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  Photoaffinity labeling analysis of the interaction of pituitary adenylate-cyclase-activating polypeptide (PACAP) with the PACAP type I receptor

Cao, Y.-J., Kojro, E., Gimpl, G., Jasionowski, M., Kasprzykowski, F., Lankiewicz, L., et al. (1997). Photoaffinity labeling analysis of the interaction of pituitary adenylate-cyclase-activating polypeptide (PACAP) with the PACAP type I receptor. European Journal of Biochemistry, 244(2), 400-406. doi:10.1111/j.1432-1033.1997.00400.x.

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 Creators:
Cao, Yong-Jiang1, Author           
Kojro, Elzbieta1, Author           
Gimpl, Gerald1, Author           
Jasionowski, Marek2, Author
Kasprzykowski, Franciszek2, Author
Lankiewicz, Leszek2, Author
Fahrenholz, Falk1, Author           
Affiliations:
1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              
2Department of Chemistry, University of Gdansk, Poland , ou_persistent22              

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Free keywords: pituitary; adenylate-cyclase-activating polypeptide; p-benzoylphenylalanine; photoaffinity labeling; peptide-hormone receptor; glycosylation
 Abstract: 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 (KA 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 W-glycosidase F1 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.

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Language(s): eng - English
 Dates: 1996-11-012004-07-161997-03-01
 Publication Status: Published in print
 Pages: 7
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1111/j.1432-1033.1997.00400.x
PMID: 9119005
 Degree: -

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Title: European Journal of Biochemistry
Source Genre: Journal
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Publ. Info: Berlin : Published by Springer-Verlag on behalf of the Federation of European Biochemical Societies
Pages: - Volume / Issue: 244 (2) Sequence Number: - Start / End Page: 400 - 406 Identifier: ISSN: 0014-2956
CoNE: https://pure.mpg.de/cone/journals/resource/111097776606040