Characterization of constitutive activation and inactivation mutants in conserved regions of the eel luteinizing hormone receptor

Abstract Background: We analyzed signal transduction of three constitutively activating mutants (designated M410T, L469R, and D590Y) and two inactivating mutants (D383N and Y546F) of the eel luteinizing hormone receptor (LHR), known to be naturally occurring in humans as LHR. The objective of the present study is to assess the functional effects of these mutations in signal transduction. Methods: Site-directed mutant receptors were transiently expressed in CHO-K1 cells and cAMP accumulation, stimulated by recombinant eelLH (rec-eelLH), was measured by homogeneous time-resolved fluorescence (HTRF) assays. Results: The cAMP response in cells expressing the wild-type eel LHR (eLHR-WT) increased in a dose-dependent manner with rec-eelLH ligand stimulation. Cells expressing the activating eelLHR mutants, M410T, L469R, and D590Y, exhibited a 4.0-, 19.1-, and 7.8-fold increase in the basal cAMP response, respectively. However, their maximal responses to agonist were approximately 73, 53, and 92%, respectively, of the maximal response of the LHR-WT. The L469R mutant exhibited a particularly marked increase in cAMP concentration in the absence of ligand. The inactivating mutations did not completely impair signal transduction. The maximal responses of the inactivating mutants, D383N and Y546F, were 32% and 24% of the LHR-WT, respectively. Conclusion: We report here the first characterization of activating and inactivating mutations in eelLHR. These results provide important data on the signal transduction of constitutively active and inactive eelLHR mutants. Keywords: eel LHR, constitutively activating/inactivating mutation