Search

The human LH receptor (hLHR) consists of a heptahelical segment prototypical of G protein-coupled receptors and a large ectodomain. The binding of hormone to the ectodomain or the presence of activating mutations stabilize the hLHR in an active conformation. Although it has been inferred that activation of the hLHR involves conformational alterations, direct evidence supporting this has not been reported. We have addressed this issue by comparing the protease sensitivity of the wild-type hLHR as compared to three activating mutants of the hLHR. Our data demonstrate that the ectodomains of the activating hLHR mutants are more susceptible to proteolysis as compared to the wild-type hLHR. As such, they provide the first experimental data demonstrating that the conformations of the active and inactive states of the hLHR are distinct and suggest that activation of the hLHR (at least as induced by mutations) involves a tighter interaction of the ectodomain with the heptahelical segment of the receptor as opposed to a release of the ectodomain from the heptahelical segment.

The lutropin/choriogonadotropin receptor (LHR) is a G protein-coupled receptor central to reproductive physiology. In addition to the complex structure formed by seven membrane-spanning helices, this receptor also contains a large amino-terminal domain whose tertiary structure is unknown. Many mutations of this receptor result in partial or complete intracellular retention of the mutant, regardless of whether the mutations are located in the extracellular domain, interhelical loops, transmembrane helices, or cytoplasmic tail. Nonetheless, as long as a mutation has not disturbed a hormone binding site, the intracellularly trapped mutant retains high affinity binding for human CG (hCG), suggesting that it is not extremely misfolded. Because temperature is known to affect protein folding, we examined the effects of reduced temperature on cell surface expression of intracellularly retained mutants of the LHR. Our studies examined three different 293 cells lines, each stably expressing a different LHR mutant t...

It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.

Previous results from this laboratory suggested that the same active conformation of the lutropin/choriogonadotropin receptor (LHR) is involved in the stimulation of G proteins and in triggering the internalization of the bound agonist. We have now analyzed two naturally occurring, constitutively active mutants of the human LHR. These mutations were introduced into the rat LHR (rLHR) and are designated L435R and D556Y. Cells expressing rLHR-D556Y bind human choriogonadotropin (hCG) with normal affinity, exhibit a 25-fold increase in basal cAMP and respond to hCG with a normal increase in cAMP accumulation. This mutation does not affect the internalization of the free receptor, but it enhances the internalization of the agonist-occupied receptors approximately 3-fold. Cells expressing rLHR-L435R also bind hCG with normal affinity, exhibit a 47-fold increase in basal cAMP, and do not respond to hCG with a further increase in cAMP accumulation. This mutation enhances the internalization of the free and agonist-occupied receptors approximately 2- and approximately 17-fold, respectively. We conclude that the state of activation of the rLHR can modulate its basal and/or agonist-stimulated internalization. Since the internalization of hCG is involved in the termination of hCG actions, we suggest that the lack of responsiveness detected in cells expressing rLHR-L435R is due to the fast rate of internalization of the bound hCG. The finding that membranes expressing rLHR-L435R (a system where internalization does not occur) respond to hCG with an increase in adenylyl cyclase activity supports this suggestion.

The LH/CG receptor (LHR) is a member of the family of G protein-coupled receptors and activates Gs when stimulated by LH or CG. Studies from other G protein-coupled receptors have implicated the carboxyl-terminal region of the third intracellular loop as being involved in the activation of G proteins. It has been suggested that the potential ability of this region to form an amphiphilic helix, with positively charged residues aligned to one face, may be important for this biological activity. To test whether the positively charged lysine residues, and thus an amphiphilic helix, in the carboxyl terminal region of the rat LHR (rLHR) are indeed important in the activation of Gs by the rLHR, a mutant rLHR was constructed in which lysines 541, 544, and 557 were simultaneously substituted with alanines. Clonal 293 cells expressing comparable numbers of cell-surface recombinant wild type rLHR or rLHR(K541,544,547A) were generated. Cells expressing the mutant receptor-bound human CG (hCG) with the same high affinity as those expressing the wild type rLHR. Since the numbers of receptors and binding affinities between the two cell lines were comparable, any changes in basal or hCG stimulated cAMP production could readily be interpreted as an alteration in the mutant receptor's ability to activate Gs. It was found, however, that basal cAMP production, the concentration of hCG required to elicit half-maximal cAMP production, and the maximal levels of cAMP produced in response to hCG were all unchanged in cells expressing rLHR(K541,544,547A).(ABSTRACT TRUNCATED AT 250 WORDS)