Agonist-Dependent Modulation of G Protein-Coupled Receptor Kinase 2 by Mitogen-Activated Protein Kinases
- Departamento de Biologı́a Molecular, Centro de Biologı́a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı́ficas-Universidad Autónoma de Madrid, Universidad Autónoma, Madrid, Spain
Abstract
A variety of G protein-coupled receptors (GPCRs) are phosphorylated by G protein-coupled receptor kinase 2 (GRK2). This event promotes the binding of regulatory proteins termed β-arrestins to GPCRs, leading to uncoupling from G proteins and receptor internalization. Recent data indicate that GRK2 and β-arrestins also play an important role in the stimulation of the extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) cascade by GPCRs. In this report, we have investigated the existence of functional interactions between GRK2 and MAPK. We show that activation of β2-adrenergic receptors (β2-AR) promotes the rapid association of GRK2 and MAPK in living cells, as assessed by coimmunoprecipitation experiments in COS-7 cells transfected with β2-AR, GRK2, and an epitope-tagged MAPK. Coimmunoprecipitation of MAPK and GRK2 is blocked by inhibition of the MAPK cascade and is not observed upon activation of MAPK in the absence of β2-AR stimulation, thus indicating that both an active MAPK and agonist occupancy of GPCR are required for the association to occur. Interestingly, we have found that purified ERK1/MAPK can directly phosphorylate the C-terminal domain of GRK2, and that the phosphorylation process is favored by the presence of Gβγ-subunits or an activated receptor. Furthermore, GRK2 phosphorylation by MAPK leads to a decreased activity of GRK2 toward GPCR. Taken together, our results suggest that stimulation of GPCRs promotes the rapid association of GRK2 and MAPK leading to modulation of GRK2 functionality, thus putting forward a new feedback mechanism for the regulation of GPCR signaling.
Footnotes
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Send reprint requests to: Dr. Federico Mayor, Jr., Centro de Biologı́a Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain. E-mail: fmayor{at}cbm.uam.es
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↵1 These authors contributed equally to this work.
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This work was supported by grants from the Ministerio de Educación y Cultura (PM95-0033 and PM98-0020), Comunidad de Madrid, and the European Union (BM H4-98-3566). The Centro de Biologı́a Molecular holds an institutional grant from the Fundación Ramón Areces.
- Abbreviations:
- GPCR
- G protein-coupled receptor
- β2-AR
- β2-adrenergic receptor
- ERK
- extracellular signal-regulated kinase
- GRK
- GPCR kinase
- GST
- glutathione S-transferase
- MAPK
- mitogen-activated protein kinase
- MEK
- MAPK kinase
- PKC
- protein kinase C
- HA
- hemagglutinin
- PAGE
- polyacrylamide gel electrophoresis
- RIPA
- radioimmune precipitation buffer
- MES
- 2-(N-morpholino)ethanesulfonic acid
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- Received October 4, 1999.
- Accepted January 4, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
