BINDING OF LOW-AFFINITY N-FORMYL PEPTIDE RECEPTORS TO G-PROTEIN - CHARACTERIZATION OF A NOVEL INACTIVE RECEPTOR INTERMEDIATE

G protein-coupled seven-transmembrane-containing receptors, such as the N-formyl peptide receptor (FPR) of neutrophils, likely undergo a conformational change upon binding of ligand, which enables the receptor to transmit a signal to G proteins. We have examined the functional significance of numerous conserved charged amino acid residues proposed to be located within or near the transmembrane domains. Whereas the wild type FPR exhibits a K-d for an agonist of 1-3 nM, which is reduced to similar to 40 nM in the presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S)) substitution of either Asp(71) or Arg(123) resulted in mutant receptors that bound ligand with only low affinity (K-d = 30-50 niM) independent of GTP gamma S. In contrast, substitution of Arg(163), predicted to be located at a similar depth within the membrane as Asp(71), had no effect on ligand binding. Replacement of residues Arg(309)-Glu(310)-Arg(311) resulted in an FPR with intermediate ligand binding characteristics. Functional analysis of the mutant receptors revealed that substitution of either Asp(71) or Arg(123) resulted in a mutant receptor that was unable to mediate calcium mobilization, whereas replacement of residues Arg(309)-Glu(310)-Arg(311) yielded a receptor with an EC(50) of 50 nM, compared with 0.5 nM for the wild type FPR. In order to determine the point of the defect in signal transduction, we performed reconstitution of the solubilized receptors with purified G proteins. The wild type FPR displayed a K-d for G protein of similar to 0.6 mu M compared with the Arg(309)/Glu(310)/Arg(311) mutant with a K-d of approximately 30 mu M. A Significant physical interaction between the Asp(71) or Arg(123) mutants and G protein was not observed. The implications of these findings for signal transduction mechanisms are discussed.