Venkatesan S, Petrovic A, Locati M, Kim Y O, Weissman D, Murphy P M
Laboratory of Molecular Microbiology and Laboratory of Host Defenses, NIAID, National Institutes of Health, Bldg. 10, Rm. 6A05, Bethesda, MD 20892, USA.
J Biol Chem. 2001 Oct 26;276(43):40133-45. doi: 10.1074/jbc.M105722200. Epub 2001 Aug 20.
We examined the structural requirements for cell surface expression, signaling, and human immunodeficiency virus co-receptor activity for the chemokine receptor, CCR5. Serial C-terminal truncation of CCR5 resulted in progressive loss of cell surface expression; mutants truncated at the 317th position and shorter were not detected at the cell surface. Alanine substitution of basic residues in the membrane-proximal domain (residues 314-322) in the context of a full-length C-tail resulted in severe reduction in surface expression. C-terminal truncation that excised the three cysteines in this domain reduced surface expression, but further truncation of upstream basic residue(s) abolished surface expression. Substituting the carboxyl-terminal domain of CXCR4 for that of CCR5 failed to rectify the trafficking defect of the tailless CCR5. In contrast, tailless CXCR4 or a CXCR4 chimera that exchanged the native cytoplasmic domain for that of wild type CCR5 was expressed at the cell surface. Deletion mutants that expressed at the cell surface responded to chemokine stimulation and mediated human immunodeficiency virus entry. Substitution of all serine and threonine residues in the C-terminal tail of CCR5 abolished chemokine-mediated receptor phosphorylation but preserved downstream signaling (Ca(2+) flux), while substitutions of tyrosine residues in the C-tail affected neither phenotype. CCR5 mutants that failed to traffic to the plasma membrane did not exhibit obvious changes in metabolic turnover and were retained in the Golgi or pre-Golgi compartments(s). Thus, the basic domain (-KHIAKRF-) and the cysteine cluster (-CKCC-) in the C-terminal tail of CCR5 function cooperatively for optimal surface expression.
我们研究了趋化因子受体CCR5在细胞表面表达、信号传导及人类免疫缺陷病毒共受体活性方面的结构要求。对CCR5进行C末端系列截短导致细胞表面表达逐渐丧失；截短至第317位及更短的突变体在细胞表面未被检测到。在全长C末端的背景下，将膜近端结构域（第314 - 322位残基）中的碱性残基替换为丙氨酸导致表面表达严重降低。切除该结构域中三个半胱氨酸的C末端截短降低了表面表达，但进一步截短上游碱性残基则消除了表面表达。用CXCR4的羧基末端结构域替换CCR5的相应结构域未能纠正无尾CCR5的转运缺陷。相反，无尾CXCR4或用野生型CCR5的天然胞质结构域替换其自身胞质结构域的CXCR4嵌合体在细胞表面表达。在细胞表面表达的缺失突变体对趋化因子刺激有反应并介导人类免疫缺陷病毒进入。将CCR5 C末端尾巴中的所有丝氨酸和苏氨酸残基替换可消除趋化因子介导的受体磷酸化，但保留下游信号传导（Ca(2+)通量），而C末端尾巴中酪氨酸残基的替换对两种表型均无影响。未能转运至质膜的CCR5突变体在代谢周转方面未表现出明显变化，并保留在高尔基体或高尔基体前区室中。因此，CCR5 C末端尾巴中的碱性结构域（-KHIAKRF-）和半胱氨酸簇（-CKCC-）协同作用以实现最佳表面表达。
