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 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.
Wang
Xinhua
X
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9040, USA.
Zeng
Weizhong
W
Soyombo
Abigail A
AA
Tang
Wei
W
Ross
Elliott M
EM
Barnes
Anthony P
AP
Milgram
Sharon L
SL
Penninger
Josef M
JM
Allen
Patrick B
PB
Greengard
Paul
P
Muallem
Shmuel
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009865:Oocytes / Q000737:chemistry; D011485:Protein Binding / Q000502:physiology; D020710:RGS Proteins / Q000378:metabolism / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014982:Xenopus laevis; D000099273:Neurabins
D002118:Calcium; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D020710:RGS Proteins; D043562:Receptors, G-Protein-Coupled; D000099273:Neurabins