An Arg present in the third transmembrane domain of all rhodopsin-like G-protein-coupled receptors is required for efficient signal transduction. Mutation of this Arg in the gonadotropin-releasing hormone receptor to Gln, His, or Lys abolished or severely impaired agonist-stimulated inositol phosphate generation, consistent with Arg having a role in receptor activation. To investigate the contribution of the surrounding structural domain in the actions of the conserved Arg, an integrated microdomain modeling and mutagenesis approach has been utilized. Two conserved residues that constrain the Arg side chain to a limited number of conformations have been identified. In the inactive wild-type receptor, the Arg side chain is proposed to form an ionic interaction with Asp^3.49(138). Experimental results for the Asp^3.49(138) → Asn mutant receptor show a modestly enhanced receptor efficiency, consistent with the hypothesis that weakening the Asp^3.49(138)-Arg^3.50(139) interaction by protonation of the Asp or by the mutation to Asn favors activation. With activation, the Asp^3.49(138)-Arg^3.50(139) ionic bond would break, and the unrestrained Arg would be prevented from orienting itself toward the water phase by a steric clash with Ile^3.54(143). The mutation Ile^3.54(143) → Ala, which eliminates this clash in simulations, causes a marked reduction in measured receptor signaling efficiency, implying that solvation of Arg^3.50(139) prevents it from functioning in the activation of the receptor. These data are consistent with residues Asp^3.49(138) and Ile^3.54(143) forming a structural motif, which helps position Arg in its appropriate inactive and active receptor conformations.

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