Characterization of a guanosine-nucleotide-binding-protein-coupled receptor for pituitary adenylate-cyclase-activating polypeptide on plasma membranes from rat brain.
Pituitary adenylate-cyclase-activating polypeptide (PACAP), a novel brain-gut hormone, was isolated from ovine hypothalami and represents the latest mammalian member of the secretin-glucagon peptide family. PACAP exists in two C-terminally amidated molecular forms, PACAP(1-27) and PACAP(1-38), comprising 27 or 38 amino acid residues, respectively. In order to identify a specific receptor for PACAP, we studied binding of 125I-labelled PACAP(1-27) to plasma membranes from rat brain. We identified a single high-affinity binding site (Kd, 340 pM and Bmax, 3.34 pmol/mg), specific for synthetic PACAP(1-38) and PACAP(1-27). Hormone binding was reversible and time, protein and temperature dependent. In contrast, neither the analogues PACAP(1-23), PACAP(18-38) and PACAP(3-25), nor vasoactive intestinal peptide (VIP), secretin and growth-hormone-releasing factor (GRF) revealed significant binding at concentrations up to 1 microM. A specific receptor protein, with an apparent molecular mass of 60 kDa, was identified by means of affinity cross-linking with disuccinimidyl suberate (DSS) and ethylene glycol disuccinimidyl suberate (EGS). PACAP receptors are associated with a GTP-binding protein as determined by the influence of different nucleotides on PACAP binding. PACAP-binding activity was solubilized with the detergents 3-[(3-cholamidopropyl)dimethylammonio]2-hydroxy-1-propane sulfonate (Chapso) or Triton X-100 and was characterized as a high-molecular-mass receptor complex (400 kDa) by non-reducing size-exclusion chromatography on Sepharose CL-6B. These data imply the following: high-affinity PACAP receptors are expressed abundantly on rat-brain plasma membranes; PACAP receptors are specific for PACAP and show no affinity for VIP, secretin and GRF; the PACAP receptor molecule has an apparent molecular mass of 60 kDa; the PACAP receptor complex is associated with a GTP-binding protein.
Schäfer
H
H
Department of Medicine, Georg-August-University, Göttingen, Federal Republic of Germany.
Schwarzhoff
R
R
Creutzfeldt
W
W
Schmidt
W E
WE
D000227:Adenine Nucleotides / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology; D007700:Kinetics; D009479:Neuropeptides / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051381:Rats; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
C495403:Adcyap1 protein, rat; D000227:Adenine Nucleotides; D003432:Cross-Linking Reagents; D006150:Guanine Nucleotides; D009479:Neuropeptides; D010455:Peptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011956:Receptors, Cell Surface; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D014660:Vasoactive Intestinal Peptide; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases