PHYSICAL COUPLING OF N-FORMYL PEPTIDE CHEMOATTRACTANT RECEPTORS TO G-PROTEIN IS UNAFFECTED BY DESENSITIZATION

Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils results in association of these receptors to the me mbrane skeleton. This is thought to be the critical event in the later al segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an inte rruption of the signaling cascade. In this study we probed the interac tion of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the pho toreactive agonist N-formyl-met-leu-phe-lys-N-epsilon[I-125]2 (p-azido salicylamido)ethyl-1,3'-dithiopropionate (fMLFK-[I-125] ASD). The inte raction of FPR with G protein was studied via a reconstitution assay a nd subsequent analysis of FPR-G protein complexes in sucrose density g radients. FPR-G protein complexes were reconstituted with solubilized FPR from partially and fully desensitized neutrophils with increasing concentrations of Gi purified from bovine brain. The respective EC(50) values for reconstitution were similar to that determined for FPR fro m unstimulated neutrophils (Bommakanti RK et al., J Biol Chem 267: 757 6-7581, 1992). We conclude, therefore, that the affinity of the intera ction of FPR with G protein is not affected by desensitization, consis tent with the model of lateral segregation of FPR and G protein as a m echanism of desensitization.