Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor

The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/ Glu-Arg-Tyr/X-X-X-IIe/Val, defines a structural "arginine cage" domain. Pre vious computational end mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this dom ain. We have investigated the functional importance of the intrahelical pos ition and orientation of the arginine cage using insertional mutagenesis. I ntroduction of a single Ala proximal to the conserved Asp-Arg of this domai n caused loss of detectable ligand binding. Inserting a second Ala, however , restored high-affinity agonist binding. Further insertion of three or fou r Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant rece ptors confirms that the structure required in this region for efficient sig naling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that s pecific orientations of this segment can stabilize high-affinity receptor c onformations that are uncoupled from signal transduction.