Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors

Abstract

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.

Homer proteins in Ca2+ signaling by excitable and non-excitable cells

“…The GAP activity of the RGS proteins is mediated by the RGS box [39], whereas the N-terminus domain mediates membrane targeting and confers receptor recognition [40]. Receptor recognition is mediated by binding of the Nterminus of RGS proteins and the third intracellular loop of GPCRs to the scaffolding protein spinophilin [41]. The divergent roles of the C-terminus are dependent on the motifs present in this domain [37,38].…”

Structural Basis for Spinophilin−Neurabin Receptor Interaction

“…Both neurabin and spinophilin contain an F-actin binding, a PP1-binding, a PDZ, and a C-terminal coiled-coil domain. In addition, neurabin, but not spinophilin [in vertebrates (7)], contains a sterile R motif (SAM) domain in its C-terminus, while spinophilin, but not neurabin, is proposed to have a dopamine receptor-R-adrenergic interacting domain in its N-terminus, possibly between spinophilin residues 200 and 400 (8,9). The highest level of primary sequence identity between neurabin and spinophilin is found in the PDZ domains (86%), the protein phosphatase 1 (PP1) binding domains (81%), and the coiled-coil domains (63%).…”

Interaction of nucleoside diphosphate kinase B with heterotrimeric G protein βγ dimers: consequences on G protein activation and stability

“…Besides the complexation of heterotrimeric G proteins with NDPK B on which this review is focused and the welldocumented interactions with GPCRs and effectors, it is meanwhile clear that the additional proteins like regulators of G proteins (for review, see Mittmann 2003, Abramow-Newerly et al 2006), scaffold proteins (Chen et al 2004;Wang et al 2005), membrane organising proteins like caveolins (Head et al 2005) and even proteins, which were, until now, believed not to be interaction partners of heterotrimeric G proteins, e.g. endothelial nitric oxide synthase (Andreeva et al 2006), a part of cellular signal transduction machineries.…”