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Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses
Andrew J Sloane1, Vic Raso, Dimiter S Dimitrov
1HIV-Protein Interactions Laboratory, Centre for Virus Research, Westmead, NSW, Australia.
Insights
The CXCR4 receptor exhibits structural and functional diversity across cells, with distinct isoforms mediating HIV-1 infection and immune cell trafficking. This heterogeneity impacts cellular responses to SDF-1alpha.
Area of Science:
- Immunology
- Cell Biology
- Virology
Background:
- Chemokine receptor CXCR4 (C-X-C chemokine receptor type 4) is crucial for immune cell trafficking and serves as a co-receptor for HIV-1 entry.
- CXCR4 exhibits significant heterogeneity in its expression and structure among different cell types.
Purpose of the Study:
- To identify and characterize the various CXCR4 isoforms expressed by cells.
- To compare the structural and functional differences of these CXCR4 isoforms.
- To investigate the role of glycosylation and oligomerization in CXCR4 heterogeneity.
Main Methods:
- Utilized sucrose-gradient-fractionated, Triton-insoluble membrane extracts for SDS-PAGE analysis of CXCR4 isoforms.
- Employed immunoprecipitation to analyze monomer and dimer forms of wild-type (WT) and N-linked glycosylation mutant CXCR4.
- Compared CXCR4 expression, isoform profiles, and functional responses (gp120 binding, Ca(++) flux, chemotaxis) in different cell lines (3T3, HEK-293, CEMT4, Nalm-6).
Main Results:
- Cells express either a predominant or multiple CXCR4 isoforms, with heterogeneity influenced by glycosylation and oligomerization.
- Immunoprecipitation revealed distinct non-glycosylated (34 kDa, 68 kDa) and glycosylated (40 kDa, 47 kDa, 73 kDa, 80 kDa) CXCR4 forms.
- Functional studies showed that a specific 83 kDa isoform in CEMT4 cells mediated gp120 binding for HIV-1 infection, while these cells lacked chemotactic responses to SDF-1alpha, unlike Nalm-6 cells expressing a single isoform.
Conclusions:
- CXCR4 exhibits significant structural and functional divergence between cell types.
- Different CXCR4 isoforms are responsible for mediating HIV-1 infection and chemotactic responses to SDF-1alpha.
- Understanding CXCR4 isoform-specific functions has implications for HIV-1 entry mechanisms and SDF-1alpha signaling, potentially revealing therapeutic targets.
Abstract:
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.
