Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily

Regulator of G protein signaling (RGS) proteins inhibit G protein signaling by activating Gα GTPase activity, but the mechanisms that regulate RGS activity are not well understood. The mammalian R7 binding protein (R7BP) can interact with all members of the R7 family of RGS proteins, and palmitoylation of R7BP can target R7 RGS proteins to the plasma membrane in cultured cells. However, whether endogenous R7 RGS proteins in neurons require R7BP or membrane localization for function remains unclear. We have identified and knocked out the only apparent R7BP homolog in Caenorhabditis elegans, RSBP-1. Genetic studies show that loss of RSBP-1 phenocopies loss of the R7 RGS protein EAT-16, but does not disrupt function of the related R7 RGS protein EGL-10. Biochemical analyses find that EAT-16 coimmunoprecipitates with RSBP-1 and is predominantly plasma membrane-associated, whereas EGL-10 does not coimmunoprecipitate with RSBP-1 and is not predominantly membrane-associated. Mutating the conserved membrane-targeting sequence in RSBP-1 disrupts both the membrane association and function of EAT-16, demonstrating that membrane targeting by RSBP-1 is essential for EAT-16 activity. Our analysis of endogenous R7 RGS proteins in C. elegans neurons reveals key differences in the functional requirements for membrane targeting between members of this protein family.

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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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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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Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle

Journal of Biological Chemistry ◽

10.1074/jbc.m112.353219 ◽

2012 ◽

Vol 287 (21) ◽

pp. 17870-17881 ◽

Cited By ~ 20

Author(s):

Swarup Roy Choudhury ◽

Corey S. Westfall ◽

John P. Laborde ◽

Naveen C. Bisht ◽

Joseph M. Jez ◽

...

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Heterotrimeric G Protein

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Cellular Regulation of RGS Proteins: Modulators and Integrators of G Protein Signaling

Pharmacological Reviews ◽

10.1124/pr.54.3.527 ◽

2002 ◽

Vol 54 (3) ◽

pp. 527-559 ◽

Cited By ~ 520

Author(s):

S. Hollinger

Keyword(s):

G Protein ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Cellular Regulation

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Probing the mutational landscape of regulators of G protein signaling proteins in cancer

Science Signaling ◽

10.1126/scisignal.aax8620 ◽

2020 ◽

Vol 13 (617) ◽

pp. eaax8620 ◽

Cited By ~ 7

Author(s):

Vincent DiGiacomo ◽

Marcin Maziarz ◽

Alex Luebbers ◽

Jillian M. Norris ◽

Pandu Laksono ◽

...

Keyword(s):

G Protein ◽

G Proteins ◽

Mammalian Cells ◽

G Protein Signaling ◽

Rgs Proteins ◽

Protein Signaling ◽

Loss Of Function ◽

Gpcr Signaling ◽

Mutational Landscape ◽

Binding Interface

The advent of deep-sequencing techniques has revealed that mutations in G protein–coupled receptor (GPCR) signaling pathways in cancer are more prominent than was previously appreciated. An emergent theme is that cancer-associated mutations tend to cause enhanced GPCR pathway activation to favor oncogenicity. Regulators of G protein signaling (RGS) proteins are critical modulators of GPCR signaling that dampen the activity of heterotrimeric G proteins through their GTPase-accelerating protein (GAP) activity, which is conferred by a conserved domain dubbed the “RGS-box.” Here, we developed an experimental pipeline to systematically assess the mutational landscape of RGS GAPs in cancer. A pan-cancer bioinformatics analysis of the 20 RGS domains with GAP activity revealed hundreds of low-frequency mutations spread throughout the conserved RGS domain structure with a slight enrichment at positions that interface with G proteins. We empirically tested multiple mutations representing all RGS GAP subfamilies and sampling both G protein interface and noninterface positions with a scalable, yeast-based assay. Last, a subset of mutants was validated using G protein activity biosensors in mammalian cells. Our findings reveal that a sizable fraction of RGS protein mutations leads to a loss of function through various mechanisms, including disruption of the G protein–binding interface, loss of protein stability, or allosteric effects on G protein coupling. Moreover, our results also validate a scalable pipeline for the rapid characterization of cancer-associated mutations in RGS proteins.

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