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J. Biol. Chem., Vol. 279, Issue 29, 30000-30008, July 16, 2004

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Ligand Selectivity and Affinity of Chemokine Receptor CXCR1

ROLE OF N-TERMINAL DOMAIN^*

Lavanya Rajagopalan and Krishna Rajarathnam

From the Department of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology, The University of Texas Medical Branch, Galveston, Texas 77555-1055

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (1 µM versus 20 µM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (3 µM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.

Received for publication, December 18, 2003 , and in revised form, March 31, 2004.

^* This work was supported by the American Heart Association, Texas Affiliate (to K. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S3.

To whom correspondence should be addressed: Dept. of Human Biological Chemistry and Genetics and Sealy Center for Structural Biology, The University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1055. Tel.: 409-772-2238; Fax: 409-772-1790; E-mail: krrajara{at}utmb.edu.

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