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. 1999 Jun 22;96(13):7496–7501. doi: 10.1073/pnas.96.13.7496

Figure 4.

Figure 4

Involvement of the first two domains of CD4 and the second extracellular loop of CCR5 in the CD4–CCR5 association. (A) Coimmunoprecipitation of CD4–CD8 hybrid molecules containing the first two domains of CD4 by an anti-CCR5 mAb. The A2.01.T4.T8 cells expressing the hybrid CD4–CD8 molecule were infected with a recombinant vaccinia virus (vvCCR5–1107) (lane 1), encoding the gene for CCR5, and a control wild-type (WR) vaccinia virus (lane 2). The anti-CCR5 mAb 5C7 was used to immunoprecipitate CCR5 and T4–4 for detection of CD4 by Western blotting. (B) The amount of CD4–CD8 molecules in A2.01.T4.T8 cells infected with the CCR5 (lane 1) or WR (lane 2) vaccinia virus was not significantly different as demonstrated by Western blot with an anti-CD4 Ab (T4–4). (C) Coimmunoprecipitation of CCR5 by an anti-CD4 mAb (OKT4) is inhibited in the presence of another anti-CD4 mAb (CG7). For comparison, lane 1 shows CCR5 immunoprecipitated by 5C7. Lanes 2, 3, and 4 represent the amount of CCR5 coimmunoprecipitated by a mixture of OKT4 (ascites fluid 3.5 μl/ml) and increasing concentrations of the anti-CD4 mAb CG7 (0, 5, and 10 μg/ml, respectively). (D) CD4 coimmunoprecipitation by 5C7 is inhibited in the presence of another anti-CCR5 mAb (2D7) (29) directed to the second extracellular loop. Equal amounts (3 μg/ml) of 5C7, which does not affect HIV entry, were mixed with increasing amounts (0, 3, and 6 μg/ml) (lanes 1, 2, and 3, respectively) of the HIV-1-blocking mAb 2D7 and used for immunoprecipitation. The sample obtained from 9 × 106 3T3.CD4.CCR5 cells was divided into two portions, and the smaller one (1/6 of total) was used for Western blot of CD4 by T4–4, and the rest were used for Western blot of CCR5 by the CKR5(C20). (E) The CCR5-terminus-specific mAb 5C7 (lane 2) immunoprecipitates CCR5 much more efficiently than the CCR5 ecl-2-specific mAb 2D7 (lane 1). Equal amounts (4 μg/ml) of these two mAbs were used for immunoprecipitation of CCR5 in 3T3.CD4.CCR5 cells. The molecular markers are shown on the right side (lane M). (F and G) Differential inhibition by two anti-CCR5 mAbs, m182 and m183 (which do not immunoprecipitate CCR5 as measured by our assay) of the CCR5 coimmunoprecipitation by the anti-CD4 mAb OKT4. Lanes 1, 2, and 3 represent 1, 2, and 4 μg/ml of m182 and m183, respectively. CCR5 was detected by Western blotting with CKR5(C20).