CHARACTERIZATION OF COMPLEX-FORMATION BETWEEN G(I2) AND OCTYL GLUCOSIDE SOLUBILIZED NEUTROPHIL N-FORMYL PEPTIDE CHEMOATTRACTANT RECEPTOR BY SEDIMENTATION-VELOCITY

The reversible formation of complexes between N-formyl peptide chemoattractant receptor (FPR) and G(12) protein was analyzed,by velocity sedimentation in linear sucrose density gradients. FPR complexed with heterotrimeric G(12), sediments at different rate than uncomplexed FPR and the two forms have apparent sedimentation coefficients of 7S and 4S, respectively. The biochemical variables important for the reconstitution of the 7S complex from the 4S receptor and G(12) were studied. The formation of 7S was saturable with G(12) and addition of excess G(i) did not cause oligomerization. The reconstituted 7S complex was stable under a variety of conditions including octyl glucoside concentrations below and above the critical micellar concentration. The optimum pH for the reconstitution is between 7 and 9, where the 4S and 7S species sedimented reproducibly, at distinct positions in the gradient. Below pH 6 both the 4S and the 7S species appear to undergo denaturation and form precipitates. Magnesium ions have no significant effect on the sedimentation of either forms of FPR. Reconstitution was stable up to a NaCl concentration of 0.2 M. At 1 M NaCl reconstitution was inhibited and at 3 M salt FPR aggregated. Since guanine nucleotides GTP, GTP gamma S, GDP beta S selectively dissociated the 7S complex in a concentration-dependent manner and adenine nucleotides had no efffect, we conclude that the FPR-G(12) system displays a vacant guanyl nucleotide binding site, the hallmark of a functional guanine nucleotide exchange complex. Moreover, our results indicate that the reconstitution of FPR-G(12) complexes is reproducible at physiologically relevant conditions, shows selectivity, specificity, and biochemically functional properties consistent with a specific and functional interaction between solubilized FPR and G protein.