The role of Asp(578) in maintaining the inactive conformation of the human lutropin/choriogonadotropin receptor

A constitutively activating mutation encoding Asp(578)-->Gly in transmembrane helix 6 of the lutropin/choriogonadotropin receptor (LHR) is the most common cause of gonadotropin-independent, male-limited precocious puberty. This mutant LHR produces a 4.5-fold increase in basal cAMP when expressed in COS-7 cells. To better understand the normal role of Asp(578) in the LHR we studied the effect of seven other amino acid substitutions at this position. No agonist binding or response was detected with the Asp(578)-->Pro mutant. Agonist binding affinity was unaffected by the other substitutions and estimated receptor concentrations ranged from 11 to 184% of wild type. Substitution of Asp(578) with Asn, a similarly sized, uncharged residue, did not produce agonist-independent activation. In contrast, replacement with Glu, Ser, or Leu caused 4.9-5.6-fold stimulation of basal cAMP. Substitution with Tyr (8.5-fold) or Phe (7.5-fold) had a greater activating effect. Only the Tyr, Phe, and Leu mutants showed constitutive activation of the inositol phosphate pathway. Our data suggest that it is the ability of the Asp(578) side chain to serve as a properly positioned hydrogen bond acceptor, rather than its negative charge, that is important for stabilizing the inactive state of the LHR. A bulky aromatic side chain at position 578 may further destabilize the inactive receptor conformation.