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

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of G alpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner(1-4). The conserved RGS domain accelerates the Ga subunit GTPase activity(5), whereas the variable amino-terminal domain participates in GPCR recognition(6). How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL)(7), which binds the third intracellualar loop (3iL) of several GPCRs(8-10), 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 (alpha AR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alpha AR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of beta AR-alpha AR chimaeras identified the (288)REKKAA(293) sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alpha AR-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.