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-125I2(p-azidosalicylamido)ethyl-1,3'-
dithiopropionate (fMLFK-125IASD). 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-125IASD 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.
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