Differences in phosphorylation of formylpeptide and C5a chemoattractant receptors correlate with differences in desensitization.
Differences in phosphorylation of formylpeptide and C5a chemoattractant receptors correlate with differences in desensitization.
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发表时间：
1993-11
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通讯作者：
Hydar Ali;R. Richardson;E. Tomhave;J. Didsbury;Ralph Snyderman
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作者：
Hydar Ali;R. Richardson;E. Tomhave;J. Didsbury;Ralph Snyderman
为明确趋化受体的调控，在大鼠嗜碱性白血病RBL-2H3细胞中稳定表达了表位标记的人甲酰肽和C5a受体cDNAs(ET-FR和ET-C5aR)。用针对“ET”的抗体(12CA5)免疫沉淀ET-FR和ET-C5aR。FMLP和C5a引起其各自受体的时间和剂量依赖性的磷酸化。在SDS-PAGE上，磷酸化的ET-FR在50-70 kDa之间呈单一的宽带迁移，而ET-C5aR既有快速迁移(39-45 kDa)，也有宽迁移(39-52 kDa)。在低浓度的C5a(0.001-0.01微米)下，或在较高浓度的C5a(0.1微米)的作用下，在早期(5-30 S)观察到快速形式的磷酸化。佛波酯、凝血酶或抗原不能引起ET-FR的磷酸化，但能特异性地刺激ET-C5aR的快速磷酸化。蛋白激酶C抑制剂星形孢子素不能抑制ET-FR的磷酸化，但阻断了磷酸化的ET-C5aR的快速迁移成分。同源脱敏与配体诱导的两种受体的磷酸化有关。值得注意的是，ET-C5aR而不是ET-FR经历了抗原、佛波酯13-醋酸酯和凝血酶的异种脱敏。这些数据表明，蛋白激酶C介导了C5aR的异源磷酸化和脱敏，而不是FR，然而，这两种受体都被星状孢子素抗性的激酶同源脱敏。
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.