Laboratoire d'Études Moléculaires et Pharmacologiques des Peptides (LEMPP), INRS-Institut Armand-Frappier, Institut National de la Recherche Scientifique, Ville de Laval, Qc, Canada.
Biochimie. 2011 Apr;93(4):669-77. doi: 10.1016/j.biochi.2010.12.010. Epub 2010 Dec 23.
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.
垂体腺苷酸环化酶激活肽（PACAP）通过与其特异性 PAC1 受体（PAC1-R）相互作用发挥许多重要的生物学功能，PAC1-R 是一种 B 类 G 蛋白偶联受体（GPCR）。为了确定 PACAP 在 PAC1-R 中的结合位点，开发了三种含有光反应性对苯甲酰苯丙氨酸（Bpa）残基的肽衍生物。这些光敏 PACAP 类似物具有完全的生物学活性，能够从 PAC1-R 上置换放射性标记的 Ac-PACAP27。随后，使用（125）I 标记的探针来锚定在中国仓鼠卵巢细胞中表达的 PAC1-R。光标记导致形成 76 和 67 kDa 的两种蛋白质复合物，代表受体的不同糖基化形式。蛋白酶和化学裂解肽-受体复合物表明，（125）I[Bpa(0), Nle(17)]PACAP27、（125）I[Bpa(6), Nle(17)]PACAP27 和（125）I[Nle(17), Bpa(22)]PACAP27 分别共价标记 Ser(98)-Met(111) 片段、Ser(124)-Glu(125) 二肽和 Ser(141)-Met(172) 片段。考虑到 PAC1-R 的拓扑结构，这些片段主要位于细胞外 N 端结构域内，表明该 PAC1-R 结构域是 PACAP27 的主要结合位点。本研究首次对 PACAP 与其特异性受体的结合域进行了特征描述，并表明了肽配体与 B 类 GPCR 结合模式的异质性。
