A Ca1DAG-GEFI/Rap1/B-Raf cassette couples M1 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 Res-independent pathway that depends largely upon Rap1, another small GTP-binding protein in the Res 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 Res. 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, (cal) under bar cium- and (d) under bari (a) under bar cylglycerol-regulated guanine nucleotide (e) under bar xchange (f) under bar actor (I) under bar (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 Ca2+ and diacylglycerol stimulate the sequential activation of CalDAG-GEFI, Rap1, and B-Raf, resulting in the activation of MEK and ERK1/2.