Involvement of Ras and Raf in the Gi-coupled acetylcholine muscarinic m2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase.

Stimulation of the acetylcholine muscarinic m2 receptor (m2R) expressed in Rat 1a fibroblasts results in the activation of the cytoplasmic mitogen-activated protein kinase (MAPK). Concomitant with carbachol stimulation of the m2R was the activation of MEK (MAPK kinase) and Raf. MEK is the dual function kinase that phosphorylates and activates MAPK. Raf is a serine/threonine kinase capable of phosphorylating and activating MEK. Carbachol stimulation of the m2R also activated Ras. Pertussis toxin treatment of Rat 1a cells inhibited the m2R-mediated activation of Ras, Raf, MEK and MAPK. In contrast, epidermal growth factor receptor-mediated activation of Ras, Raf, MEK and MAPK was pertussis toxin-insensitive. m2R activation of Ras, Raf, and MAPK was insensitive to inhibition by genistein, while the epidermal growth factor receptor-induced responses were inhibited by genistein. The findings demonstrate that both Ras and Raf can be regulated by seven-membrane-spanning receptors that selectively couple to Gi proteins.