Chemokines are proinflammatory cytokines that attract and activate specific types of leukocyte1. There are two main chemokine families, based on the position of the first two cysteine residues: the CC and the CXC chemokines1. Chemokines mediate their effects through interactions with seven-transmembrane-spanning glyco-protein receptors coupled to a G-protein signalling pathway1. Chemokine receptors normally undergo a ligand-mediated homodimerization process, which is required for Ca2+ flux and chemotaxis2. Here we show that in the chemokine response it is possible for heterodimerization, rather than homodimerization, to occur between a mutant form of the CCR2 receptor (the CCR2V64I receptor), which helps to delay the development of AIDS in HIV-1-infected individuals, and the CCR5 or CXCR4 chemokine receptor, which are used by HIV to gain entry into cells. These results may explain why AIDS takes longer to develop in HIV-1-infected individuals carrying the CCR2V64I mutation3.
The discovery that certain chemokine receptors can also act as receptors for HIV-1, HIV-2 and simian immunodeficiency virus has shed light on the mechanisms underlying viral entry and tropism4. The virus strains responsible for transmission, which are the predominant virus type isolated from asymptomatic HIV-positive individuals, use CCR5 as a receptor, whereas viruses that emerge later in the course of infection use CXCR4 to gain entry into the cell, either instead of or in addition to CCR5 (ref. 5).
The CCR2V64I polymorphism of CCR2, in which a valine at position 64 is replaced by an isoleucine, occurs at an allele frequency of 10-25%, depending on the ethnic group of the population, and is associated with a delay of 2-4 years in the progression to AIDS3. Relatively few viral strains have been reported that can use CCR2 in conjunction with CD4 to infect cells, so the mechanism underlying this protective effect is not clear6.
After binding to their specific receptors, chemokines induce receptor homodimerization and activate the receptor-associated JAK kinase, possibly by transphosphorylation of tyrosine residues2,7. This creates SH2 docking sites, leading to the recruitment of STAT transcription factors. In another member of the seven-transmembrane-spanning receptor family, the response to the neurotransmitter GABA requires hetero-dimerization of the GBR1 and GBR2 receptors8. The interaction between GBR1 and GRB2 seems to be essential for the activation of potassium channels.
One possibility is that the protective effect of CCR2V64I is due to its ability to heterodimerize with the CCR5 and/or CXCR4 receptor. We tested this idea by transfecting HEK-293 cells, which constitutively express the CXCR4 receptor, with the CCR2V64I mutant (Fig. 1a) and monitoring the response to the chemokines MCP-1 and SDF-1
. Equivalent expression of both receptors was verified by flow cytometry analysis and by their ability to respond in chemotaxis and in Ca2+ flux to either MCP-1 or SDF-1. In these cells, MCP-1 and SDF-1 sensitize responses to the homologous, but not the heterologous, chemokine (results not shown).
To test for heterodimerization of the CCR2V64I and CXCR4 receptors following MCP-1 and SDF-1 binding, cells were first crosslinked by using disuccinimidyl suberate and then lysed. Anti-CCR2 immuno-precipitates of cell lysates were developed in a western blot by using anti-CXCR4 or anti-CCR2 antibodies. In CCR2-derived immunoprecipitates, CXCR4 receptors were observed after MCP-1 and SDF-1 activation, but not in the presence of either chemokine alone (Fig. 1b, left). The membrane was then stripped and reprobed with anti-CCR2 antibody. As expected, MCP-1 was found to induce dimerization of the CCR2 receptor (Fig. 1b, right), as shown by the presence of dimers or complexes of higher relative molecular mass.
In contrast, CCR2/CXCR4 co-transfected receptors do not undergo heterodimerization in response to stimulation by MCP-1 and SDF-1 (not shown). This indicates that the isoleucine at position 64 is required to promote heterodimerization between the CCR2V64I and CXCR4 receptors. Isoleucine is also found at position 64 in the CCR5 receptor. In similar experiments with CCR5-transfected HEK-293 cells, we also detected CCR2V64I in CCR5 immunoprecipitates when cells were stimulated simultaneously with the chemokines RANTES and MCP-1 (data not shown). Finally, the CCR2V64I mutant receptor and the wild-type CCR2 receptor also heterodimerize with the CCR5 receptor (results not shown).
Our data showing that CXCR4 can dimerize with the CCR2V64I mutant, but not with wild-type CCR2, indicate that Val 64 may be critical for dimer stabilization. We have previously suggested that chemokine-receptor dimerization may help to prevent HIV-1 infection9. The ability of CCR2V64I to dimerize with CXCR4 may reduce the amount of CXCR4 on peripheral blood mononuclear cells. Together with the ability of CCR2V64I to heterodimerize with CCR5, this might explain the delayed progression to seroconversion in HIV-infected donors carrying this mutation1.
These findings may help us to understand the molecular mechanisms responsible for chemokine-receptor signalling and to explain why some chemokines block HIV-1 infection. One implication of receptor clustering following chemotactic responses is that the activity of one receptor could influence that of its neighbours, so a ligand might be able to act in trans on a receptor for which it is not specific.
A chemokine-driven posit
