Marchis-Mouren G, Martin J M, Luis J, el Battari A, Muller J M, Marvaldi J, Pichon J
Institut de Chimie Biologique, CNRS UA202, Université d'Aix-Marseille, France.
Biochimie. 1988 May;70(5):663-71. doi: 10.1016/0300-9084(88)90251-9.
HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
HT 29是一种源自人结肠腺癌的细胞系，对血管活性肠肽（VIP）高度敏感，表现为细胞内cAMP增加100多倍（Ka = 0.3 nM）、蛋白激酶A受到刺激（Ka = 0.1 nM）以及低Km的cAMP磷酸二酯酶被激活（Ka = 40 nM）。值得注意的是，腺苷酸环化酶、cAMP依赖性激酶和cAMP特异性磷酸二酯酶以一种相继的方式被激活。用[125I]标记的VIP进行的结合研究表明存在一个高亲和力位点，其Kd值（0.5 nM）接近这三种酶的激活常数（Ka）值。通过免疫学和化学方法研究了VIP受体的分子结构。一种单克隆抗体（mAb 109 - 10 - 16）可部分降低VIP与其受体的结合，从而鉴定出分子量为53,000和48 - 49,000的多肽。通过四种双功能试剂对完整的HT 29细胞进行共价交联，更精确地鉴定了VIP受体的蛋白质成分：二硫代双（琥珀酰亚胺丙酸酯）及其不可裂解的类似物辛二酸双琥珀酰亚胺酯、可光活化的叠氮苯乙二醛和二甲基庚二酸亚胺酯。SDS - 聚丙烯酰胺凝胶电泳分析表明，无论使用哪种交联剂，都有一条主要的分子量为67,000的条带。在使用HT 29细胞粗膜组分的实验中也发现了相同的条带和一个分子量为49,000的条带。假设每条多肽连接一个VIP分子（分子量 = 3326），这些观察结果表明分子量为64,000的物质属于VIP特异性质膜受体。该蛋白质含有一个分子量为20,000的富含N - 连接唾液酸的寡糖部分。（摘要截于250字）
