Orlandi ◽

Ekaterina Posokhova ◽

Ikuo Masuho ◽

Thomas A. Ray ◽

Nazarul Hasan ◽

...

Keyword(s):

G Protein ◽

Night Vision ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Gpcr Signaling ◽

Signaling Specificity ◽

Orphan Gpcrs ◽

G Protein Coupled

The extent and temporal characteristics of G protein–coupled receptor (GPCR) signaling are shaped by the regulator of G protein signaling (RGS) proteins, which promote G protein deactivation. With hundreds of GPCRs and dozens of RGS proteins, compartmentalization plays a key role in establishing signaling specificity. However, the molecular details and mechanisms of this process are poorly understood. In this paper, we report that the R7 group of RGS regulators is controlled by interaction with two previously uncharacterized orphan GPCRs: GPR158 and GPR179. We show that GPR158/179 recruited RGS complexes to the plasma membrane and augmented their ability to regulate GPCR signaling. The loss of GPR179 in a mouse model of night blindness prevented targeting of RGS to the postsynaptic compartment of bipolar neurons in the retina, illuminating the role of GPR179 in night vision. We propose that the interaction of RGS proteins with orphan GPCRs promotes signaling selectivity in G protein pathways.

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Regulation of Protease-activated Receptor 1 Signaling by the Adaptor Protein Complex 2 and R4 Subfamily of Regulator of G Protein Signaling Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m113.528273 ◽

2013 ◽

Vol 289 (3) ◽

pp. 1580-1591 ◽

Cited By ~ 9

Author(s):

Buxin Chen ◽

David P. Siderovski ◽

Richard R. Neubig ◽

Mark A. Lawson ◽

JoAnn Trejo

Keyword(s):

G Protein ◽

Protein Complex ◽

Adaptor Protein ◽

Surface Expression ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Wild Type ◽

Protease Activated Receptor 1 ◽

G Protein Coupled

The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of “regulator of G protein signaling” (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 420AKKAA424 mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

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A newly identified complex of spinophilin and the tyrosine phosphatase, SHP-1, modulates platelet activation by regulating G protein–dependent signaling

Blood ◽

10.1182/blood-2011-10-387910 ◽

2012 ◽

Vol 119 (8) ◽

pp. 1935-1945 ◽

Cited By ~ 42

Author(s):

Peisong Ma ◽

Aleksandra Cierniewska ◽

Rachel Signarvic ◽

Marcin Cieslak ◽

Hong Kong ◽

...

Keyword(s):

G Protein ◽

Platelet Activation ◽

Tyrosine Phosphatase ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Normal Hemostasis ◽

The Impact ◽

Constitutive Phosphorylation

Abstract Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI2). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI2. After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.

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Established and Emerging Fluorescence-Based Assays for G-Protein Function: Heterotrimeric G-Protein Alpha Subunits and Regulator of G-Protein Signaling (RGS) Proteins

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/138620703106298491 ◽

2003 ◽

Vol 6 (4) ◽

pp. 399-407 ◽

Cited By ~ 24

Author(s):

Randall Kimple ◽

Miller Jones ◽

Adam Shutes ◽

Benjamin Yerxa ◽

David Siderovski ◽

...

Keyword(s):

G Protein ◽

Protein Function ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Heterotrimeric G Protein ◽

Alpha Subunits

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A Regulator of G Protein Signaling-containing Kinase Is Important for Chemotaxis and Multicellular Development inDictyostelium

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0550 ◽

2003 ◽

Vol 14 (4) ◽

pp. 1727-1743 ◽

Cited By ~ 17

Author(s):

Binggang Sun ◽

Richard A. Firtel

Keyword(s):

Protein Kinase ◽

G Protein ◽

Kinase Activity ◽

Site Directed Mutagenesis ◽

Membrane Localization ◽

Pleckstrin Homology Domain ◽

G Protein Signaling ◽

Protein Signaling ◽

Wild Type ◽

Gene Encoding

We have identified a gene encoding RGS domain-containing protein kinase (RCK1), a novel regulator of G protein signaling domain-containing protein kinase. RCK1 mutant strains exhibit strong aggregation and chemotaxis defects. rck1 null cells chemotax ∼50% faster than wild-type cells, suggesting RCK1 plays a negative regulatory role in chemotaxis. Consistent with this finding, overexpression of wild-type RCK1 reduces chemotaxis speed by ∼40%. On cAMP stimulation, RCK1 transiently translocates to the membrane/cortex region with membrane localization peaking at ∼10 s, similar to the kinetics of membrane localization of the pleckstrin homology domain-containing proteins CRAC, Akt/PKB, and PhdA. RCK1 kinase activity also increases dramatically. The RCK1 kinase activity does not rapidly adapt, but decreases after the cAMP stimulus is removed. This is particularly novel considering that most other chemoattractant-activated kinases (e.g., Akt/PKB, ERK1, ERK2, and PAKa) rapidly adapt after activation. Using site-directed mutagenesis, we further show that both the RGS and kinase domains are required for RCK1 function and that RCK1 kinase activity is required for the delocalization of RCK1 from the plasma membrane. Genetic evidence suggests RCK1 function lies downstream from Gα2, the heterotrimeric G protein that couples to the cAMP chemoattractant receptors. We suggest that RCK1 might be part of an adaptation pathway that regulates aspects of chemotaxis in Dictyostelium.

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Regulators of G protein Signaling (RGS) proteins (version 2020.4) in the IUPHAR/BPS Guide to Pharmacology Database

IUPHAR/BPS Guide to Pharmacology CITE ◽

10.2218/gtopdb/f891/2020.4 ◽

2020 ◽

Vol 2020 (4) ◽

Author(s):

Katelin E. Ahlers-Dannen ◽

Mohammed Alqinyah ◽

Christopher Bodle ◽

Josephine Bou Dagher ◽

Bandana Chakravarti ◽

...

Keyword(s):

G Protein ◽

G Protein Coupled Receptors ◽

Signaling Cascades ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Heterotrimeric G Proteins ◽

Gtp Hydrolysis ◽

Rgs Domain ◽

G Protein Coupled

Regulator of G protein Signaling, or RGS, proteins serve an important regulatory role in signaling mediated by G protein-coupled receptors (GPCRs). They all share a common RGS domain that directly interacts with active, GTP-bound Gα subunits of heterotrimeric G proteins. RGS proteins stabilize the transition state for GTP hydrolysis on Gα and thus induce a conformational change in the Gα subunit that accelerates GTP hydrolysis, thereby effectively turning off signaling cascades mediated by GPCRs. This GTPase accelerating protein (GAP) activity is the canonical mechanism of action for RGS proteins, although many also possess additional functions and domains. RGS proteins are divided into four families, R4, R7, R12 and RZ based on sequence homology, domain structure as well as specificity towards Gα subunits. For reviews on RGS proteins and their potential as therapeutic targets, see e.g. [160, 377, 411, 415, 416, 512, 519, 312, 6].

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