Sloane Andrew J, Raso Vic, Dimitrov Dimiter S, Xiao Xiaodong, Deo Shivashni, Muljadi Nick, Restuccia David, Turville Stuart, Kearney Christine, Broder Christopher C, Zoellner Hans, Cunningham Anthony L, Bendall Linda, Lynch Garry W
HIV-Protein Interactions Laboratory, Centre for Virus Research, Westmead, NSW, Australia.
Immunol Cell Biol. 2005 Apr;83(2):129-43. doi: 10.1111/j.1440-1711.2004.01304.x.
CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS-PAGE using sucrose-gradient-fractionated, triton-insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild-type (WT) and dual N-linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK-293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non-glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV-1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive Nalm-6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca(++) flux or a chemotactic response to SDF-1alpha. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV-1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV-1 entry and SDF-1alpha responses and may indicate therapeutic possibilities.
CXCR4是SDF-1α的趋化细胞受体，对免疫细胞迁移和HIV感染至关重要。CXCR4具有显著的异质性，本研究旨在更好地鉴定细胞表达的异构体，并比较它们的结构和功能。我们发现细胞要么表达一种主要的异构体，要么表达多种异构体。使用蔗糖梯度分级分离的、Triton不溶性膜提取物在SDS-PAGE上能最好地分辨这些异构体。我们推测糖基修饰可能是这种异质性的部分原因，且细胞异构体差异可能突出了CXCR4的多种细胞功能。对在3T3和HEK-293细胞中表达的野生型（WT）和双N-连接糖基化位点突变体N11A/N176A的CXCR4进行比较，结果表明几乎一半的异构体中糖基化和寡聚化存在差异。CXCR4的免疫沉淀显示，N11A/N176A突变体中有34 kDa和68 kDa的非糖基化单体和二聚体形式，而WT中有糖基化的40 kDa、47 kDa、73 kDa和80 kDa形式。异构体作用的功能特异性也得到了证实，因为尽管CEMT4细胞表达高水平的CXCR4和11种不同的异构体，但发现只有一种83 kDa的形式能结合gp120以实现HIV-1 IIIB感染。此外，比较研究发现，与表达相似水平单一异构体的对SDF-1α有反应的Nalm-6细胞不同，CEMT4细胞对SDF-1α没有显示出Ca(++)通量或趋化反应。因此，CXCR4在细胞之间的结构和功能可能存在差异，HIV-1感染和趋化作用显然由不同的异构体介导。这种结构和功能的分离对于理解HIV-1进入和SDF-1α反应具有重要意义，可能暗示了治疗的可能性。
