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 membrane skeleton. This is thought to be the critical event in the lateral segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an interruption of the signaling cascade. In this study we probed the interaction of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamino)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). The interaction of FPR with protein was studied via a reconstitution assay and subsequent analysis of FPR-G protein complexes in sucrose density gradients. 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 EC50 values for reconstitution were similar to that determined for FPR from unstimulated neutrophils (Bommakanti RK et al., J Biol Chem 267: 7576-7581, 1992). We conclude, therefore, that the affinity of the interaction of FPR with G protein is not affected by desensitization, consistent with the model of lateral segregation of FPR and G protein as a mechanism of desensitization.
Klotz
K N
KN
Department of Microbiology, Montana State University, Bozeman 59717.
Jesaitis
A J
AJ
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002417:Cattle; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives; D009504:Neutrophils / Q000737:chemistry / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000737:chemistry / Q000378:metabolism*; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C049721:N-formyl-methionylleucyl-phenylalanyl-N(epsilon)-(2-(4-azidosalicylamido)ethyl-1,3'-dithiopropionyl)lysine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019204:GTP-Binding Proteins