R. Bommakanti, G. M. Bokoch, J. O. Tolley, R. E. Schreiber, D. W. Siemsen, Karl-Norbert Klotz, A. J. Jesaitis

• 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. Tbe 7 S form can be converted to the 4 S form by guanosine 5' -0- (3-thiotriphosphate) (GTP-yS) with an EC&o of -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. 0., Bokoch, G. M., and Allen, R. A. (1989) J. CellPhotoaffinity-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. Tbe 7 S form can be converted to the 4 S form by guanosine 5' -0- (3-thiotriphosphate) (GTP-yS) with an EC&o of -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. 0., 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-yS-treated neutrophil plasma membranes, was incubated with purified (>95%) G. protein from bovine brain (containing both G\(_{ia1}\) and G\(_{ia2}\)) or with neutrophil G protein (G\(_a\)), and formation of the 7 S complex was analyzed on sucrose density gradients. The EC\(_{50}\) of 7 S complex formation induced by the two G proteins was 70 \(\pm\) 25 and 170 \(\pm\) 40 DM for G\(_a\) and G\(_1\), respectively. No complexation was measurable when bovine transducin (G\(_t\)) was used up to 30 times the EC\(_{50\) for G\(_a\). The EC\(_{50}\) for G\(_t\) was the same for receptors, obtained from formyl peptide-stimulated or unstimulated cells. The addition of 10 \(\mu\)M GTP-yS to the reconstituted 7 S complex caused a complete reversion of the receptor to the 4 S form, and anti-G\(_1\) peptide antisera immunosedimented the 7 S form. ADP-ribosylation of Gt prevented formation of the 7 S form even at 20 times the concentration of unribosylated G. normally used to attain 50% conversion to the 7 S form. These observations suggest that the 7 S species is a pbysical complex containing N-formyl chemotactic peptide receptor and G protein.…