Cloning and expression of a complementary DNA encoding the bovine receptor for pituitary adenylate cyclase-activating polypeptide (PACAP).
A cDNA encoding a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was cloned from a bovine brain cDNA library using a synthetic oligonucleotide probe corresponding to the partial N-terminal amino acid sequence of the PACAP receptor purified from the bovine brain. The cloned cDNA encoded a polypeptide of 513 amino acid residues with seven putative transmembrane domains. The deduced amino acid sequence exactly matched the N-terminal amino acid sequence of the purified PACAP receptor. It also shared an apparent similarity with the vasoactive intestinal peptide (VIP), secretin, growth hormone releasing hormone, calcitonin, and glucagon receptors, suggesting that the PACAP receptor is a member of the secretin receptor subfamily of the guanine nucleotide-binding regulatory protein-coupled receptor family. Northern blot analysis showed that the size of the major mRNA band which hybridized with the cDNA was about 7 kb in the bovine cerebral-cortex and hippocampus. An expression vector containing the cloned cDNA for the PACAP receptor was introduced into Chinese hamster ovary (CHO) cells. The affinity of PACAP receptors expressed on the transfected CHO cells was quite similar to that of natural PACAP receptors on the bovine brain membranes. Competitive binding experiments showed that PACAP38 displaced the binding of 125I-labeled PACAP27 to the receptors on the CHO cells more efficiently than PACAP27, while VIP was less effective. In addition, both of PACAP27 and PACAP38 elevated the levels of cAMP and inositol phosphates in the transformed CHO cells. These results indicate that the PACAP receptors encoded by the cloned cDNA are identical to the purified PACAP receptors, and that they can stimulate dual signaling cascades.
Miyamoto
Y
Y
Discovery Research Laboratories I, Takeda Chemical Industries, Ltd., Ibaraki, Japan.
Habata
Y
Y
Ohtaki
T
T
Masuda
Y
Y
Ogi
K
K
Onda
H
H
Fujino
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002417:Cattle; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism*; D015723:Gene Library; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism*; D015345:Oligonucleotide Probes; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D010902:Pituitary Gland / Q000378:metabolism*; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone / Q000096:biosynthesis* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D018076:DNA, Complementary; D007295:Inositol Phosphates; D009479:Neuropeptides; D015345:Oligonucleotide Probes; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D011994:Recombinant Proteins; D000242:Cyclic AMP