The hop cassette of the PAC1 receptor confers coupling to Ca2+ elevation required for pituitary adenylate cyclase-activating polypeptide-evoked neurosecretion

We have identified the single PAC1 receptor variant responsible for Ca2+ mobilization from intracellular stores and influx through voltage-gated Ca2+ channels in bovine chromaffin cells and the domain of this receptor variant that confers coupling to Ca2+]i elevation. This receptor (bPAC1hop) contains a 28-amino acid "hop" insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1hop receptor in NG108-15 cells, which lack endogenous PAC1 receptors, reconstituted high affinity PACAP binding and PACAP-dependent elevation of both cAMP and intracellular Ca2+ concentrations (Ca2+]i). Removal of the hop domain and expression of this receptor (bPAC1null) in NG108-15 cells reconstituted high affinity PACAP binding and PACAP-dependent cAMP generation but without a corresponding Ca2+]i elevation. PC12-G cells express sufficient levels of PAC1 receptors to provide PACAP-saturable coupling to adenylate cyclase and to drive PACAP-dependent differentiation but do not express PAC1 receptors at levels found in postmitotic neuronal and endocrine cells and do not support PACAP-mediated neurosecretion. Expression of bPAC1hop, but not bPAC1(null), at levels comparable with those of bPAC1hop in bovine chromaffin cells resulted in acquisition by PC12-G cells of PACAP-dependent Ca2+]i increase and extracellular Ca2+ influx. In addition, PC12-G cells expressing bPAC1hop acquired the ability to release 3H]norepinephrine in a Ca2+ influx-dependent manner in response to PACAP. Expression of PACAP receptors in neuroendocrine rather than nonneuroendocrine cells reveals key differences between PAC1hop and PAC1null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca2+ signaling in neuroendocrine cells.