IDENTIFICATION OF THE MAJOR PHOSPHORYLATION SITES IN HUMAN C5A ANAPHYLATOXIN RECEPTOR IN-VIVO
IDENTIFICATION OF THE MAJOR PHOSPHORYLATION SITES IN HUMAN C5A ANAPHYLATOXIN RECEPTOR IN-VIVO
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DOI：
10.1074/jbc.270.32.19166
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发表时间：
1995-08-11
影响因子：
4.8
通讯作者：
BOULAY, F
中科院分区：
文献类型：
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作者：
GIANNINI, E;BROUCHON, L;BOULAY, F
人C5a过敏毒素与细胞表面受体相互作用介导细胞激活和受体脱敏，C5a或佛波酯(PMA)处理分化的HL60细胞或瞬时转染的COS-7细胞导致C5aR迅速过度磷酸化。为了更深入地了解磷酸化在C5aR脱敏中的作用，我们启动了实验，以确定在C5a或PMA刺激细胞后，在氨基酸水平上的磷受体位置。在这份报告中，我们表明C5aR在分化的HL60和转基因的COS-7细胞中仅在丝氨酸残基上被磷酸化，而与使用的刺激无关。溴化氰裂解磷酸化的C5aR后的肽图表明，尽管存在蛋白激酶C的共同基序，但当细胞受到C5a或PMA的攻击时，第三个细胞质环不被磷酸化。因此，无论是用C5a还是PMA刺激细胞，磷酸化位点似乎都局限于羧基尾部的丝氨酸残基。磷酸氨基酸分析表明，C5aR在Ser(314)、Ser(317)、Ser(327)、Ser(332)、Ser(334)和Ser(338)处发生依赖于C5a的磷酸化，达到受体的最大化学计量比6摩尔/摩尔。同时将332、334和338位的丝氨酸残基替换为丙氨酸，既不影响C5a的结合，也不影响突变体的细胞表面表达，但导致C5a和PMA介导的磷酸化比野生型受体显著减少(超过80%)。这一结果表明，从Ser(332)到Ser(338)的片段上的磷酸化是C5aR羧基末端尾部随后的磷酸化所必需的。
Interaction of human C5a anaphylatoxin with cell surface receptors mediates cell activation and receptor desensitization, Treatment of differentiated HL60 cells or transiently transfected COS-7 cells with C5a or phorbol 12-myristate 13-acetate (PMA) results in rapid hyperphosphorylation of the C5aR. In an attempt to gain more insight into the function of phosphorylation in the desensitization of C5aR, we have initiated experiments to identify phosphoacceptor sites at the amino acid level after stimulation of cells with either C5a or PMA. In this report we show that C5aR is phosphorylated exclusively on serine residues in both differentiated HL60 and transfected COS-7 cells irrespective of the stimulus used. Peptide mapping after cyanogen bromide cleavage of phosphorylated C5aR indicates that despite the presence of a protein kinase C consensus motif the third cytoplasmic loop is not phosphorylated when cells are challenged with either C5a or PMA. Thus, whether the cells are stimulated with C5a or PMA, the phosphorylation sites appear to be restricted to serine residues in the carboxyl tail. Phosphoamino acid analysis of a series of mutants in which an individual serine residue was replaced by a threonine residue indicates that the C5aR undergoes C5a-dependent phosphorylation to the maximal stoichiometry of 6 mol of PO4/mol of receptor at Ser(314), Ser(317), Ser(327), Ser(332), Ser(334), and Ser(338). Simultaneous substitution of serine residues by alanine at positions 332, 334, and 338 affected neither the binding of C5a nor the cell surface expression of the mutant, but resulted in a dramatic reduction (more than 80%) of both C5a- and PMA-mediated phosphorylation as compared to the wild type receptor. This result suggests that phosphorylation on the segment extending from Ser(332) to Ser(338) is required for the subsequent phosphorylation of the carboxyl-terminal tail of C5aR.