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

Nature Cell Biology volume 7, pages 405–411 (2005)Cite this article

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.

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Figure 1: Interaction of RGS2 with spinophilin (SPL).
Figure 2: Effect of SPL and RGS2 on Ca2+ signalling by WT-αAR and αARA293E.
Figure 3: The αAR 3iL sequence 288REKKAA293 mediates interaction with SPL.
Figure 4: Role of SPL in α-adrenergic receptor Ca2+signaling in native cells.
Figure 5: Binding of RGS2 to SPL and the αAR 3iL.

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Acknowledgements

We thank T. Südhof, UT Southwestern, Dallas, TX, for the brain library and S. Cotecchia, Institute of Pharm, Lausanne, Switzerland, for clones. This work was supported by the National Institutes of Health grants DE12309 and DK38938, the Peter Jay Sharp Foundation and the F.M. Kirby Foundation, Inc.

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Author notes

  1. Xinhua Wang and Weizhong Zeng: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390-9040, TX, USA

    Xinhua Wang, Weizhong Zeng, Abigail A. Soyombo & Shmuel Muallem

  2. Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390-9040, TX, USA

    Wei Tang & Elliott M. Ross

  3. The Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, CB7090, Chapel Hill, 27599-7090, NC, USA

    Anthony P. Barnes & Sharon L. Milgram

  4. IMBA (Institute of Molecular Biotechnology of the Austrian Academy of Sciences), Dr Bohr-gasse3-5, Vienna, A-1030, Austria

    Josef M. Penninger

  5. Department of Psychiatry, Yale University School of Medicine, New Haven, 06508, CT, USA

    Patrick B. Allen

  6. Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, 10021, New York, USA

    Paul Greengard

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Correspondence to Shmuel Muallem.

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Wang, X., Zeng, W., Soyombo, A. et al. Spinophilin regulates Ca2+ signalling by binding the N-terminal domain of RGS2 and the third intracellular loop of G-protein-coupled receptors. Nat Cell Biol 7, 405–411 (2005). https://doi.org/10.1038/ncb1237

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