RT Journal Article SR Electronic T1 An Anionic Residue at Position 564 Is Important for Maintaining the Inactive Conformation of the Human Lutropin/Choriogonadotropin Receptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 894 OP 901 VO 53 IS 5 A1 Shinji Kosugi A1 Toru Mori A1 Andrew Shenker YR 1998 UL http://molpharm.aspetjournals.org/content/53/5/894.abstract AB Gonadotropin-independent, male-limited precocious puberty is caused by a variety of mutations in the lutropin/choriogonadotropin receptor (LHR) that produce constitutive receptor activation. Two of these mutations encode replacement of conserved aspartate residues at positions 564 and 578 with glycine. We previously used site-directed mutagenesis to study the functional role of the Asp578 side chain in transmembrane helix 6, and concluded that it is its ability to serve as a properly positioned interhelical hydrogen bond acceptor, rather than its negative charge, that is important for stabilizing the inactive state of the LHR. We now report the effects of substituting seven different amino acids for the Asp564 residue located at the carboxyl terminus of the third intracellular loop. Glycine, alanine, valine, leucine, phenylalanine, and asparagine produced constitutive activation in a COS-7 cell expression system (3–5-fold increase in basal cAMP), but glutamate did not, indicating that a negative charge at position 564 may be important for maintaining the inactive LHR conformation. Characterization of double-mutant receptors showed that certain substitutions at Asp564 and Asp578 have a cumulative effect on basal receptor activity, perhaps because they mimic different aspects of the activation process normally triggered by hormone binding. The American Society for Pharmacology and Experimental Therapeutics