A CalDAG-GEFI/Rap1/B-Raf cassette couples M(1) muscarinic acetylcholine receptors to the activation of ERK1/2.

In this study we examine signaling pathways linking the M(1) subtype of muscarinic acetylcholine receptor (M(1) mAChR) to activation of extracellular signal-regulated kinases (ERK) 1 and 2 in neuronal PC12D cells. We first show that activation of ERK1/2 by the M(1) mAChR agonist carbachol takes place primarily via a Ras-independent pathway that depends largely upon Rap1, another small GTP-binding protein in the Ras family. Rap1 in turn activates B-Raf, an upstream activator of ERK1/2. Consistent with these results, carbachol was found to activate Rap1 more potently than Ras. Similar to other small GTP-binding proteins, activation of Rap1 requires a guanine nucleotide exchange factor (GEF) to promote its conversion from the GDP- to GTP-bound form. Using specific antibodies, we show that a recently identified Rap1 GEF, calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), is expressed in PC12D cells and that carbachol stimulates the formation of a complex containing CalDAG-GEFI, Rap1, and activated B-Raf. Finally, we show that expression of CalDAG-GEFI antisense RNA largely blocks carbachol-stimulated activation of hemagglutinin (HA)1-tagged B-Raf and formation of the CalDAG-GEFI/Rap1/HA1-tagged B-Raf complex. Together, these data define a novel signaling pathway for M(1) mAChR, where increases in Ca(2+) and diacylglycerol stimulate the sequential activation of CalDAG-GEFI, Rap1, and B-Raf, resulting in the activation of MEK and ERK1/2.