Abstract
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.
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