An intracellular loop (IL2) residue confers different basal constitutive activities to the human lutropin receptor and human thyrotropin receptor through structural communication between IL2 and helix 6, via helix 3

The human lutropin receptor (hLHR) and human TSH receptor (hTSHR) are G protein-coupled receptors that play key roles in reproductive and thyroid physiology, respectively. We show using a quantitative assessment of cAMP production as a function of cell surface receptor expression that the hTSHR possesses greater basal constitutive activity than the hLHR. Further studies were undertaken to test the hypothesis that different potential Gs-coupling motifs identified in IL2 of the hTSHR and hLHR contribute to their different basal constitutive activities. Although mutating the receptors to interchange their potential Gs-coupling motifs reversed their relative activities, we show this to be due to the swapping of one IL2 residue (Q476 in the hLHR; R531 in the hTSHR). Molecular dynamics simulations show that the effect of the hLHR(Q476R) mutation, switching the structural features of the hLHR toward those of the hTSHR, is greater than the switching effect of the hTSHR(R531Q) mutant toward the hLHR. The structural model of the hLHR(Q476R) mutant can be considered as a hybrid of wild-type (wt) hTSHR and constitutively active mutant hLHR forms. In this hLHR(Q476R) mutant, IL2 adopts a structure similar to IL2 of the wt hTSHR, but it shares with the hLHR constitutively active mutant the solvent exposure and the reciprocal arrangement of helices 3, 5, and 6, including the weakening of the wt native R3.50-D6.30 interaction. Our results suggest a H3-mediated structural connection between IL2 and the cytosolic extension of H6. Thus, IL2 contributes significantly to the inactive and active state ensembles of these G protein-coupled receptors.