Open AccessPhotoaffinity Labeling Analysis of the Interaction of Pituitary Adenylate‐Cyclase‐Activating Polypeptide (PACAP) with the PACAP Type I Receptor
Author(s) -
Cao YongJiang,
Kojro Elzbieta,
Gimpl Gerald,
Jasionowski Marek,
Kasprzykowski Franciszek,
Lankiewicz Leszek,
Fahrenholz Falk
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00400.x
Subject(s) - photoaffinity labeling , cyclase , peptide , receptor , pituitary adenylate cyclase activating peptide , adenylate kinase , chemistry , affinity label , biochemistry , stereochemistry , peptide hormone , ligand (biochemistry) , affinity labeling , binding site , vasoactive intestinal peptide , neuropeptide
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 photo‐reactive 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‐(l‐27)‐peptide (K A 1.3 nM) retained the high binding affinity of PACAP‐(1–27)‐peptide (K d 0.5 nM), wheras Bz‐Phe substitution of Tyr22 reduced the affinity about tenfold (K d 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 125 I‐labeled photoreactive analogues specifically label a 66000‐M r protein band. Photoaffinity labeling of the rat brain PACAP receptor expressed in COS cells resulted in two specifically photolabeled proteins: a major band of M r 58000 and a minor band of M r 78000. By treatment of photolabeled membranes with W‐glycosidase F 1 both of the polypeptide bands were converted to a single polypeptide band of M r 54000, which corresponds to the deglycosylated PACAP receptor. Despite its lower receptor affinity, [Bz‐Phe22]‐PACAP‐(l‐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‐(l–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 α‐helical C‐terminal region of PACAP is directly involved in receptor binding.