Purification of the Receptor for Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) using Biotinylated Ligands

Abstract

The pituitary adenylate cyclase-activating polypeptide (PACAP), including PACAP27 and PACAP38, stimulates adenylate cyclase via specific PACAP receptors that are coupled to G proteins. The present study describes the application of avidin-biotin technology to the purification of the PACAP receptor from bovine brain membranes. The PACAP receptor was solubilized from the membranes with digitonin and partially purified by DEAE-Toyopearl and hydroxylapatite chromatography for removal of avidin-binding proteins. The partially purified PACAP receptor was mixed with a biotinylated ligand (PACAP27Cys(biotin)-NH2) to form a receptor-ligand complex, adsorbed onto avidinagarose, and then eluted with 1.0 M NaCl-acetate buffer (pH 4.0). The affinity-purified receptor was further purified by hydroxylapatite and gel-filtration chromatography. Analysis of the purified preparation by SDS-polyacrylamide gel electrophoresis and silver staining showed a single broad protein band with an M r = 55 000 to 60 000. The specific activity of the purified receptor (17.2 nmol/mg) was close to the theoretical value. The dissociation constant (K d = 25.8 pM) was similar to that of the crude solubilized PACAP receptor. Thus, the PACAP receptor was purified to near homogeneity in a fully active form preserving high affinity for PACAP. The present study demonstrates that affinity chromatography using biotinylated ligands is a promising method for the purification of the PACAP receptor.