Characterization of complex formation between Gi2 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 Gi2 protein was analyzed by velocity sedimentation in linear sucrose density gradients. FPR complexed with heterotrimeric Gi2, 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 Gi2 were studied. The formation of 7S was saturable with Gi2 and addition of excess Gi 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 effect, we conclude that the FPR-Gi2 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-Gi2 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.
Bommakanti
R K
RK
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717.
Dratz
E A
EA
Siemsen
D W
DW
Jesaitis
A J
AJ
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003902:Detergents / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D005960:Glucosides / Q000737:chemistry*; D006150:Guanine Nucleotides / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009504:Neutrophils / Q000737:chemistry*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry*; D018000:Receptors, Peptide / Q000737:chemistry*; D012995:Solubility
D003902:Detergents; D005960:Glucosides; D006150:Guanine Nucleotides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C018619:octyl-beta-D-glucoside; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins; D008274:Magnesium