Purification and characterization of the receptor for pituitary adenylate cyclase-activating polypeptide.
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide (PACAP38) or a truncated peptide with the same 27 amino-terminal residues (PACAP27). The PACAP receptor was solubilized from bovine brain membranes with digitonin and purified 30-fold by the combination of DEAE-Toyopearl and hydroxylapatite chromatographic analyses. The partially purified PACAP receptors were mixed with biotinylated PACAP27 to form receptor-ligand complexes and then adsorbed onto avidin-agarose. The adsorbed PACAP receptors were eluted with an acidic buffer containing 1.0 M NaCl (pH 4.0). The eluted receptors were purified further by hydroxylapatite and gel filtration chromatography. A single protein band with a M(r) = 55,000-60,000 was found in the final preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Affinity labeling of the purified receptors with 125I-PACAP27 labeled the M(r) 55,000-60,000 protein specifically. The dissociation constant and the specific activity of the purified receptors were 25.8 pM and 17.2 nmol of ligand binding per mg of protein, respectively. Inhibitory constants determined by competitive binding experiments were 30.0 pM for PACAP27, 4.6 pM for PACAP38, and 37.3 nM for vasoactive intestinal peptide. Therefore, the purified PACAP receptor retained high affinity and ligand specificity. The sequence of the amino-terminal 29 residues was derived from the purified receptor.
Ohtaki
T
T
Discovery Research Laboratories I, Takeda Chemical Industries, Ltd., Ibaraki, Japan.
Masuda
Y
Y
Ishibashi
Y
Y
Kitada
C
C
Arimura
A
A
Fujino
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D001360:Avidin; D001710:Biotin