A unique constitutively activating mutation in third transmembrane helix of luteinizing hormone receptor causes sporadic male gonadotropin-independent precocious puberty

Several constitutively activating mutations have been demonstrated in the sixth transmembrane helix of the human LH receptor (hLHR) in boys with gonadotropin-independent precocious puberty. In the current study, we examined two unrelated Brazilian boys with gonadotropin-independent precocious puberty caused by two different heterozygous activating mutations of the hLHR. Direct sequencing of the entire exon 11 of the hLHR revealed a heterozygous substitution oft for G at nucleotide 1370, that converts Leu 457 to Arg in the third transmembrane helix of the hLHR in one affected boy. His biological parents had a normal hLHR gene sequence, establishing the sporadic nature of this novel Leu457Arg mutation. Human embryonic 293 cells expressing hLHR mutant (L457R) or hLHR wild-type bound CG with high affinity. However, cells expressing hLHR(L457R) exhibited significantly higher basal levels of cAMP (7- to 14-fold) than cells expressing the wild-type receptor, indicating constitutive activation of hLHR(L457R). Basal levels of cAMP in hLHR(L457R)- expressing cells were, nonetheless, not as great as the levels of cAMP produced by hLHR wild-type-expressing cells incubated with a saturating concentration of CG. Furthermore, cells expressing hLHR(L457R) were unresponsive to further stimulation by CG. This finding was confirmed in the patient by lack of an increase in serum testosterone after CG stimulation. These results suggest that the conformation of hLHR(L457R) mutant represents a different activated receptor state (R*) than the agonist-occupied wild- type receptor. We also identified the previously described Ala568Val mutation in the third intracellular loop of the LHR in the other affected African- Brazilian boy and his normal prepubertal sister, suggesting the inherited form of precocious puberty in this boy. We conclude that the third transmembrane helix is a potential area for activating mutations of the hLHR that cause male precocious puberty