Human interferon-inducible 10-kDa protein and human interferon-inducible Tcell alpha chemoattractant are allotopic ligands for human CXCR3: Differential binding to receptor states
Ma. Cox et al., Human interferon-inducible 10-kDa protein and human interferon-inducible Tcell alpha chemoattractant are allotopic ligands for human CXCR3: Differential binding to receptor states, MOLEC PHARM, 59(4), 2001, pp. 707-715
The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (
monokine induced by human interferon-gamma), and I-TAC (interferon-inducibl
e T cell alpha chemoattractant) attract lymphocytes through activation of C
XCR3. In the studies presented here, we examined interaction of these chemo
kines with human CXCR3 expressed in recombinant cells and human peripheral
blood lymphocytes (PBL). IP-10, MIG, and I-TAC were agonists in stimulating
[S-35]GTP gammaS binding in recombinant cell and PBL membranes but had no
effect in the absence of hCXCR3 expression. I-125-IP-10 and I-125-I-TAC bou
nd hCXCR3 with high affinity, although the I-125-I-TAC B-max value in satur
ation bindings was 7- to 13-fold higher than that measured with I-125-IP-10
. Coincubation with unlabeled chemokines decreased I-125-IP-10 binding with
a single discernible affinity. However, with I-125-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 IC50 values did not
increase predictably with increased I-125-I-TAC concentration in competitio
n bindings, suggesting that these chemokines are noncompetitive (i.e., allo
topic) ligands. Uncoupling of hCXCR3 eliminated I-125-IP-10 binding but onl
y decreased I-125-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 e
xamine chemokine binding to R*, we tested the effect of anti-hCXCR3 antibod
y on I-TAC- and IP-10-stimulated [S-35]GTP gammaS binding. The antibody att
enuated [S-35]GTP gammaS binding in response to IP-10 but not to I-TAC, sug
gesting that the two chemokines bind differently to R*. Moreover, increased
occupancy of R* with a > 75-fold increase in I-125-IP-10 concentration did
not increase the I-TAC binding IC50 value, and I-TAC increased the dissoci
ation rate of I-125-IP-10. From these data, we conclude that the binding of
IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.