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J Biol Chem, Vol. 274, Issue 47, 33259-33266, November 19, 1999

Quantitative Analysis of Formyl Peptide Receptor Coupling to G_i1, G_i2, and G_i3

Katharina Wenzel-Seifert^§, John M. Arthur, Hui-Yu Liu^§, and Roland Seifert^§

From the  Higuchi Biosciences Center and the ^§ Department of Pharmacology and Toxicology, The University of Kansas, Lawrence, Kansas 66045-2505 and the  Department of Medicine, Kidney Disease Program, University of Louisville, Louisville, Kentucky 40202

The human formyl peptide receptor (FPR) is a prototypical G_i protein-coupled receptor, but little is known about quantitative aspects of FPR-G_i protein coupling. To address this issue, we fused the FPR to G_i1, G_i2, and G_i3 and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to G ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPS) binding and GTP hydrolysis and photolabeling of G, we demonstrate highly efficient coupling of the FPR to fused G_i1, G_i2, and G_i3 without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G_i isoforms. The K_d values of high affinity agonist binding were ~100-fold lower than the EC₅₀ (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B_max values of agonist saturation binding and ligand-regulated GTPS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G_i proteins. Analysis of agonist saturation binding, ligand-regulated GTPS saturation binding and quantitative immunoblotting with membranes expressing FPR-G_i fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G_i1, G_i2, and G_i3 with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G_i proteins linearly and not catalytically.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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