REGULATORY INTERACTION OF N-FORMYL PEPTIDE CHEMOATTRACTANT RECEPTORS WITH THE MEMBRANE SKELETON IN HUMAN NEUTROPHILS

The cytoskeleton and/or membrane skeleton has been implicated in the r egulation of N-formyl peptide receptors. The coupling of these chemota ctic receptors to the membrane skeleton was investigated in plasma mem branes from unstimulated and desensitized human neutrophils using the photoreactive agonist ]2(p-azidosalicylamido)ethyl-1,3'-dithiopropiona te (fMLFK-[I-125]ASD). When membranes of unstimulated cells were solub ilized in Triton-X 100, a detergent that does not disrupt actin filame nts, only 50% of the photoaffinity-labeled receptors were solubilized sedimenting in sucrose density gradients at a rate consistent with pre vious reports. The remainder were found in the pellet fraction along w ith the membrane skeletal actin. Solubilization of the membranes in th e presence of p-chloromercuriphenylsulfonic acid, elevated concentrati ons of KCl, or deoxyribonuclease I released receptors in parallel with actin. When membranes from neutrophils, desensitized by incubation wi th fMLFK-[I-125]ASD at 15-degrees-C, were solubilized, nearly all rece ptors were recovered in the pellet fraction. Incubation of cells with the ligand at 4-degrees-C inhibited desensitization partially and prev ented the conversion of a significant fraction of receptors to the for m associated with the membrane skeletal pellet. In these separations t he photoaffinity-labeled receptors not sedimenting to the pellet cosed imented with actin. Approximately 25% of these receptors could be immu nosedimented with antiactin antibodies suggesting that N-formyl peptid e receptors may interact directly with actin. These results are consis tent with a regulatory role for the interaction of chemotactic N-formy l peptide receptors with actin of the membrane skeleton.