Human interferon-inducible 10-kDa protein and human interferon-inducible T cell alpha chemoattractant are allotopic ligands for human CXCR3: differential binding to receptor states.
The human CXC chemokines IP-10 (10-kDa interferon-inducible protein), MIG (monokine induced by human interferon-gamma), and I-TAC (interferon-inducible T cell alpha 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 [(35)S]GTP gamma S binding in recombinant cell and PBL membranes but had no effect in the absence of hCXCR3 expression. (125)I-IP-10 and (125)I-I-TAC bound hCXCR3 with high affinity, although the (125)I-I-TAC B(max) value in saturation bindings was 7- to 13-fold higher than that measured with (125)I-IP-10. Coincubation with unlabeled chemokines decreased (125)I-IP-10 binding with a single discernible affinity. However, with (125)I-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 IC(50) values did not increase predictably with increased (125)I-I-TAC concentration in competition bindings, suggesting that these chemokines are noncompetitive (i.e., allotopic) ligands. Uncoupling of hCXCR3 eliminated (125)I-IP-10 binding but only decreased (125)I-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 [(35)S]GTP gamma S binding. The antibody attenuated [(35)S]GTP gamma S binding in response to IP-10 but not to I-TAC, suggesting that the two chemokines bind differently to R*. Moreover, increased occupancy of R* with a >75-fold increase in (125)I -IP-10 concentration did not increase the I-TAC binding IC(50) value, and I-TAC increased the dissociation rate of (125)I-IP-10. From these data, we conclude that the binding of IP-10 and I-TAC to the R* state of hCXCR3 is allotopic.
Department of Immunology, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA.
M A
MA
Cox
C H
CH
Jenh
W
W
Gonsiorek
J
J
Fine
S K
SK
Narula
P J
PJ
Zavodny
R W
RW
Hipkin
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D018925:Chemokines / Q000494:pharmacology; D019743:Chemokines, CXC / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008214:Lymphocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*
D000906:Antibodies; C515202:CXCL11 protein, human; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D018925:Chemokines; D019743:Chemokines, CXC; C515203:Cxcl11 protein, mouse; C515188:Cxcr3 protein, mouse; D008024:Ligands; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine; D016244:Guanosine 5'-O-(3-Thiotriphosphate)