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

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Abstract
Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Gα subunits and act in a G-protein-coupled receptor (GPCR)-specific manner1,2,3,4. The conserved RGS domain accelerates the G subunit GTPase activity5, whereas the variable amino-terminal domain participates in GPCR recognition6. 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 GPCRs8,9,10, 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 α-adrenergic receptor (αAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant αARA293E (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of βAR–αAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, αAR-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.