Characterization and purification of the solubilized pituitary adenylate‐cyclase‐activating polypeptide‐1 receptor from porcine brain using a biotinylated ligand

Abstract

A specific receptor for the brain-gut neuropeptide pituitary adenylate-cyclase-activating polypeptide (PACAP-1 receptor) was solubilized with Chapso from porcine brain plasma membranes and purified. Binding of '251-PACAP(1 -27) to the solubilized material was reversed equipotently by unlabeled PACAP(1-27) and PACAP(1-38). Soluble receptors retained the binding affinities and specificities of the plasma membrane fraction. Scatchard analysis of equilibrium-binding data indicated the existence of a single high-affinity binding site (Kd = 0.23 nM, Bmax = 1.2 pmol/mg protein). Binding of 1251-PACAP(1 -27) to solubilized receptors was not affected by guanosine nucleotides, suggesting that solubilization dissociates the PACAP-1 receptor/guanosine-nucleotidebinding protein complex. Affinity cross-linking of lZ51-PACAP(1 -27) to soluble PACAP-1 receptors identified a specifically labeled 60-kDa protein. Enzymic deglycosylation of soluble affinitylabeled receptors reduced the apparent molecular mass by 10 kDa. The solubilized receptor glycoprotein was purified 4-5-fold by lectin-adsorption chromatography on wheatgerm agglutinin immobilized on agarose. S-Biotinyl[Ala28 -34, Cys35]PACAP(1-35) was synthesized, immobilized on streptavidin-coated magnetic Sepharose beads and used to further affinity-purify wheatgerm-agglutinin-eluted receptor material. This more than 6000-fold enriched PACAP-1-receptor-preparation retained single-class high-affinity binding and consisted of an almost homogenous 55 -60-kDa protein identified by silver staining. In conclusion, we established a rapid method for purification of PACAP-1 receptors, allowing further studies to be performed by protein chemistry.PACAP represents a novel neuropeptide that exists as two C-terminally amidated molecular forms; PACAP(1-38) and PACAP vasoactive intestinal peptide (VIP). Therefore, PACAP is regarded as a member of the secretinlglucagonNIP family of structurally related regulatory peptides. Yet, several biological effects of PACAP on pituitary [5-71, pancreatic [8, 91 and intestinal function [lo-131 have been described, qualifying PACAP as a brain-gut peptide. PACAP is widely expressed in gastrointestinal and nervous tissues [14-161 and binds specifically to at least two receptor types; PACAP-1 receptors present in brain [17, 181 and gastrointestinal muscles [ 11 -131 and PACAP-2NIP receptors, similar or identical to the previously described VIP receptor. PACAP-1 receptors have been identified in various mammalian tissues as a 60-kDa protein [19-221, linked to a GTP-binding protein. To gain insight into the mechanisms by which PACAP exerts its action as neuromodulatory peptide, biochemical and molecular characterization of the PACAP-1 receptor is highly desirable. Therefore, we investigated the binding properties of the solubilized PACAP-1 receptor from porcine brain and developed a simple straight-forward strategy for receptor purification based on lectin-adsorption chromatography on immobilized wheatgerm agglutinin and ligand-affinity chromatography usi...

Identification and biochemical characterization of the human brain galanin receptor

“…As recently shown for the porcine brain Gal receptor, extraction with CHAPS was similarly successful (Chen et al 1992). We recently demonstrated that the G protein-coupled receptor for pituitary adenylate cyclase-activating peptide could be purified 7000-fold using a combination of solubilization with CHAPSO and affinity chromatography (Schäfer & Schmidt, 1993). Similar results have been reported for the affinity purification of the porcine Gal receptor (Chen et al 1993).…”

“…Never¬ theless, the overall yield was rather low compared with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulphonate) or CHAPSO ex¬ tracts of other hormone receptors (Feldman et al 1990;Schäfer et al 1991;Schäfer & Schmidt, 1993), suggesting that the hGal receptor is sensitive to treatment with detergent. As recently shown for the porcine brain Gal receptor, extraction with CHAPS was similarly successful (Chen et al 1992).…”

Interaction of Amino Acid Residues at Positions 8–15 of Secretin with the N‐Terminal Domain of the Secretin Receptor

“…C-terminally shortened peptides had a reduced affinity but, when interacting with the receptor, behaved as full agonists. Secretin fragment 1 -15 was active on guinea pig secretin receptor [25], PACAP fragment 1-23 had a reduced potency on the PACAP receptor [28], and VIP fragment 1-25 did not bind to the guinea pig tracheal receptor [29].…”

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 38 and PACAP27 Activate Common and Distinct Intracellular Signaling Pathways to Stimulate Growth Hormone Secretion from Porcine Somatotropes1

“…Initial studies suggested that the response of somatotropes to PACAP was mediated by VPAC 2 or VPAC 1 , but not PAC 1 (2,23,27,28). Although the type of PVRs expressed in porcine pituitary has not yet been determined, a type I PACAP-binding site, analogous to the rat PAC 1 , has been isolated from pig brain (49,50). Although the type of PVRs expressed in porcine pituitary has not yet been determined, a type I PACAP-binding site, analogous to the rat PAC 1 , has been isolated from pig brain (49,50).…”