Contribution of the extracellular domain to agonist-induced and constitutive LH receptor activation

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Glycoprotein hormone receptors like the LH and FSH receptor are G-protein-coupled receptors characterized by a large N-terminally extracellular domain. While it is well established that the ectodomain is responsible for specific hormone binding, the activation mechanism is still not understood. To investigate the functional role of the ectodomain, we N-terminally deleted various portions of the LH receptor ectodomain. Upon addition of amino acids 1–39 of the V2 vasopressin receptor to the N-termini of the truncated LHRs, these mutants transiently expressed in COS-7 cells revealed cell surface targeting comparable to the wildtype receptor. However, mutant LHRs did not display constitutive cAMP formation, nor revealed activation by hCG. To exclude that this lack to hCG response was due to negligible agonist affinity, single-chain hCG was fused to the wildtype or truncated receptors. While the wildtype LHR fused to hCG exhibited constitutive cAMP formation after transient expression, and could further be stimulated by exogenous hormone, none of the truncated receptors displayed elevated basal activity indicating that hCG does not functionally interact with the heptahelical LHR portion and that the entire intact ectodomain is required for hormone-induced receptor activation. To examine if truncated LHRs are fundamentally misfolded and principally unable to interact with Gs, several naturally occurring activating mutations were introduced into the transmembrane helices of a truncated LHR. While all mutations induced constitutive activity in the wildtype LHR, only one mutation evoked agonist-independent cAMP formation via the truncation mutant. The activity of most non-active mutations could be recovered by N-terminal linking of the TSHR or FSHR ectodomain to the transmembrane helices of the LHR, whereas linking of the hLGR7 ectodomain was ineffective. These data highlight an as yet unappreciated structural role of the LHR ectodomain for the stabilization of a distinct conformation of the heptahelical portion poised for activation by agonist or activating mutations.