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 aden ylate-cyclase-activating polypeptide (PACAP) that interact with the ty pe 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 the se peptide analogues as photolabels were tested for pig brain PACAP re ceptors. [Bz-Phe6]-PACAP-(1-27)-peptide (K-d 1.3 nM) retained the high binding affinity of PACAP-(1-27)-peptide (K-d 0.5 nM), whereas Bz-Phe substitution of Tyr22 reduced the affinity about tenfold (K-d 4.4 nM) thus demonstrating the importance of Tyr22 for receptor binding. Mono iodination of the photoreactive analogues did not change the binding a ffinity of the photoreactive analogues, Photoaffinity labeling using p ig brain membrane demonstrated that the I-125-labeled photoreactive an alogues specifically label a 66 000-M(r) protein band. Photoaffinity l abeling of the rat brain PACAP receptor expressed in COS cells resulte d in two specifically photolabeled proteins: a major band of M(r) 58 0 00 and a minor band of M(r) 78 000. By treatment of photolabeled membr anes with N-glycosidase F, both of the polypeptide bands were converte d to a single polypeptide band of M, 54000, which corresponds to the d eglycosylated 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)-pept ide is located in or close to the hormone-binding site of the PACAP ty pe I receptor. The results provide evidence that the alpha-helical C-t erminal region of PACAP is directly involved in receptor binding.