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  • Title: Mutational analyses of the extracellular domain of the full-length lutropin/choriogonadotropin receptor suggest leucine-rich repeats 1-6 are involved in hormone binding.
    Author: Thomas D, Rozell TG, Liu X, Segaloff DL.
    Journal: Mol Endocrinol; 1996 Jun; 10(6):760-8. PubMed ID: 8776736.
    Abstract:
    Previous studies have demonstrated that the amino-terminal extracellular domain of the lutropin/choriogonadotropin receptor (LHR) is sufficient for conferring high affinity binding and binding specificity. The present study was undertaken to further delineate those regions involved in hormone binding. Since the extracellular domains of the gonadotropin receptors are defined by multiple leucine-rich repeat motifs, LHR deletion mutants were constructed in which individual or multiple leucine-rich repeats were deleted from the full-length receptor. These mutants were transiently expressed in mammalian 293 cells, assayed for protein expression by Western blotting of cell extracts, and then tested for hormone binding in both intact cells and detergent-solubilized cell extracts. Western blot analyses confirmed the expression of all LHR deletion mutant proteins in the transfected cells. Although human (h) CG binding activity was not detected for any of the mutants when intact cells were assayed, mutants in which leucine-rich repeats 9-14 were collectively deleted, or repeats 7 or 8 were individually deleted, expressed binding activity when the cells were first solubilized in detergent. Of significance, rat (r) LHR(delta LRR 9-14) exhibited high affinity hCG and hLH binding, comparable to wild type rLHR. Detergent extracts from cells expressing rLHR(delta LRR 8) and rLHR(delta LRR 7) bound hCG and hLH, albeit with reduced affinities compared with the wild type receptor. All three of these deletion mutants, rLHR(delta LRR 9-14), rLHR(delta LRR 8), and rLHR(delta LRR 7), exhibited normal binding specificity as they did not bind hFSH even at very high concentrations. Thus, deletion of these regions in the carboxyl portion of the extracellular domain of the LHR did not remove any potentially inhibitory elements that would normally prevent hFSH binding. Deletion of either the 11 amino-terminal acids before LRR 1 or LRRs 1, 2, 3, 4, 5, or 6 individually from the full-length rLHR resulted in the total absence of detectable hCG binding activity in detergent-solubilized extracts, in spite of stable protein expression. These results suggest that the amino terminus and LRRs 1-6 are absolutely essential for gonadotropin binding to the rLHR.
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