Molecular structure analysis of the pituitary adenylate cyclase activating polypeptide type I receptor from pig brain

Abstract

In pig brain a type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP) has been identified and structurally characterized. Scatchard analysis of the equilibrium binding data indicates a single class of binding sites with K d of 0.50 nM and B max of 2.52 pmol/mg. The receptors could be efficiently solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (Chapso) without altering their ligand binding parameters ( K d = 0.60 nM; B max = 2.46 pmol/mg). In both preparations the ligand-receptor complex was identified as a 68 kDa polypeptide by affinity crosslinking with 125I-PACAP-27 and disuccinimidyl suberate. Employment of nonreducing conditions decreased the electrophoretic mobility of the complex to a 60 kDa species suggesting that the native PACAP receptor exists in a compact conformation stabilized by intramolecular disulfide bridges. Disulfide bonds are also important for the ligand binding activity of the PACAP receptor since pretreatment of the membranes with dithiothreitol led to complete inhibition of PACAP binding and to 61% dissociation of the ligand-receptor complex. Among different sulfhydryl selective reagents tested, p-chloromercuriphenylsulfonic acid was found to inhibit PACAP binding to its receptor in a dose-dependent fashion ( IC 50 = 0.5 mM) suggesting that one or more sulfhydryl groups are located close to the ligand binding domain of the receptor. Desialylation of the affinity-labeled PACAP receptor with neuraminidase revealed a 61 kDa protein, whereas deglycosylation with N-glycosidase F decreased the M r to 52000. Chromatography on a series of lectin agaroses showed the highest affinity for wheat germ agglutinin (WGA). WGA was also effective in dose-dependent inhibition of PACAP binding activity. Our results characterize the pig PACAP receptor as a tri- or tetraantennary non- or low fucosylated complex type glycoprotein with a protein core of about 49 kDa containing several terminal sialyl residues.