Pituitary Adenylyl Cyclase-activating Peptide Stimulates Extracellular Signal-regulated Kinase 1 or 2 (ERK1/2) Activity in a Ras-independent, Mitogen-activated Protein Kinase/ERK Kinase 1 or 2-dependent Manner in PC12 Cells

Abstract

Sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is critical for initiating differentiation of the PC12 cell to a sympathetic-like neurone. The neuropeptide, pituitary adenylyl cyclase-activating peptide (PACAP), has been demonstrated to cause cells to adopt a neuronal phenotype, although the mechanism of this activity is unclear. PACAP through its type I receptor stimulates a biphasic activation of ERK1/2; a >10-fold increase within 5 min, followed by a >5-fold increase that is sustained for 60 min. An equivalent stimulation is seen in PC12 cells expressing a dominant negative Ras mutant. However, the mitogen-activated kinase/ERK kinase 1/2 (MEK1/2) inhibitor PD98059 blocked both PACAP-induced stimulation of ERK1/2 activity and neurite outgrowth. Thus, the activation signal from the PACAP type I receptor on the ERK1/2 cascade pathway is received downstream of Ras, either at Raf or MEK. Down-regulation of protein kinase C or its inhibition by calphostin C blocked the ability of PACAP to stimulate ERK1/2. We conclude that activation of PACAP type I receptor activates protein kinase C, which then activates the ERK1/2 cascade in a Ras-independent manner at either Raf or MEK1/2.The two forms of pituitary adenylyl cyclase-activating polypeptide 1 are neuropeptides of the secretin/glucagon/vasoactive intestinal peptide/growth hormonereleasing hormone family. They share the same 27 aminoterminal amino acids and arise from a precursor peptide by post-translational processing (1). Two receptor subtypes have been identified for PACAP; both are G protein-coupled receptors. The PACAP type I receptor (found in hypothalamus, brain stem, pituitary, adrenal gland, and testes) is specific for PACAP, having a K d of 0.5-2.0 nM. There are several splice variants that demonstrate different abilities to activate adenylyl cyclase and phospholipase C (2). The type II receptor does not discriminate between PACAP and VIP and is only positively coupled to adenylyl cyclase.PACAP38 potently stimulates neuritogenesis of neuroblastoma cells (3), neonatal chromaffin cells (4), corticotrope cells (5), and PC12 cells (6 -8).The MAP kinases ERK1/2 are thought to be key players in the control of gene transcription events that lead to proliferation or differentiation in PC12 cells in response to epidermal growth factor or NGF, respectively. It has been suggested that the determinant of the nature of the response of PC12 cells (proliferation or differentiation) correlates with the duration of ERK1/2 activation and its translocation to the nucleus (9). ERK1/2 activation following agonist stimulation of G protein-coupled receptors has been reported (reviewed in Ref. 10), although various effectors are employed to couple these receptors to the MAP kinase cascade. In light of the neurite-stimulatory role of PACAP38 in pituitary neural cells and neuroblastoma cell lines, we have investigated the relationship of PACAP38 stimulation to ERK1/2 activation and compared this to that for NGF. In this study, we have demonstrated that ...

Stimulation of the ERK Pathway by GTP-loaded Rap1 Requires the Concomitant Activation of Ras, Protein Kinase C, and Protein Kinase A in Neuronal Cells

“…This finding will hopefully help to solve some of the "apparent discrepancies" (52) found in the literature on the mechanism of stimulation of the ERK pathway by Rap1 in neuronal cells. These data also reconciled previous reports with respect to the role of the cAMP pathway in PACAP-versus forskolin-induced ERK activation in PC12 cells (5,6,36,58). Finally, our data shed new light on the control of Rap1 activity by GPCRs, an important event in the context of neuronal differentiation.…”

“…Pac1 displays a complex pattern of alternative splicing that modulates its ligand binding and signaling properties (31)(32)(33)(34) resulting in the modulation of PACAP physiological effects (28,35). We and others (21,31) showed that Pac1 potently stimulated the AC and PLC activities as well as the ERK pathway that was implicated in its neurotrophic activity (23,36). In this context, we selected the PC12 cell line to assess the involvement of the small GTP-binding proteins Rap1 and Ras in the signaling pathways responsible for PACAP-induced ERK activation in this system.…”

Soluble Adenylyl Cyclase Mediates Nerve Growth Factor-induced Activation of Rap1

“…In PC12 cells, tmACs can be activated by pituitary adenylyl cyclase-activating peptide (PACAP) via its specific G protein-coupled receptor PAC1 (12). Similar to NGF, PACAP activates Rap1 and elicits neurite outgrowth (13)(14)(15), but all attempts to link NGF or its receptors to tmAC activation have been unsuccessful. We have identified an additional, ubiquitously expressed (17,18) source of cAMP in mammalian cells, "soluble" adenylyl cyclase (sAC) (16).…”

Molecular Physiology

“…In Drosophila muscle, PACAP induces synaptic currents through the co-activation of adenylyl cyclase and p21 ras (Zhong, 1995). In contrast, PC12 neurite outgrowth is independent of cAMP-dependent protein kinase (PKA) and p21 ras (Barrie et al 1997). This study investigated the signalling pathways coupling PACAP receptors to Ca2+ influx in PC12 cells.…”

“…This study investigated the signalling pathways coupling PACAP receptors to Ca2+ influx in PC12 cells. Experiments employed PC12 cells and a dominant negative Ras cell line (PC12asn17-W7) (Barrie et al 1997). Cytosolic [Ca2+] was recorded from cell suspensions loaded with 6 J1M fura-2 AM.…”