Abstract

The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-γ), and I-TAC (interferon-inducible T cell α chemoattractant) attract lymphocytes through activation of CXCR3. In the studies presented here, we examined interaction of these chemokines with human CXCR3 expressed in recombinant cells and human peripheral blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating [³⁵S]GTPγS binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. ¹²⁵I-IP-10 and¹²⁵I-I-TAC bound hCXCR3 with high affinity, although the¹²⁵I-I-TAC B _max value in saturation bindings was 7- to 13-fold higher than that measured with¹²⁵I-IP-10. Coincubation with unlabeled chemokines decreased ¹²⁵I-IP-10 binding with a single discernible affinity. However, with ¹²⁵I-I-TAC, competition with IP-10 or MIG was incomplete, and multiple binding affinities were evident. Moreover, in contrast to I-TAC, IP-10 and MIG binding IC₅₀values did not increase predictably with increased¹²⁵I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated ¹²⁵I-IP-10 binding but only decreased ¹²⁵I-I-TAC binding 30 to 80%, indicating that unlike IP-10, I-TAC binds with high affinity to uncoupled (R) and coupled (R*) hCXCR3. To examine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibody on I-TAC- and IP-10-stimulated [³⁵S]GTPγS binding. The antibody attenuated [³⁵S]GTPγS binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently toR*. Moreover, increased occupancy of R* with a >75-fold increase in ¹²⁵I -IP-10 concentration did not increase the I-TAC binding IC₅₀ value, and I-TAC increased the dissociation rate of¹²⁵I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.