Covalent affinity labeling, detergent solubilization, and fluid-phase characterization of the rabbit neutrophil formyl peptide chemotaxis receptor

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Abstract
The formyl peptide chemotaxis receptor of rabbit neutrophils and purified rabbit neutrophil plasma membranes was identified by several affinity labeling techniques: covalent affinity cross-linking of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys (125I-hexapeptide) to the membrane-bound receptor with either dimethyl suberimidate of ethylene glycol bis(succinimidyl succinate) and photoactivation of N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-N.epsilon.-[6-[(4-azido-2-nitrophenyl)amino]hexanoyl]Lys(125I-PAL). These techniques specifically identify the receptor as a polypeptide that migrates as a broad band on sodium dodecyl sulfate-polyacrylamide electrophoresis, with MW 50,000-65,000. The receptor was solubilized in active form from rabbit neutrophil membranes with the detergents 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and from whole cells with CHAPS. Chemotaxis receptor activity was measured by the ability of the solubilized membrane material to bind 125I-hexapeptide or fMet-Leu-[3H]Phe with gel filtration or rapid filtration through poly(ethylenimine)- (PEI) treated filters as assay systems. 125I-PAL was specifically cross-linked to the same MW material in the CHAPS and digitonin solubilized extract, but no specific labeling of the receptor was seen when membranes were extracted with Nonidet P-40 and Triton X-100. Although a large number of detergents are able to solubilize the receptor, it appears that some release the receptor in an inactive form. The ligand binding characteristics of fMet-Leu-[3H]Phe to the CHAPS-solubilized receptor shared properties with the membrane-bound formyl peptide receptor, both of which showed curvilinear, concave-upward Scatchard plots. Computer curve fitting with NONLIN and statistical analyses of the binding data indicated that for both the membrane-bound and solubilized receptors a 2 saturable sites model fitted the data significantly better (P < 0.01) than did a one saturable site model. The characteristics of the 2 saturable sites model for the soluble receptor were a high-affinity site with a KD value of 1.25 .+-. 0.45 nM and a low-affinity site with a KD value of 19.77 .+-. 3.28 nM. A total of 35% of the 2 sites detected was of the higher affinity. In addition, a Hill coefficient of 0.61 .+-. 0.12 was observed.