title: Identification by photoaffinity labeling of the extracellular N-terminal domain of PAC1 receptor as the major binding site for PACAP.
abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts many crucial biological functions through the interaction with its specific PAC1 receptor (PAC1-R), a class B G protein-coupled receptor (GPCR). To identify the binding sites of PACAP in the PAC1-R, three peptide derivatives containing a photoreactive p-benzoyl-phenylalanine (Bpa) residue were developed. These photosensitive PACAP analogs were fully biologically active and competent to displace radiolabeled Ac-PACAP27 from the PAC1-R. Subsequently, the (125)I-labeled photoprobes were used to anchor the PAC1-R expressed in Chinese hamster ovary cells. Photolabeling led to the formation of two protein complexes of 76 and 67 kDa, representing different glycosylated forms of the receptor. Proteinase and chemical cleavages of the peptide-receptor complexes revealed that (125)I[Bpa(0), Nle(17)]PACAP27, (125)I[Bpa(6), Nle(17)]PACAP27 and (125)I[Nle(17), Bpa(22)]PACAP27 covalently labeled the Ser(98) - Met(111) segment, the Ser(124) - Glu(125) dipeptide and the Ser(141) - Met(172) fragment, respectively. Taking into account the topology of the PAC1-R, these segments are mainly located within the extracellular N-terminal domain, indicating that this PAC1-R domain is the major binding site of PACAP27. The present study constitutes the first characterization of the binding domains of PACAP to its specific receptor and suggests heterogeneity within the binding mode of peptide ligands to class B GPCRs.
authors.affiliation: Laboratoire d'Études Moléculaires et Pharmacologiques des Peptides (LEMPP), INRS-Institut Armand-Frappier, Institut National de la Recherche Scientifique, Ville de Laval, Qc, Canada.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Dejda
authors.forename: Steve
authors.initials: S
authors.lastname: Bourgault
authors.forename: Ngoc Duc
authors.initials: ND
authors.lastname: Doan
authors.forename: Myriam
authors.initials: M
authors.lastname: Létourneau
authors.forename: Alain
authors.initials: A
authors.lastname: Couvineau
authors.forename: Hubert
authors.initials: H
authors.lastname: Vaudry
authors.forename: David
authors.initials: D
authors.lastname: Vaudry
authors.forename: Alain
authors.initials: A
authors.lastname: Fournier
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001577:Benzophenones / Q000032:analysis; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006031:Glycosylation; D006801:Humans; D007456:Iodine Isotopes / Q000737:chemistry; D008969:Molecular Sequence Data; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000032:analysis; D019920:Photoaffinity Labels / Q000737:chemistry; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C488060:4-benzoylphenylalanine; D001577:Benzophenones; D007456:Iodine Isotopes; D019920:Photoaffinity Labels; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D010649:Phenylalanine