Characterization of site-directed antibodies to the LH receptor in functionally active gonadal cells and their differential effects on LH-stimulated signal transduction in Leydig tumour (MA10) cells

Abstract

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 μg/ml)) also inhibited 125I-hCG binding to a greater extent than the C-terminal antibody (antibody 3 (10 μg/ml)). Antibody 1 (1 and 10 μg/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 μg/ml), whereas at the concentrations tested antibody 3 exerted no greater inhibition than that yielded by pre-immune IgG. At 0.1 μg/ml antibody 1 significantly inhibited and at 10 μg/ml completely inhibited LH-stimulated cAMP and progesterone production by MA10 cells. With antibody 2, 10 μg/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 gondal cells.