G Protein-Coupled Receptor-Mediated Mitogen-Activated Protein Kinase Activation through Cooperation of Gα_q and Gα_i Signals

Abstract

G protein-coupled receptors (GPCRs) have been shown to stimulate extracellular regulated kinases (ERKs) through a number of linear pathways that are initiated by G_q/11 or G_iproteins. We studied signaling to the ERK cascade by receptors that simultaneously activate both G protein subfamilies. In HEK293T cells, bradykinin B₂ receptor (B₂R)-induced stimulation of ERK2 and transcriptional activity of Elk1 are dependent on Gα_q-mediated protein kinase C (PKC) and on Gα_i-induced Ras activation, while they are independent of Gβγ subunits, phosphatidylinositol 3-kinase, and tyrosine kinases. Similar results were obtained with m₁ and m₃muscarinic receptors in HEK293T cells and with the B₂R in human and mouse fibroblasts, indicating a general mechanism in signaling toward the ERK cascade. Furthermore, the bradykinin-induced activation of ERK is strongly reduced in Gα_q/11-deficient fibroblasts. In addition, we found that constitutively active mutants of Gα_q/11 or Gα_i proteins alone poorly stimulate ERK2, whereas a combination of both led to synergistic effects. We conclude that dually coupled GPCRs require a cooperation of Gα_i- and G_q/11-mediated pathways for efficient stimulation of the ERK cascade. Cooperative signaling by multiple G proteins thus might represent a novel concept implicated in the regulation of cellular responses by GPCRs.

ACKNOWLEDGMENTS

We thank all scientists who generously provided reagents used in this study. We acknowledge A. Pizard and R. M. Rajerison (INSERM U367, Paris, France) for the initial characterization of B₂R expression in HEK293T cells.

A. Barac was supported by a scholarship from the Swedish Institute, M. J. Cross is supported by a Marie Curie TMR fellowship, and A. Blaukat is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. I. Dikic is a research fellow of the Boehringer Ingelheim Fonds.