Chemokines play important roles in immune defense by directing migration of leukocytes and serve as key promoters of tumorigenesis and metastasis. This study explores the molecular mechanisms of recognition and activation of two homologous chemokine receptors, CXCR1 and CXCR2, using CXCL8 analogues with residue substitutions in the conserved Glu4Leu5Arg6 (ELR) triad. Analysis of the binding of CXCL8 analogues to CXCR1 is consistent with the two-site model for signal recognition of CXCR1, whereas analysis of the binding of CXCL8 analogues to CXCR2 supported a single-site model for signal recognition of CXCR2. The CXCL8-Arg6His analogue stimulated calcium release, phosphorylation of ERK1/2, and chemotaxis in cells expressing CXCR1. However, CXCL8-Arg6His failed to stimulate calcium release and chemotaxis in cells expressing CXCR2, although it stimulated phosphorylation of ERK1/2, indicating that CXCL8-Arg6His operated as a classical CXCR2 biased agonist. The CXCL8-Glu4AlaLeu5AlaArg6His analogue was inactive in cells expressing CXCR1 and CXCR2. These findings suggest that the Glu4Leu5 motif in CXCL8 is essential for activation of CXCR1 and CXCR2. Importantly, CXCL8-Glu4AlaLeu5AlaArg6His blocked specifically the calcium release and chemotaxis of cells expressing CXCR1 but not of cells expressing CXCR2. CXCL8-Glu4AlaLeu5AlaArg6His was identified as the first specific CXCR1 antagonist. The binding of CXCL8-ELR6H to CXCR1 created a Zn 2+ coordination site at the receptor activation domain responsible for calcium release, as ZnCl 2 specifically blocked CXCL8-Arg6His-induced calcium release without affecting CXCL8-induced calcium release. This work provides the basis for further exploration of the activation mechanisms of chemokine receptors and will assist in the design of the next generation of modulators of CXCR1 and CXCR2.
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