title: Regulatory interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton in human neutrophils.
abstract: The cytoskeleton and/or membrane skeleton has been implicated in the regulation of N-formyl peptide receptors. The coupling of these chemotactic receptors to the membrane skeleton was investigated in plasma membranes from unstimulated and desensitized human neutrophils using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamido)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). When membranes of unstimulated cells were solubilized in Triton-X 100, a detergent that does not disrupt actin filaments, only 50% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with previous reports. The remainder were found in the pellet fraction along with the membrane skeletal actin. Solubilization of the membranes in the presence of p-chloromercuriphenylsulfonic acid, elevated concentrations of KCl, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation with fMLFK-[125I]ASD at 15 degrees C, were solubilized, nearly all receptors were recovered in the pellet fraction. Incubation of cells with the ligand at 4 degrees C inhibited desensitization partially and prevented the conversion of a significant fraction of receptors to the form associated with the membrane skeletal pellet. In these separations the photoaffinity-labeled receptors not sedimenting to the pellet cosedimented with actin. Approximately 25% of these receptors could be immunosedimented with antiactin antibodies suggesting that N-formyl peptide receptors may interact directly with actin. These results are consistent with a regulatory role for the interaction of chemotactic N-formyl peptide receptors with actin of the membrane skeleton.
authors.affiliation: Department of Microbiology, Montana State University, Bozeman 59717.
authors.forename: K N
authors.initials: KN
authors.lastname: Klotz
authors.forename: K L
authors.initials: KL
authors.lastname: Krotec
authors.forename: J
authors.initials: J
authors.lastname: Gripentrog
authors.forename: A J
authors.initials: AJ
authors.lastname: Jesaitis
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000648:ultrastructure*; D002474:Cell-Free System; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000031:analogs & derivatives / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism* / Q000648:ultrastructure; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D012995:Solubility
chemical_list: D000199:Actins; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine