Pallikaros Z, Schulster D, Baldwin S A, Helliwell R J, Michael A E, Cooke B A
Department of Biochemistry and Molecular Biology, Royal Free Hospital School of Medicine, University of London, UK.
Mol Cell Endocrinol. 1995 Oct 30;114(1-2):57-68. doi: 10.1016/0303-7207(95)03642-k.
In this study site-directed antibodies have been used to investigate the structure/activity relationships of the LH receptor in functionally active gonadal cells. Polyclonal antibodies were raised in rabbits against synthetic peptides corresponding to regions within both the extracellular N-terminal domain (antibodies 1 and 2 against residues 48-65 and 187-206, respectively) and the cytoplasmic C-terminal domain (antibody 3 against residues 622-636) of the LH receptor. Following affinity purification by chromatography on columns of immobilised peptides the antibodies were demonstrated to be peptide specific both by ELISA and by dot-blotting assays. On Western blots of membranes proteins prepared from superovulated rat ovaries, mouse Leydig tumour (MA10) cells, and rat testes, all three antibodies recognised a single broad band of apparent M(r) 95,000-100,000 corresponding to the putative LH receptor. The protein of apparent M(r) 95,000-100,000 also bound 125I-hCG on ligand blots, and binding was displaced by excess unlabelled hCG. The binding of 125I-hCG in the ligand blots was completely inhibited by excess unlabelled hCG. The two N-terminal antibodies (antibodies 1 and 2 (10 micrograms/ml)) also inhibited 125I-hCG binding to a greater extent than the C-terminal antibody (antibody 3 (10 micrograms/ml)). Antibody 1 (1 and 10 micrograms/ml) also potently inhibited the binding of 125I-hCG to MA10 cells. A lesser but still significant inhibition of binding was produced by antibody 2 (with 10 micrograms/ml), whereas at the concentrations tested antibody 3 exerted no greater inhibition than that yielded by pre-immune IgG. At 0.1 micrograms/ml antibody 1 significantly inhibited and at 10 micrograms/ml completely inhibited LH-stimulated cAMP and progesterone production by MA10 cells. With antibody 2, 10 micrograms/ml was required to give a significant inhibition, whereas neither antibody 3 nor pre-immune IgG had a significant effect. The antibodies had no effect on cAMP or progesterone production when added to the MA10 cells in the absence of LH. These results indicate that binding of antibody 1 and, to a lesser extent, antibody 2 interferes with ligand binding which consequently affects signal transduction. In view of the ability of the antibodies to recognise the LH receptors both in the ovary and the testis and in more than one rodent species, and their greater apparent potency than previously available antisera, the anti-peptide antibodies raised in the present study will therefore be useful to study LH receptors in normal, functionally active gonadal cells.
在本研究中，已使用定点抗体来研究功能活跃的性腺细胞中促黄体生成素（LH）受体的结构/活性关系。针对与LH受体细胞外N端结构域（分别针对第48 - 65位和第187 - 206位残基的抗体1和抗体2）以及细胞质C端结构域（针对第622 - 636位残基的抗体3）内区域相对应的合成肽，在兔体内制备多克隆抗体。通过固定化肽柱进行亲和纯化后，通过酶联免疫吸附测定（ELISA）和斑点印迹分析证明抗体具有肽特异性。在由超排卵大鼠卵巢、小鼠睾丸间质细胞瘤（MA10）细胞和大鼠睾丸制备的膜蛋白的蛋白质印迹（Western blot）上，所有三种抗体均识别出一条单一的宽条带，其表观相对分子质量（M(r)）为95,000 - 100,000，对应于假定的LH受体。表观M(r)为95,000 - 100,000的蛋白质在配体印迹上也结合125I - 人绒毛膜促性腺激素（hCG），并且结合被过量未标记的hCG取代。配体印迹中125I - hCG的结合被过量未标记的hCG完全抑制。两种N端抗体（抗体1和抗体2（10微克/毫升））比C端抗体（抗体3（10微克/毫升））在更大程度上抑制125I - hCG结合。抗体1（1和10微克/毫升）也有效抑制125I - hCG与MA10细胞的结合。抗体2（10微克/毫升）产生较小但仍显著的结合抑制，而在所测试的浓度下，抗体3产生的抑制作用不超过免疫前IgG。在0.1微克/毫升时，抗体1显著抑制，在10微克/毫升时完全抑制MA10细胞中LH刺激的环磷酸腺苷（cAMP）和孕酮的产生。对于抗体2，需要10微克/毫升才能产生显著抑制，而抗体3和免疫前IgG均无显著作用。当在不存在LH的情况下添加到MA10细胞中时，这些抗体对cAMP或孕酮的产生没有影响。这些结果表明，抗体1的结合以及在较小程度上抗体2的结合会干扰配体结合，从而影响信号转导。鉴于这些抗体能够识别卵巢和睾丸以及多种啮齿动物物种中的LH受体，并且它们的表观效力比以前可用的抗血清更高，因此本研究中制备的抗肽抗体将有助于研究正常功能活跃的性腺细胞中的LH受体。
