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Human interferon-inducible protein 10 (IP-10; HGMW-approved gene symbol CXCL10) is an ELR(-) CXC chemokine that contains binding domains for both the chemokine receptor CXCR3 and glycosaminoglycans. IP-10 has been recently demonstrated to be a potent angiostatic protein in vivo. Whether IP-10 exerts its angiostatic function through binding to CXCR3, glycosaminoglycans, or both, is not clear. To clarify this issue, we created expression constructs for mutants of IP-10 that exhibit partial (IP-10C) or total (IP-10C22) loss of binding to CXCR3 or loss of binding to glycosaminoglycans (IP-10H and IP-10C22H). The A375 human melanoma cell line was transfected with these expression vectors, and stable clones were selected and inoculated subcutaneously into nude mice. As expected, tumor cells secreting wild-type IP-10 showed remarkable reduction in tumor growth compared to control vector-transfected tumor cells. Surprisingly, mutation of IP-10 resulting in partial loss of receptor binding (IP-10C), or loss of GAG binding (IP-10H), did not significantly alter the ability to inhibit tumor growth. This tumor growth inhibition was associated with a reduction in microvessel density, leading to the observed increase in both tumor cell apoptosis and necrosis. In contrast, expression of the IP-10C22 mutant failed to inhibit melanoma tumor growth. These data suggest that CXCR3 receptor binding, but not glycosaminoglycan binding, is essential for the tumor angiostatic activity of IP-10. We conclude that the arginine 22 amino acid residue of IP-10 is essential for both CXCR3 binding and angiostasis.