PAC1‐Gs Coupling Through AC6 Mediates NCS/Rapgef2‐dependent ERK Activation in Neuroendocrine Cells

Abstract

The neuritogenic cAMP sensor (NCS), the protein product of the Rapgef2 gene, couples GPCR‐Gs‐dependent elevation of cAMP to ERK activation required for neuritogenesis, while additional aspects of cAMP‐dependent cell differentiation are mediated through PKA (pro‐survival effects) and Epac2 (growth arrest) (Emery et al., JBC 289: 10126, 2014). Functional full‐length Rapgef2 is expressed in cell lines of neuroendocrine lineage, but not in several non‐neuroendocrine cell lines commonly used for high‐throughput GPCR ligand and antagonist screening (Emery et al., Sci. Sig. 6: ra51, 2013). In a series of NS‐1 cell sublines into which Gs‐coupled GPCRs were introduced as single‐copy genes under the control of the CMV promoter, some (e.g. ADCYAP1R1, ADBR1, DRD1), but not others (ADBR2, ADORA2A), engage NCS/Rapgef2 and induce neuritogenesis when stimulated with the appropriate ligand. All are coupled to adenylate cyclase and cause stimulation of Epac and PKA. To determine the mechanism of coupling specificity between GPCRs and NCS/Rapgef2, we are evaluating the membranous adenylate cyclase (AC) specificity for NCS/Rapgef2 coupling. In PC12 cells, which express ACs 3, 4, 6, 7, and 9, PACAP‐initiated signaling through ADCYAP1R1 (cAMP elevation, ERK phosphorylation, and neuritogenesis) is greatly attenuated by knockdown of AC6, but not AC7, achieved by stable lentiviral‐mediated shRNA expression, with no significant effects of either knock‐down on forskolin‐induced downstream cAMP‐dependent signaling events (ERK activation and neuritogenesis). These data suggest that the ADCYAP1R1 (PAC1) receptor is preferentially coupled to AC6, and this AC preference is linked to engagement of NCS/Rapgef2, ERK activation, and neuritogenesis.