Grigoriadis D E, Zaczek R, Pearsall D M, De Souza E B
Laboratory of Neurobiology, Neuroscience Branch, National Institute on Drug Abuse, Baltimore, Maryland 21224.
Endocrinology. 1989 Dec;125(6):3068-77. doi: 10.1210/endo-125-6-3068.
The binding characteristics of CRF receptors in rat frontal cerebral cortex membranes solubilized in 1% digitonin were determined. The binding of [125I]Tyro-ovine CRF ([125I]oCRF) to solubilized membrane proteins was dependent on incubation time, temperature, and protein concentration, was saturable and of high affinity, and was absent in boiled tissue. The solubilized receptors retained their high affinity for [125I] oCRF in the solubilized state, exhibiting a dissociation constant (KD) of approximately 200 pM, as determined by direct binding saturation isotherms. Solubilized CRF receptors maintained the rank order of potencies for various related and unrelated CRF peptides characteristic of the membrane CRF receptor: rat/human CRF congruent to ovine CRF congruent to Nle21,38-rat CRF greater than alpha-helical oCRF-(9-41) greater than oCRF-(7-41) much greater than vasoactive intestinal peptide, arginine vasopressin, or the substance-P antagonist. Furthermore, the absolute potencies (Ki values) for the various CRF-related peptides in solubilized receptors were almost identical to those observed in the membrane preparations, indicating that the CRF receptor retained its high affinity binding capacity in the digitonin-solubilized state. Chemical affinity cross-linking of digitonin-solubilized rat cortical membrane proteins revealed a specifically labeled protein with an apparent mol wt of 58,000 which was similar to the labeled protein in native membrane homogenates. Although solubilized CRF receptors retained their high affinity for agonists, their sensitivity for guanine nucleotide was lost. Size exclusion chromatography substantiated these results, demonstrating that in the presence or absence of guanine nucleotides, [125I]oCRF labeled the same size receptor complex. These data suggest that either the guanine nucleotide-binding protein (Ns) is tightly associated with the CRF receptor after solubilization and is insensitive to guanine nucleotides, or that high affinity binding for soluble CRF receptors is not dependent on the coupling of a guanine nucleotide-binding protein. The solubilization of CRF receptors from membranes in digitonin should allow for the more complete molecular and functional characterization of CRF-mediated events and purification of the receptor.
测定了溶解于1%洋地黄皂苷中的大鼠额叶大脑皮质膜中促肾上腺皮质激素释放因子（CRF）受体的结合特性。[125I]酪氨酸-羊CRF（[125I]oCRF）与溶解的膜蛋白的结合取决于孵育时间、温度和蛋白浓度，具有饱和性和高亲和力，且在煮沸的组织中不存在这种结合。溶解的受体在溶解状态下对[125I]oCRF仍保持高亲和力，通过直接结合饱和等温线测定，其解离常数（KD）约为200 pM。溶解的CRF受体维持了膜CRF受体对各种相关和不相关CRF肽的效价顺序：大鼠/人CRF等同于羊CRF等同于Nle21,38-大鼠CRF大于α-螺旋oCRF-(9-41)大于oCRF-(7-41)远大于血管活性肠肽、精氨酸加压素或P物质拮抗剂。此外，溶解受体中各种CRF相关肽的绝对效价（Ki值）与膜制剂中观察到的几乎相同，表明CRF受体在洋地黄皂苷溶解状态下仍保持其高亲和力结合能力。对洋地黄皂苷溶解的大鼠皮质膜蛋白进行化学亲和交联，发现一种表观分子量为58,000的特异性标记蛋白，这与天然膜匀浆中的标记蛋白相似。虽然溶解的CRF受体对激动剂仍保持高亲和力，但其对鸟嘌呤核苷酸的敏感性丧失。尺寸排阻色谱证实了这些结果，表明在存在或不存在鸟嘌呤核苷酸的情况下，[125I]oCRF标记相同大小的受体复合物。这些数据表明，要么鸟嘌呤核苷酸结合蛋白（Ns）在溶解后与CRF受体紧密结合且对鸟嘌呤核苷酸不敏感，要么可溶性CRF受体的高亲和力结合不依赖于鸟嘌呤核苷酸结合蛋白的偶联。从膜中用洋地黄皂苷溶解CRF受体应有助于更全面地对CRF介导的事件进行分子和功能表征以及受体的纯化。
