Functional molecular complexes of human N-formyl chemoattractant
receptors and actin
J Immunol, 151 (10):
5653-65 (November 1993)Jesaitis, A J Erickson, R W Klotz, K N Bommakanti, R K Siemsen, D
W 5R01-AI22735/AI/NIAID NIH HHS/United States Research Support, Non-U.S.
Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support,
U.S. Gov't, P.H.S. United states Journal of immunology (Baltimore,
Md. : 1950) J Immunol. 1993 Nov 15;151(10):5653-65..Abstract
When human neutrophils become desensitized to formyl peptide chemoattractants,
the receptors (FPR) for these peptides are converted to a high affinity,
GTP-insensitive form that is associated with the Triton X-100-insoluble
membrane skeleton from surface membrane domains. These domains are
actin and fodrin-rich, but G protein-depleted suggesting that FPR
shuttling between G protein-enriched and depleted domains may control
signal transduction. To determine the molecular basis for FPR interaction
with the membrane skeleton, neutrophil subcellular fractions were
screened for molecules that could bind photoaffinity-radioiodinated
FPR solubilized in Triton X-100. These receptors showed a propensity
to bind to a 41- to 43-kDa protein band on nitrocellulose overlays
of SDS-PAGE-separated cytosol and plasma membrane fractions of neutrophils.
This binding, as well as FPR binding to purified neutrophil actin,
was inhibited 50% by 0.6 microM free neutrophil cytosolic actin.
Addition of greater than 1 microM G-actin to crude or lectin-purified
Triton X-100 extracts of FPR from neutrophil membranes increased
the sedimentation rate of a significant fraction of FPR two to three
fold as measured by velocity sedimentation in Triton X-100-containing
linear sucrose density gradients. Addition of anti-actin antibodies
to FPR extracts caused a concentration-dependent immunoprecipitation
of at least 65% of the FPR. More than 40% of the immunoprecipitated
FPR was specifically retained on protein A affinity matrices. Membrane
actin was stabilized to alkaline washing when membranes were photoaffinity
labeled. Conversely, when purified neutrophil cytosolic actin was
added to membranes or their digitonin extracts, after prior depletion
of actin by an alkaline membrane wash, photoaffinity labeling of
FPR was increased two- to fourfold with an EC50 of approximately
0.1 microM actin. We conclude that FPR from human neutrophils may
interact with actin in membranes to form Triton X-100-stable physical
complexes. These complexes can accept additional G-actin monomers
to form higher order molecular complexes. Formation of FPR-actin
complexes in the neutrophil may play a role in the regulation of
chemoattractant-induced activation or actin polymerization.