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J. Biol. Chem., Vol. 282, Issue 11, 8079-8091, March 16, 2007

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The Hop Cassette of the PAC1 Receptor Confers Coupling to Ca²⁺ Elevation Required for Pituitary Adenylate Cyclase-activating Polypeptide-evoked Neurosecretion^*

Tomris Mustafa¹, Maurizio Grimaldi, and Lee E. Eiden²

From the Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, Maryland 20892 and the Laboratory of Neuropharmacology, Department of Biochemistry, Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35205

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 (bPAC1_hop) contains a 28-amino acid "hop" insertion in the third intracellular loop, with a full-length 171-amino acid N terminus. Expression of the bPAC1_hop 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 (bPAC1_null) 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 bPAC1_hop, but not bPAC1_null, at levels comparable with those of bPAC1_hop 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 bPAC1_hop 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 PAC1_hop and PAC1_null coupling, indicating an important and previously unrecognized role of the hop cassette in PAC1-mediated Ca²⁺ signaling in neuroendocrine cells.

Received for publication, October 12, 2006 , and in revised form, December 28, 2006.

^* This work was supported by a grant from the National Institute of Mental Health-Intramural Research Program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A Julius Axelrod Fellow in the National Institute of Mental Health Intramural Research program.

² To whom correspondence should be addressed: Section on Molecular Neuroscience, Laboratory of Cellular and Molecular Regulation, National Institutes of Mental Health, Bldg. 49, Rm. 5A-68, 9000 Rockville Pike, Bethesda, MD 20892. Tel.: 301-496-4110; Fax: 301-496-1748; E-mail: eidenl{at}mail.nih.gov.

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