Selective Regulation of Gq Signaling by G Protein-Coupled Receptor Kinase 2: Direct Interaction of Kinase N Terminus with Activated Gαq

  1. Michele Sallese1,2,
  2. Stefania Mariggiò1,1,
  3. Etrusca D'Urbano1,
  4. Luisa Iacovelli1 and
  5. Antonio De Blasi1,2
  1. 1Department of Molecular Pharmacology and Pathology, Consorzio Mario Negri Sud, Istituto di Ricerche Farmacologiche, “Mario Negri”, Santa Maria Imbaro (M.S., S.M., E.D., L.I., A.D.B.), and 2Istituto Neurologico Mediterraneo Neuromed, Pozzilli, Italy (M.S., A.D.B.)

    Abstract

    In this study, we investigated the regulation of different G protein-coupled receptor (GPCR)-stimulated signaling pathways by GPCR kinase 2 (GRK2). We used thyrotropin receptor, which is coupled to different G proteins, to investigate the regulation of Gαs- and Gαq-mediated signaling (assessed by cAMP and inositol phosphate production, respectively). In transfected cells, both pathways were desensitized by GRK2. However a kinase-dead GRK2 mutant (GRK2-K220R) only decreased inositol phosphate production, indicating that GRK2 could regulate Gαq signaling through a phosphorylation-independent mechanism. Similar results were obtained with serotonin receptor 5-hydroxytryptamine2C, which is coupled to Gαq. This effect was mimicked by the N-terminal domain of GRK2 (GRK2-Nter), but not by the C-terminal domain. In cells transfected with Gαq, direct activation of Gαq signaling (by AlF4−) was desensitized by GRK2-Nter, indicating an effect at the Gα-level. For comparison, in parallel samples we studied a protein regulator of G protein signaling RGS4 and we found a similar regulatory profile. We therefore hypothesized that the GRK2-Nter could directly interact with the Gαq subunit to regulate its signaling, as demonstrated for several RGS proteins. This hypothesis is further supported by the presence, within the GRK2-Nter, of an RGS homology domain. In direct binding experiments, we found that GRK2-Nter interacts with Gαq (only when activated) but not with Gαs and Gαo. We conclude that GRK2, besides desensitizing the GPCR by phosphorylation, is able to selectively bind to Gαq and to regulate its signaling.

    Footnotes

    • Send reprint requests to: Antonio De Blasi, Consorzio Mario Negri Sud, via Nazionale 66030 S. Maria Imbaro, Italy. E-mail:deblasi{at}cmns.mnegri.it

    • ↵1 Recipient of a fellowship granted by Progetto Speciale Ricerca Scientifica e Applicata nel Mezzogiorno PS35–93/IND.

    • This study was supported by Telethon-Italy (Grant 1238), the Associazione Italiana per la Ricerca sul Cancro, Consiglio Nazionale delle Richerche Target Project on Biotechnology, and EC Biomed 2 program-PL 963566.

    • Abbreviations:
      GPCR
      G protein-coupled receptor
      GRK
      G protein-coupled receptor kinase
      RGS
      regulator of G protein signaling
      GST
      glutathione S-transferase
      GST-Nter
      GST fusion protein with N-terminal domain of GRK2
      GRK2-Nter
      N-terminal domain of GRK2
      GRK2-Cter
      C-terminal domain of GRK2
      TSHr
      thyrotropin receptor
      5-HT
      5-hydroxytryptamine
      HEK
      human embryonic kidney
      IP
      inositol phosphate
      HBSS
      Hanks' balanced salt solution
      DTT
      dithiothreitol
      GRK2-K220R
      kinase-dead GRK2 mutant
      • Received October 21, 1999.
      • Accepted January 3, 2000.
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    1. Molecular Pharmacology April 1, 2000 vol. 57 no. 4 826-831
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