Specific antibody recognition of rat pituitary adenylate cyclase activating polypeptide receptors.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a new member of the secretin/VIP family of peptides. The specific receptor for PACAP has been cloned in rat, human, and bovine tissues. The distribution of the transcripts of PACAP receptor genes has been studied in various tissues using in situ hybridization. However, the unavailability of a specific antibody against the PACAP receptor has hampered further study of the expression of receptor proteins. In the present study, rabbit antisera were generated against a synthetic 25-residue peptide corresponding to the C-terminal intracellular domain of the rat PACAP receptor. To validate the specificity of the antisera, CHO cells and cells stably transfected with rat PACAP receptor cDNA were prepared. Using one of these antisera, the membrane and soluble fractions of the transformants were examined by Western blot analysis. Three bands were observed in subcellular fractions from the transfected CHO cells, but no bands were found in similar preparations from the nontransfected cells. A distinct 57-kDa band, which corresponds to the size of cloned rat PACAP receptor, was detected. In addition, a less intense band, larger than 57 kDa, and a very weakly stained band, smaller than 57 kDa, were demonstrated. All of these bands disappeared or were considerably diminished when the antiserum was preabsorbed with the synthetic immunogen peptide. This suggests that these bands are PACAP receptor-related proteins. The membranes from the transfected CHO cells bound to [125I]PACAP27. The size of the ligand/protein crosslinked product approximated 60 kDa, corresponding to the combined size of the PACAP receptor and PACAP27. No additional bands were observed, indicating that the immunopositive proteins larger or smaller than 57 kDa do not bind to the ligand and are not functional. Unlabeled PACAP27 and PACAP38, but not VIP, displaced the binding, suggesting that the receptors expressed in CHO cells are specific for PACAP. Solubilized membrane fractions prepared from rat brains were used for an immunoprecipitation study with [125I]PACAP27 and [125I]VIP. The PACAP receptor antiserum recognized [125I]PACAP-, but not [125I]VIP-bound proteins in the solubilized brain membrane fractions. Immunohistochemistry using this antiserum showed a distribution of PACAP receptor-like immunoreactivities similar to the distribution of the mRNA of PACAP receptor in the rat brain. Thus, the PACAP receptor antiserum is sufficiently specific to be used as a tool for studying the expression of PACAP receptors and related proteins.