Structure-function studies of the C3a-receptor:: C-terminal serine and threonine residues which influence receptor internalization and signaling
Structure-function studies of the C3a-receptor:: C-terminal serine and threonine residues which influence receptor internalization and signaling
复制标题
DOI：
10.1002/eji.200323293
复制
发表时间：
2003-04-01
影响因子：
5.4
通讯作者：
Klos, A
中科院分区：
文献类型：
--
作者：
Settmacher, B;Rheinheimer, C;Klos, A
过敏毒素肽C3 a是在补体活化期间产生的促炎介质，其特异性G蛋白偶联受体在粒细胞、单核细胞、肥大细胞、活化的淋巴细胞和神经组织中表达。我们已经产生了RBL-2 H3细胞克隆稳定表达突变体的人C3 a-受体（C3 aR）与组合丙氨酸（Ala）取代的10个C-末端丝氨酸（Ser）或苏氨酸（Thr）残基，这可能代表假定的磷酸化位点，以表征其在配体诱导的C3 aR内化和信号转导的作用。Ser 475/479和Thr 480/481以及Ser 449似乎不参与配体诱导的受体内化。直接或通过构象变化，它们甚至“抑制”C3 aR内化。与此相反，突变体与Ala取代在Ser 465/470和Thr 463/466是很差的内化，和Thr 463似乎是最重要的C-末端的Thr或Ser残基直接影响受体内化。然而，它是可能的，其他C3 aR区域另外参与这种负反馈机制，因为即使是突变体与多个Ala取代仍然内化到有限的程度。有趣的是，在Ser 449单次交换为Ala的突变体中，通过Ca 2+测定和分别与G-α 16或G-α 0瞬时共转染的HEK细胞上的[S-35] GTP γ S结合评估的信号转导严重受损，表明C3 aR的该残基参与G蛋白偶联。
The anaphylatoxic peptide C3a is a pro-inflammatory mediator generated during complement activation, whose specific G protein coupled receptor is expressed on granulocytes, monocytes, mast cells, activated lymphocytes, and in the nervous tissue. We have generated RBL-2H3 cell clones stably expressing mutants of the human C3a-receptor (C3aR) with combined alanine (Ala) substitutions of ten C-terminal serine (Ser) or threonine (Thr) residues, which may represent putative phosphorylation sites to characterize their role in ligand-induced C3aR internalization and signaling. Ser475/479 and Thr480/481 as well as Ser449 seemed not to be involved in ligand-induced receptor internalization. Either directly or by a conformational change they even "inhibit" C3aR internalization. In contrast, mutants with Ala substitutions at Ser465/470 and Thr463/466 were poorly internalized, and Thr463 seemed to be the most important C-terminal Thr or Ser residue directly effecting receptor internalization. However, it is likely that other C3aR regions additionally participate in this negative feed-back mechanism since even mutants with multiple Ala substitutions still internalized to a limited degree. Interestingly, in a mutant with a single exchange of Ser449 to Ala, the signal transduction assessed by a Ca2+ assay and [S-35]GTPgammaS-binding on HEK cells transiently cotransfected with G-alpha 16 or G-alpha 0, respectively, was severely impaired, indicating that this residue of C3aR is involved in G protein coupling.