Differences in phosphorylation of formylpeptide and C5a chemoattractant receptors correlate with differences in desensitization.
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.
Ali
H
H
Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
Richardson
R M
RM
Tomhave
E D
ED
Didsbury
J R
JR
Snyderman
R
R
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000941:Antigens / Q000494:pharmacology; D002478:Cells, Cultured; D015936:Complement C5a / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology
D000470:Alkaloids; D000941:Antigens; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate