PURIFICATION AND CHARACTERIZATION OF THE RECEPTOR FOR PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-ami no acid peptide (PACAP38) or a truncated peptide with the same 27 amin o-terminal residues (PACAP27). The PACAP receptor was solubilized from bovine brain membranes with digitonin and purified 30-fold by the com bination of DEAE-Toyopearl and hydroxylapatite chromatographic analyse s. The partially purified PACAP receptors were mixed with biotinylated PACAP27 to form receptor-ligand complexes and then adsorbed onto avid in-agarose. The adsorbed PACAP receptors were eluted with an acidic bu ffer containing 1.0 M NaCl (pH 4.0). The eluted receptors were purifie d further by hydroxylapatite and gel filtration chromatography. A sing le protein band with a M(r) 55,000-60,000 was found in the final prepa ration by sodium dodecyl sulfate-polyacrylamide gel electrophoresis an d silver staining. Affinity labeling of the purified receptors with I- 125-PACAP27 labeled the M(r) 55,000-60,000 protein specifically. The d issociation constant and the specific activity of the purified recepto rs were 25.8 pM and 17.2 nmol of ligand binding per mg of protein, res pectively. Inhibitory constants determined by competitive binding expe riments were 30.0 pM for PACAP27, 4.6 pM for PACAP38, and 37.3 nM for vasoactive intestinal peptide. Therefore, the purified PACAP receptor retained high affinity and ligand specificity. The sequence of the ami no-terminal 29 residues was derived from the purified receptor.