Photoaffinity labeling analysis of the interaction of pituitary adenylate-cyclase-activating polypeptide (PACAP) with the PACAP type I receptor.
To identify residues and domains of the peptide hormone pituitary adenylate-cyclase-activating polypeptide (PACAP) that interact with the type I receptor, two photoreactive analogues of PACAP-(1-27)-peptide were synthesized using solid-phase peptide synthesis. Phe6 or Tyr22 within the PACAP sequence were replaced by p-benzoyl-L-phenylalanine (Bz-Phe) thus creating two PACAP derivatives with a photoreactive amino acid in either the disordered N-terminal or the helical C-terminal part of the peptide. The ligand-binding properties and the efficiencies of these peptide analogues as photolabels were tested for pig brain PACAP receptors. [Bz-Phe6]-PACAP-(1-27)-peptide (Kd 1.3 nM) retained the high binding affinity of PACAP-(1-27)-peptide (Kd 0.5 nM), wheras Bz-Phe substitution of Tyr22 reduced the affinity about tenfold (Kd 4.4 nM) thus demonstrating the importance of Tyr22 for receptor binding. Monoiodination of the photoreactive analogues did not change the binding affinity of the photoreactive analogues. Photoaffinity labeling using pig brain membrane demonstrated that the 125I-labeled photoreactive analogues specifically label a 66000-Mr protein band. Photoaffinity labeling of the rat brain PACAP receptor expressed in COS cells resulted in two specifically photolabeled proteins: a major band of Mr 58000 and a minor band of Mr 78000. By treatment of photolabeled membranes with N-glycosidase F, both of the polypeptide bands were converted to a single polypeptide band of Mr 54000, which corresponds to the deglycosylated PACAP receptor. Despite its lower receptor affinity, [Bz-Phe22]-PACAP-(1-27)-peptide labeled the PACAP type I receptor in pig brain membranes and the rat receptor expressed in COS cells with much higher efficiency (20-fold for the pig receptor) than [Bz-Phe6]-PACAP-(1-27)-peptide. These findings suggest that Tyr22 in PACAP-(1-27)-peptide is located in or close to the hormone-binding site of the PACAP type I receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.
Cao
Y J
YJ
Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
Kojro
E
E
Gimpl
G
G
Jasionowski
M
M
Kasprzykowski
F
F
Lankiewicz
L
L
Fahrenholz
F
F
D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D015870:Gene Expression; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010777:Photochemistry; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011869:Radioligand Assay; D051381:Rats; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine
C495403:Adcyap1 protein, rat; D000345:Affinity Labels; D009479:Neuropeptides; 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