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Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by G?i1 and G?i-linked G protein-coupled receptors.

ABSTRACT: Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active G?(i/o)-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive G?(i1/3)-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive G?(i1)-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive G?(i1)-GDP but not active G?(i1)-GTP. Active H-Ras(G/V) interacts with a native RGS14·G?(i1) complex in brain lysates, and co-expression of RGS14 and G?(i1) in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the G?(i)-linked ?(2A)-adrenergic receptor induces a conformational change in the G?(i1)·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive G?(i1)-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.

SUBMITTER: Vellano CP

PROVIDER: S-EPMC3561580 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by Gαi1 and Gαi-linked G protein-coupled receptors.

Vellano Christopher P CP Brown Nicole E NE Blumer Joe B JB Hepler John R JR

The Journal of biological chemistry 20121217 5

Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα(i/o)-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα(i1/3)-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 p ...[more]

PMID: 23250758

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Project description:Bone homeostasis intimately relies on the balance between osteoblasts (OBs) and osteoclasts (OCs). Our previous studies have revealed that regulator of G protein signaling protein 12 (Rgs12), the largest protein in the Rgs super family, is essential for osteoclastogenesis from hematopoietic cells and OC precursors. However, how Rgs12 regulates OB differentiation and function is still unknown. To understand that, we generated an OB-targeted Rgs12 conditional knockout (CKO) mice model by crossing Rgs12fl/fl mice with Osterix (Osx)-Cre transgenic mice. We found that Rgs12 was highly expressed in both OB precursor cells (OPCs) and OBs of wild-type (WT) mice, and gradually increased during OB differentiation, whereas Rgs12-CKO mice (OsxCre/+ ; Rgs12fl/fl ) exhibited a dramatic decrease in both trabecular and cortical bone mass, with reduced numbers of OBs and increased apoptotic cell population. Loss of Rgs12 in OPCs in vitro significantly inhibited OB differentiation and the expression of OB marker genes, resulting in suppression of OB maturation and mineralization. Further mechanism study showed that deletion of Rgs12 in OPCs significantly inhibited guanosine triphosphatase (GTPase) activity and cyclic adenosine monophosphate (cAMP) level, and impaired Calcium (Ca2+ ) oscillations via restraints of major Ca2+ entry sources (extracellular Ca2+ influx and intracellular Ca2+ release from endoplasmic reticulum), partially contributed by the blockage of L-type Ca2+ channel mediated Ca2+ influx. Downstream mediator extracellular signal-related protein kinase (ERK) was found inactive in OBs of OsxCre/+ ; Rgs12fl/fl mice and in OPCs after Rgs12 deletion, whereas application of pertussis toxin (PTX) or overexpression of Rgs12 could rescue the defective OB differentiation via restoration of ERK phosphorylation. Our findings reveal that Rgs12 is an important regulator during osteogenesis and highlight Rgs12 as a potential therapeutic target for bone disorders. © 2018 American Society for Bone and Mineral Research.

Dataset Information

Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by G?i1 and G?i-linked G protein-coupled receptors.

Publications

Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by Gαi1 and Gαi-linked G protein-coupled receptors.

Similar Datasets

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