A CalDAG-GEFI/Rap1/B-Raf Cassette Couples M1Muscarinic Acetylcholine Receptors to the Activation of ERK1/2

Abstract

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.

Generation of Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate by CD38 for Ca2+ Signaling in Interleukin-8-treated Lymphokine-activated Killer Cells

“…Rap activation is important for lymphocyte migration in vivo (53,54). The Rap-dependent signaling pathway activated by Epac is known to be cell-specific (55,56), and there is evidence for Rap-mediated action of Epac to stimulate mitogen-activated protein kinases (p38, extracellular signal-regulated kinase 1/2) in neurons, endocrine cells, and T-cells (57)(58)(59). Rap1 was reported to be activated by PKA through the phosphorylation of Rap1GAP (60), consistent with our finding that treatment of LAK cells with IL-8 or PKA activator did not show Rap1 phosphorylation.…”

G Protein regulation of MAPK networks

“…In HEK293 cells, a 1 -adrenergic receptor-mediated activation of ERK1/2 appears to involve PKC-as well as Ca 2 þ -mediated pathways (Della Rocca et al, 1997). In addition to these typical Ga q -mediated pathways, it has also been shown that Ga q can activate ERK through a novel mechanism involving a DAG-as well as Ca 2 þ -dependent Rap-1-GEF (Guo et al, 2001). Using a variant of PC12 cells, PC12D, it has been shown that m 1 -muscarinic receptor stimulation of ERK1/2 modules is independent of Ras but dependent on Ga q -stimulated PLCb.…”

Glucose-dependent Insulinotropic Polypeptide Activates the Raf-Mek1/2-ERK1/2 Module via a Cyclic AMP/cAMP-dependent Protein Kinase/Rap1-mediated Pathway

“…In support of this hypothesis, other GPCRs have also been shown to regulate ERK1/2 activity via B-Raf and Rap1. For example, the adenosine receptor (A 2A ), the prototypical ␤ 2 -adrenergic receptor, and the M 1 muscarinic receptor have all been found to stimulate ERK1/2 via Rap1 and B-Raf in CHO, HEK, and PC12 cells, respectively (51)(52)(53). These cells all express the Raf isoform B-Raf and are therefore able to activate ERK1/2 via cAMP signaling.…”