The amino-terminal fragment of the adenylate cyclase activating polypeptide (PACAP) receptor functions as a high affinity PACAP binding domain.
The PACAP receptor represents a member of a novel subfamily of G-protein coupled receptors with a common structurally conserved extracellular domain of about 150 amino acids. We have addressed the question whether this extracellular amino-terminus of the PACAP type I receptor can solely function as a PACAP binding domain. For that purpose a cDNA was constructed that encodes the membrane-anchored amino-terminus of the rat PACAP receptor including the decapeptide epitope EQKLISEEDL for immunodetection. COS-7 cells were transfected with this cDNA and a comparable construct of the wild-type receptor. Binding analysis showed that the amino-terminal fragment of the PACAP receptor bound PACAP with high-affinity (Kd = 3.8 nM; Bmax = 12.8 pmol/mg protein). In comparison to the full-length receptor (Kd = 0.2 nM; Bmax = 1.96 pmol/mg protein) its affinity was reduced by a factor of about 20. The results suggest that the amino-terminus of the PACAP receptor functions as the major binding site for its ligand.
Cao
Y J
YJ
Max-Planck-Institut für Biophysik, Kennedyallee, Germany.
Gimpl
G
G
Fahrenholz
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011487:Protein Conformation; D051381:Rats; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D014162:Transfection
C495403:Adcyap1 protein, rat; D018076:DNA, Complementary; D009479:Neuropeptides; D010446:Peptide Fragments; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D011994:Recombinant Proteins