AN ANIONIC RESIDUE AT POSITION-564 IS IMPORTANT FOR MAINTAINING THE INACTIVE CONFORMATION OF THE HUMAN LUTROPIN CHORIOGONADOTROPIN RECEPTOR/

Gonadotropin-independent, male-limited precocious puberty is caused by a variety of mutations in the lutropin/choriogonadotropin receptor (L HR) that produce constitutive receptor activation. Two of these mutati ons encode replacement of conserved aspartate residues at positions 56 4 and 578 with glycine. We previously used site-directed mutagenesis t o 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 p ositioned interhelical hydrogen bond acceptor, rather than its negativ e charge, that is important for stabilizing the inactive state of the LHR. We now report the effects of substituting seven different amino a cids for the Asp564 residue located at the carboxyl terminus of the th ird intracellular loop. Glycine, alanine, valine, leucine, phenylalani ne, and asparagine produced constitutive activation in a COS-7 cell ex pression system (3-5-fold increase in basal cAMP), but glutamate did n ot, indicating that a negative charge at position 564 may be important for maintaining the inactive LHR conformation. Characterization of do uble-mutant receptors showed that certain substitutions at Asp564 and Asp578 have a cumulative effect on basal receptor activity, perhaps be cause they mimic different aspects of the activation process normally triggered by hormone binding.