Reconstitution of a physical complex between the N-formyl chemotactic peptide receptor and G protein. Inhibition by pertussis toxin-catalyzed ADP ribosylation.
Photoaffinity-labeled N-formyl chemotactic peptide receptors from human neutrophils solubilized in octyl glucoside exhibit two forms upon sucrose density gradient sedimentation, with apparent sedimentation coefficients of approximately 4 and 7 S. The 7 S form can be converted to the 4 S form by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with an EC50 of approximately 20 nM, suggesting that the 7 S form may represent a physical complex of the receptor with endogenous G protein (Jesaitis, A. J., Tolley, J. O., Bokoch, G. M., and Allen, R. A. (1989) J. Cell Biol. 109, 2783-2790). To probe the nature of the 7 S form, we reconstituted the 7 S form from the 4 S form by adding purified G protein. The 4 S form, obtained by solubilizing GTP gamma S-treated neutrophil plasma membranes, was incubated with purified (greater than 95%) Gi protein from bovine brain (containing both Gi alpha 1 and Gi alpha 2) or with neutrophil G protein (Gn), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC50 of 7 S complex formation induced by the two G proteins was 70 +/- 25 and 170 +/- 40 nM for Gn and Gi, respectively. No complexation was measurable when bovine transducin (Gt) was used up to 30 times the EC50 for Gn. The EC50 for Gi was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 microM GTP gamma S to the reconstituted 7 S complex caused a complete revision of the receptor to the 4 S form, and anti-Gi peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gi prevented formation of the 7 S form even at 20 times the concentration of unribosylated Gi normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a physical complex containing N-formyl chemotactic peptide receptor and G protein.
Bommakanti
R K
RK
Department of Chemistry and Biochemistry, Montana State University, Bozeman 59717.
Bokoch
G M
GM
Tolley
J O
JO
Schreiber
R E
RE
Siemsen
D W
DW
Klotz
K N
KN
Jesaitis
A J
AJ
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D001921:Brain / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D037342:Pertussis Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins