EXTENSIVE CONTACT BETWEEN G(I2) AND N-FORMYL PEPTIDE RECEPTOR OF HUMAN NEUTROPHILS - MAPPING OF BINDING-SITES USING RECEPTOR-MIMETIC PEPTIDES

The N-formyl peptide receptor (FPR) of human neutrophils is a member of the G protein-coupled receptor (GPCR) superfamily, Sites on agonist-occupied FPR involved in binding the G(i2) protein were investigated by competition with synthetic receptor-mimetic peptides. Twenty-three synthetic FPR-mimetic and control peptides were tested for their ability to disrupt functionally active complexes of FPR and G(i2) in octyl glucoside, assayed by changes in sedimentation rates of FPR in detergent-containing sucrose gradients. GPCRs are thought to contain seven transmembrane segments with three cytoplasmic connecting loops and a cytoplasmic tail. Only certain peptides from regions in or adjoining each of the four predicted cytoplasmic domains of the 350 amino acid FPR, including the first cytoplasmic loop, were able to disrupt the reconstituted FPR-G(i2) complex. The IC(50)s Of the peptides that were able to fully disrupt the FPR-G(i2) complex ranged from 20 mu M (C2W 122-134) to 1.4 mM (C3A 230-246), a range similar to peptide inhibition of other G protein-coupled receptor-G protein interactions. Detergent concentrations above and below the critical micelle concentration had no effect on the activity of even the most hydrophobic peptide, C3B, and there was no apparent correlation of activity with hydrophobic moment, hydrophilic index, or net charge of the peptides. Control peptides from irrelevant proteins with similar physical properties and FPR extracellular domains did not dissociate the reconstituted FPR-G(i2) complex up to 5 mM, the highest concentration tested. These results suggest that the structural interface between FPR and G(i2) is extensive, requiring at least six interfacial, intramembrane, and cytoplasmic domains of FPR but without favorable interaction of the hydrophilic cytoplasmic loops connecting predicted transmembrane helices 3-7.