title: Regulation of chemoattractant receptor interaction with transducing proteins by organizational control in the plasma membrane of human neutrophils.
abstract: Isolated purified plasma membrane domains from unstimulated human neutrophils were photoaffinity labeled with F-Met-Leu-Phe-N epsilon-(2-(p-azido-[125I]salicylamido)ethyl- 1,3'-dithiopropionyl)-Lys also referred to as FMLPL-SASD[125I]. Most of the photoaffinity-labeled N-formyl peptide receptors were found in light plasma membrane fraction (PM-L) which has been previously shown to be enriched in guanyl nucleotide binding proteins and the plasma membrane marker alkaline phosphatase (Jesaitis, A. J., G. M. Bokoch, J. O. Tolley, and R. A. Allen. 1988. J. Cell Biol. 107:921-928). Furthermore, the heavy plasma membrane fraction (PM-H), which is enriched in actin and fodrin, was depleted in receptors. Solubilization of PM-L and PM-H in divalent cation-free buffer containing octylglucoside and subsequent sedimentation at 180,000 g in detergent-containing sucrose gradients revealed two receptor forms. The major population, found in PM-L sedimented as a globular protein with an apparent sedimentation coefficient of 6-7S, while a minor fraction found in the PM-H fraction sedimented as a 4S particle. In addition, the 6-7S form could be converted to the 4S form by inclusion of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in the extraction buffer (ED50 = 10-30 nM). ATP was not effective at doses of up to 10 microM. In contrast, isolation and solubilization of receptors from desensitized cells (photoaffinity labeled after a 15 degrees C incubation with FMLPL-SASD[125I]) revealed that the majority of receptors (greater than 60-90%), which are found in PM-H, sedimented as 4S particles. A minor fraction of receptors found in the PM-L sedimented as 6-7S species. The receptors in the PM-H fraction, however, were still capable of interacting with G-proteins, since addition of unlabeled PM-L membrane fraction as a G-protein source reconstituted a more rapidly sedimenting form showing sensitivity to GTP gamma S. These results suggest that receptors in unstimulated human neutrophils have a higher probability of interacting with G-proteins because they are in the light plasma membrane domain. The results also suggest that receptors that have been translocated to the heavy plasma membrane domain during the process of desensitization or response termination have a lower probability of interacting with G-protein. Since the latter receptors are still capable of forming G protein associations, then their lateral segregation would represent a mechanism of controlling of receptor G-protein interactions. This reorganization of the plasma membrane, therefore, may form the molecular basis for response termination or homologous desensitization in human neutrophils.
authors.affiliation: Department of Chemistry, Montana State University, Bozeman 59717.
authors.forename: A J
authors.initials: AJ
authors.lastname: Jesaitis
authors.forename: J O
authors.initials: JO
authors.lastname: Tolley
authors.forename: G M
authors.initials: GM
authors.lastname: Bokoch
authors.forename: R A
authors.initials: RA
authors.lastname: Allen
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000097:blood* / Q000302:isolation & purification / Q000502:physiology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000302:isolation & purification / Q000378:metabolism*; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins