Provow S, Veliçelebi G
Endocrinology. 1987 Jun;120(6):2442-52. doi: 10.1210/endo-120-6-2442.
Receptors for vasoactive intestinal peptide (VIP) were characterized in rat lung membranes by binding and covalent cross-linking of [125I]VIP using ethylene glycolbis-(succinimidylsuccinate). Binding studies indicated the presence of two classes of binding sites for VIP in rat lung membranes: 0.28 +/- 0.11 pmol/mg protein high affinity receptors (Kd = 79.2 +/- 26.4 pM) and 3.3 +/- 0.9 pmol/mg protein lower affinity receptors (Kd = 4.8 +/- 2.1 nM). Furthermore, binding of [125I]VIP to rat lung receptors was inhibited by micromolar concentrations of GTP analogs, guanosine-5'-O-(3-thiotriphosphate) GTP gamma S), and guanylylimidodiphosphate, suggesting that VIP receptors in rat lung membranes were tightly coupled to the guanine nucleotide regulatory protein (Ns). Scatchard analysis of VIP binding in the presence of GTP gamma S revealed selective inhibition of binding to high affinity sites. A 58K band was specifically labeled when membranes covalently labeled with [125I]VIP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The apparent size of this species was not altered when sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis was carried out in the absence of reducing agent. Unlabeled VIP inhibited the labeling, with an IC50 of about 1 nM. A related peptide, GH-releasing factor-(1-40)OH, exhibited a much lower binding affinity, and two unrelated peptides, insulin and atrial natriuretic factor, did not inhibit labeling of the 58K species, even at micromolar concentrations. Labeling of the 58K species was inhibited in a GTP gamma S-dependent manner, suggesting the involvement of this species in the coupling to Ns. These data collectively indicated that the 58K species was a VIP-binding unit of VIP receptors in rat lung membranes. Several nondenaturing detergents were tested for extraction of labeled receptors from the membrane; the best extraction was obtained using 1% n-octyl-beta-D-glucopyranoside.
通过使用乙二醇双（琥珀酰亚胺琥珀酸酯）对[125I]血管活性肠肽（VIP）进行结合和共价交联，对大鼠肺膜中的VIP受体进行了表征。结合研究表明，大鼠肺膜中存在两类VIP结合位点：0.28±0.11 pmol/mg蛋白质的高亲和力受体（Kd = 79.2±26.4 pM）和3.3±0.9 pmol/mg蛋白质的低亲和力受体（Kd = 4.8±2.1 nM）。此外，微摩尔浓度的GTP类似物、鸟苷-5'-O-（3-硫代三磷酸）（GTPγS）和鸟苷酰亚胺二磷酸可抑制[125I]VIP与大鼠肺受体的结合，这表明大鼠肺膜中的VIP受体与鸟嘌呤核苷酸调节蛋白（Ns）紧密偶联。在存在GTPγS的情况下对VIP结合进行Scatchard分析，结果显示对高亲和力位点的结合有选择性抑制。当在还原条件下通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析用[125I]VIP共价标记的膜时，一条58K的条带被特异性标记。在不存在还原剂的情况下进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析时，该条带的表观大小没有改变。未标记的VIP可抑制标记，IC50约为1 nM。一种相关肽，生长激素释放因子-（1-40）OH，表现出低得多的结合亲和力，而两种不相关的肽，胰岛素和心房利钠因子，即使在微摩尔浓度下也不抑制58K条带的标记。58K条带的标记以GTPγS依赖的方式被抑制，这表明该条带参与了与Ns的偶联。这些数据共同表明，58K条带是大鼠肺膜中VIP受体的VIP结合单元。测试了几种非变性去污剂从膜中提取标记受体的效果；使用1%正辛基-β-D-吡喃葡萄糖苷获得了最佳提取效果。
