Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III

Whereas numerous mutations of the human lutropin receptor (hLHR) and human TSH receptor (hTSHR) have been shown to cause constitutive activation of th ese receptors, it has been suggested that either the hFSHR as a whole, or t he i3/TM VI region of the hFSHR, is less susceptible to mutation-induced co nstitutive activation. However, as shown herein, substitution of a highly c onserved leucine residue in transmembrane III (TM III) of the I hFSHR (Leu III.18) with arginine causes a B-fold increase in basal cAMP in transfected cells, consistent with a strong constitutive activation of the hFSHR. Inte restingly, this mutant is unresponsive to further hormonal stimulation. Sub stitutions of hFSHR(L460) with lysine, alanine, or aspartate show that only arginine causes constitutive activation. However, all result in decreased FSH responsiveness, suggesting a role for L460 in FSH-stimulated cAMP produ ction by the hFSHR. Because Leu III.18 is highly conserved in rhodopsin-lik e G protein-coupled receptors (GPCRs), we tested the effects of substitutio n of the comparable leucine in the human beta(2)-adrenergic receptor (h bet a(2)-AR). Substitution of L124 in the h beta(2)-AR with arginine, lysine, o r alanine resulted in constitutive activation as evidenced by increased bas al levels of cAMP that could be attenuated by an inverse agonist. In all ca ses, isoproterenol-stimulated cAMP was unaffected. Taken altogether, our da ta support a model whereby Leu III.18 may play a general role in GPCRs by s tabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu III.18 as well as the introduction of a specific r esidue that serves to stabilize the active state of the receptor.