Quantitative analysis of formyl peptide receptor coupling to Giα1 Gi,α2, and Giα3

The human formyl peptide receptor (FPR) is a prototypical Gi 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(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to G alpha 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) (GTP gamma S) binding and GTP hydrolysis and photolabeling of G alpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K-d values of high affinity agonist binding were similar to 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 GTP gamma S binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several Gi proteins. Analysis of agonist saturation binding, ligand-regulated GTP gamma S saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha 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(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) 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 Gi proteins linearly and not catalytically.