J. Biol. Chem., Vol. 268, Issue 32, 24247-24254, 11, 1993
Differences in phosphorylation of formylpeptide and C5a chemoattractant receptors correlate with differences in desensitization
H Ali, RM Richardson, ED Tomhave, JR Didsbury and R Snyderman
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
To define the regulation of chemoattractant receptors, epitope-tagged human
formyl peptide and C5a receptor cDNAs (ET-FR and ET-C5aR) were stably
expressed in rat basophilic leukemia, RBL-2H3 cells. An antibody (12CA5)
specific to "ET" was used to immunoprecipitate ET-FR and ET- C5aR. fMLP and
C5a caused time- and dose-dependent phosphorylation of their respective
receptors. Phosphorylated ET-FR migrated as a single broad band between 50
and 70 kDa on SDS-polyacrylamide gel electrophoresis, whereas ET-C5aR
exhibited both fast (39-45 kDa) and broadly (39-52 kDa) migrating forms.
Fast form phosphorylation alone was observed at low concentrations of C5a
(0.001-0.01 microM), or at early times (5-30 s) with a higher concentration
of C5a (0.1 microM). Phorbol 12-myristate 13-acetate, thrombin, or antigen
caused no phosphorylation of ET-FR but stimulated exclusively fast form
phosphorylation of ET-C5aR. The protein kinase C inhibitor staurosporine
did not inhibit phosphorylation of ET-FR but blocked the fast migrating
component of phosphorylated ET-C5aR. Homologous desensitization correlated
with ligand-induced phosphorylation of both receptors. Of note, ET-C5aR but
not ET-FR underwent heterologous desensitization by antigen, phorbol
12-myristate 13-acetate, and thrombin. The data suggest that protein kinase
C mediates heterologous phosphorylation and desensitization of C5aR but not
FR, yet, both receptors are homologously desensitized by a
staurosporine-resistant kinase.
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