Lateral segregation of neutrophil chemotactic receptors into actin- and fodrin-rich plasma membrane microdomains depleted in guanyl nucleotide regulatory proteins.
Subcellular fractions were prepared from human neutrophils desensitized at 15 degrees C with stimulatory doses of the photoaffinity derivative F-Met-Leu-Phe-N epsilon-(2-(rho-azido[125I]salicylamido)ethyl-1,3'- dithio-propionyl)-Lys. The covalently labeled receptors were found in a membrane fraction of higher density than those from cells preexposed to ligand at 4 degrees C but not desensitized. The denser fraction (rho approximately equal to 1.155 g/cc) was the cellular locus of the membrane associated cytoskeletal proteins, actin, and fodrin, as detected immunologically on western blots. The light fraction (rho approximately equal to 1.135), cosedimented with neutrophil plasma membrane markers, plasma membrane guanyl nucleotide regulatory proteins, and several characteristic polypeptides identified by SDS-PAGE, including a major 72-kD species. The photoaffinity-labeled species in either case showed the same mobility on SDS-PAGE (Mr = 50,000-70,000) corresponding to previously reported values for N-formyl chemotactic receptors. These labeled receptors were sensitive to proteolysis after exposure of the intact photoaffinity-labeled cells to papain at 4 degrees C. We conclude that (a) the fractions isolated are probably derived from different lateral microdomains of the surface of human neutrophils; (b) the higher density fraction contains occupied N-formyl-chemotactic receptors previously shown to have been converted, to a high affinity, slowly dissociating form coisolating with neutrophil cytoskeleton and implicated in the termination of formyl peptide-induced neutrophil activation; and (c) the translocation of receptors to these microdomains may serve to compartmentalize receptors and perhaps regulate the interaction of the receptor/G-protein transduction pair.
D000199:Actins / Q000032:analysis; D001345:Autoradiography; D002352:Carrier Proteins / Q000032:analysis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000032:analysis / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D003599:Cytoskeleton / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis*; D006801:Humans; D007118:Immunoassay; D008024:Ligands; D008840:Microfilament Proteins / Q000032:analysis; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000032:analysis* / Q000648:ultrastructure; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000032:analysis* / Q000378:metabolism; D013049:Spectrin
D000199:Actins; D002352:Carrier Proteins; D019204:GTP-Binding Proteins; D008024:Ligands; D008840:Microfilament Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013049:Spectrin