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 ~40 nM in the presence of guanosine 5'-3-O-(thio)triphosphate (GTPγS), substitution of either Asp or Arg resulted in mutant receptors that bound ligand with only low affinity (K(d) = 30-50 nM) independent of GTPγS. In contrast, substitution of Arg, predicted to be located at a similar depth within the membrane as Asp, had no effect on ligand binding. Replacement of residues Arg-Glu-Arg resulted in an FPR with intermediate ligand binding characteristics. Functional analysis of the mutant receptors revealed that substitution of either Asp or Arg resulted in a mutant receptor that was unable to mediate calcium mobilization, whereas replacement of residues Arg-Glu-A yielded a receptor with an EC 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 ~0.6 μM compared with the Arg/Glu/Arg mutant with a K(d) of approximately 30 μM. A significant physical interaction between the Asp or Arg mutants and G protein was not observed. The implications of these findings for signal transduction mechanisms are discussed.