Corticotropin-releasing factor binding to the anterior pituitary receptor is modulated by divalent cations and guanyl nucleotides.
Corticotropin-releasing factor binding to the anterior pituitary receptor is modulated by divalent cations and guanyl nucleotides.
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DOI：
10.1210/endo-118-3-1171
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发表时间：
1986-03
期刊：
影响因子：
4.8
通讯作者：
M. Perrin;Yaira Haas;J. Rivier;W. Vale
中科院分区：
文献类型：
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作者：
M. Perrin;Yaira Haas;J. Rivier;W. Vale
用放射性配体[Nle21， m-125I Tyr32]表征了绵羊CRF与牛垂体前膜的结合。二价阳离子Mg2+、Ca2+和Mn2+的毫摩尔浓度增加了CRF的特异性结合，而一价阳离子Na+、Ki+和Li+不影响CRF的特异性结合。特异性结合的增加不是由亲和力的改变引起的，而是由于高亲和力结合位点的明显增加。在10 mM Mg2+的存在下，CRF的结合是饱和的、特异性的和高亲和力的。在室温下，在最佳条件下，结合在70 min内达到平衡，稳定至少4 h，被多余的未标记肽可逆，Kd为1.3 nM(0.74-2.4)，总位点R度为90 fmol/mg蛋白（55-145）。对牛垂体前膜的亲和力与对大鼠垂体前膜匀浆的亲和力相同，且位点浓度相似。所选择的激动剂和拮抗剂在牛和大鼠系统中对CRF受体的相对结合亲和力相似，并且都与体外培养的大鼠垂体前叶细胞中分别刺激或拮抗ACTH释放的相对效力具有良好的相关性。不可水解的GTP类似物5'-鸟磺酰二磷酸[Gpp(NH)p]对结合的放射性配体产生剂量依赖性抑制，EC50约为0.5微米。在1微米Gpp(NH)p存在下，高亲和力的crf结合位点数量减少，但亲和力不变。在5微米Gpp(NH)p存在的情况下，没有检测到高亲和力结合，并且先前结合的放射配体的解离率增加。其他鸟嘌呤核苷酸，GTP和GDP，抑制结合但程度较轻，而GMP和（Bu）2cGMP无效。这种抑制作用对鸟腈核苷酸具有特异性。鉴于鸟苷核苷酸和二价阳离子对腺苷酸环化酶连接受体的作用，这些数据支持腺苷酸环化酶系统参与了CRF对垂体前叶的作用机制。
The binding of ovine CRF to bovine anterior pituitary membranes was characterized with the radioligand [Nle21, m-125I Tyr32]ovine CRF. The specific binding of CRF was increased by millimolar concentrations of the divalent cations Mg2+, Ca2+, and Mn2+, but was unaffected by the monovalent cations Na+, Ki+, and Li+. The increase in specific binding was not caused by a change in the affinity, but resulted from an apparent increased number of high affinity binding sites. In the presence of 10 mM Mg2+, CRF binding was saturable, specific, and of high affinity. At room temperature, under optimum conditions, binding reached equilibrium within 70 min, remained stable for at least 4 h, was reversible by excess unlabeled peptide, and was characterized by a Kd of 1.3 nM (0.74-2.4) and a total number of sites, R degree, of 90 fmol/mg protein (55-145). The affinity for the bovine membranes was the same as that for rat anterior pituitary membrane homogenates, and the concentrations of sites were similar. The relative binding affinities for the CRF receptor of selected agonists and antagonists in the bovine and rat systems were similar, and both showed good correlation with the relative in vitro potencies at stimulating or antagonizing, respectively, ACTH release from cultured rat anterior pituitary cells. The nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p], caused a dose-dependent inhibition of bound radioligand, with an EC50 of about 0.5 microM. In the presence of 1 microM Gpp(NH)p, the number of high affinity CRF-binding sites was reduced, but the affinity was unchanged. In the presence of 5 microM Gpp(NH)p, there was no detectable high affinity binding, and the rate of dissociation of previously bound radioligand was increased. The other guanyl nucleotides, GTP and GDP, inhibited binding but to a lesser extent, whereas GMP and (Bu)2cGMP were ineffective. The inhibition was specific for guanyl nucleotides. In view of the established effects of guanyl nucleotides and divalent cations on adenylate cyclase-linked receptors, these data support the involvement of the adenylate cyclase system in the mechanism of action of CRF on the anterior pituitary.