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 μM. 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.