Differential intracellular signaling through PAC1 isoforms as a result of alternative splicing in the first extracellular domain and the third intracellular loop.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a pleiotropic neuropeptide, performs a variety of physiological functions. The PACAP-specific receptor PAC1 has several variants that result mainly from alternative splicing in the mRNA regions encoding the first extracellular (EC1) domain and the third intracellular cytoplasmic (IC3) loop. The effects on downstream signaling produced by combinations of alternative splicing events in the EC1 domain and IC3 loop have not yet been clarified. In this study, we have used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine the tissue distributions of four PAC1 isoforms in mice. We then established cell lines constitutively expressing each of the PAC1 isoforms and characterized the binding properties of each isoform to PACAP-38, vasoactive intestinal polypeptide (VIP), and the PAC1-specific agonist maxadilan, as well as the resulting effects on two major intracellular signaling pathways: cAMP production and changes in the intracellular calcium concentration. The results demonstrate that the variants of the IC3 loop affect the binding affinity of the ligands for the receptor, whereas the variants of the EC1 domain primarily affect the intracellular signaling downstream of PAC1. Accordingly, this study indicates that the combination of alternative splicing events in the EC1 domain and the IC3 loop create a variety of PAC1 isoforms, which in turn may contribute to the functional pleiotropism of PACAP. This study not only contributes to the understanding of the multiple functions of PACAP but also helps to elucidate the relationship between the structures and functions of G-protein-coupled receptors.