Zhang S B, Dattatreyamurty B, Reichert L E
Department of Biochemistry, Albany Medical College, New York 12208.
Mol Endocrinol. 1988 Feb;2(2):148-58. doi: 10.1210/mend-2-2-148.
In a previous study we reported that FSH receptors in bovine testes membranes are physically and functionally associated with a guanine nucleotide-binding protein (N protein). In this study we examined the mechanism whereby GTP binding to N protein regulates FSH binding to its receptors. Binding of FSH to receptors decreased in the presence of GTP in a dose-dependent and noncompetitive manner. This effect did not require the presence of Mg+2 and is in contrast to the reported requirement for Mg+2 for GTP effects on human CG binding to ovarian receptors. Equilibrium binding experiments indicated that decreased hormone binding in the presence of GTP was not due to a decrease in the number of FSH receptors per se; rather, the altered binding isotherm was the result of a decrease in affinity of receptors for FSH. Moreover, the dissociation of [125I]human FSH from preformed FSH-receptor complex was rapid in onset and significantly accelerated in the presence of GTP. In a series of nucleotides, GTP was most effective in causing this effect. Evidently, occupancy of GTP binding sites on the N protein, including low affinity and high capacity sites, is necessary for GTP regulation of FSH binding to receptors. The fact that pretreatment of bovine testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminates the GTP effect on FSH binding to its receptors suggests that the GTP regulatory binding protein mediating the GTP regulation of FSH binding is probably Ns and not Ni. Further characterization of FSH receptor sensitivity to GTP, however, indicated that the N protein involved does not exhibit all of the characteristics reported for Ns. For example, the affinity of GTP for N protein is relatively low even under conditions where GTP hydrolysis has a minimal effect in reducing the total concentration of GTP. Also, the absence of a requirement for Mg+2 in high affinity FSH receptor-N protein coupling is different from the requirement for Mg+2 seen with the beta-adrenergic receptor and Ns. Moreover, the N protein which mediates GTP regulation of FSH-receptor binding appears to be relatively insensitive to N-ethylmaleimide, unlike the N-ethylmaleimide sensitivity of the turkey erythrocyte Ns. These results suggest that differences may exist in the structure-function features of GTP regulatory binding protein associated with different types of hormone ligands and receptors.
在先前的一项研究中，我们报道了牛睾丸膜中的促卵泡激素（FSH）受体在物理和功能上与一种鸟嘌呤核苷酸结合蛋白（N蛋白）相关联。在本研究中，我们研究了GTP与N蛋白结合调节FSH与其受体结合的机制。在GTP存在的情况下，FSH与受体的结合以剂量依赖性和非竞争性方式减少。这种效应不需要Mg+2的存在，这与报道的GTP对人绒毛膜促性腺激素（hCG）与卵巢受体结合的效应需要Mg+2的情况相反。平衡结合实验表明，在GTP存在下激素结合减少并非由于FSH受体数量本身的减少；相反，结合等温线的改变是受体对FSH亲和力降低的结果。此外，[125I]人FSH从预先形成的FSH-受体复合物中的解离起效迅速，并且在GTP存在下显著加速。在一系列核苷酸中，GTP在引起这种效应方面最有效。显然，N蛋白上GTP结合位点的占据，包括低亲和力和高容量位点，对于GTP调节FSH与受体的结合是必要的。用霍乱毒素加NAD预处理牛睾丸膜，但不用百日咳毒素加NAD预处理，可消除GTP对FSH与其受体结合的效应，这一事实表明介导GTP对FSH结合调节的GTP调节结合蛋白可能是Ns而不是Ni。然而，对FSH受体对GTP敏感性的进一步表征表明，所涉及的N蛋白并不表现出报道的Ns的所有特征。例如，即使在GTP水解对降低GTP总浓度影响最小的条件下，GTP对N蛋白的亲和力也相对较低。此外，高亲和力FSH受体-N蛋白偶联中不需要Mg+2与β-肾上腺素能受体和Ns对Mg+2的需求不同。而且，介导GTP对FSH-受体结合调节的N蛋白似乎对N-乙基马来酰亚胺相对不敏感，这与火鸡红细胞Ns对N-乙基马来酰亚胺的敏感性不同。这些结果表明，与不同类型的激素配体和受体相关的GTP调节结合蛋白的结构-功能特征可能存在差异。
