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J. Biol. Chem., Vol. 282, Issue 9, 6906-6915, March 2, 2007

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CXCR1 and CXCR2 Activation and Regulation

ROLE OF ASPARTATE 199 OF THE SECOND EXTRACELLULAR LOOP OF CXCR2 IN CXCL8-MEDIATED RAPID RECEPTOR INTERNALIZATION^*

Mohd W. Nasser, Sandeep K. Raghuwanshi, Kimberly M. Malloy, Pavani Gangavarapu, Joong-Youn Shim, Krishna Rajarathnam, and Ricardo M. Richardson¹

From the Julius L. Chambers Biomedical/Biotechnology Research Institute and the Department of Biology, North Carolina Central University, Durham, North Carolina 27707 and the Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555-1055

CXCL8 (interleukin-8) interacts with two receptors, CXCR1 and CXCR2, to activate leukocytes. Upon activation, CXCR2 internalizes very rapidly relative to CXCR1 (90% versus 10% after 5 min). The C termini of the receptors have been shown to be necessary for internalization but are not sufficient to explain the distinct kinetics of down-regulation. To determine the structural determinant(s) that modulate receptor internalization, various chimeric and point mutant receptors were generated by progressively exchanging specific domains or amino acids between CXCR1 and CXCR2. The receptors were stably expressed in rat basophilic leukemia 2H3 cells and characterized for receptor binding, intracellular Ca²⁺ mobilization, phosphoinositide hydrolysis, phosphorylation, internalization, and MAPK activation. The data herein indicate that the second extracellular loop (2ECL) of the receptors is critical for the distinct rate of internalization. Replacing the 2ECL of CXCR2 with that of CXCR1 (B_2ECLA) or Asp¹⁹⁹ with its CXCR1 valine counterpart (B_D199VA) delayed CXCR2 internalization similarly to CXCR1. Replacing Asp¹⁹⁹ with Asn (B_D199N) restored CXCR2 rapid internalization. Structure modeling of the 2ECL of the receptors also suggested that Asp¹⁹⁹ plays a critical role in stabilizing and modulating CXCR2 rapid internalization relative to CXCR1. B_D199N internalized rapidly but migrated as a single phosphorylated form like CXCR1 (75 kDa), whereas B_2ECLA and B_D199VA showed slow and fast migrating forms like CXCR2 (45 and 65 kDa, respectively) but internalized like CXCR1. These data further undermine the role of receptor oligomerization in CXCL8 receptor internalization. Like CXCR1, B_D199VA also induced sustained ERK activation and cross-desensitized Ca²⁺ mobilization to CCR5 relative to B_D199N and CXCR2. Altogether, the data suggest that the 2ECL of the CXCL8 receptors is important in modulating their distinct rate of down-regulation and thereby signal length and post-internalization activities.

Received for publication, November 3, 2006 , and in revised form, December 26, 2006.

^* This work was supported by National Institutes of Health Grants AI-38910 (to R. M. R.) and AI-069152 (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.

¹ To whom correspondence should be addressed: Julius L. Chambers Biomedical/Biotechnology Research Inst., North Carolina Central University, 1801 Fayetteville St., Durham, NC 27707. Tel.: 919-530-6421; Fax: 919-530-7780; E-mail: mrrichardson{at}nccu.edu.

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