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J. Biol. Chem., Vol. 283, Issue 3, 1257-1266, January 18, 2008

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Novel Roles in Human MD-2 of Phenylalanines 121 and 126 and Tyrosine 131 in Activation of Toll-like Receptor 4 by Endotoxin^*

Athmane Teghanemt, Fabio Re, Polonca Prohinar, Richard Widstrom, Theresa L. Gioannini^¶||, and Jerrold P. Weiss^**1

From the Department of Internal Medicine and The Inflammation Program, the ^**Department of Microbiology, and the ^||Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, the ^¶Veterans' Administration Medical Center, Iowa City, Iowa 52246, and the Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, Tennessee 38163

Potent mammalian cell activation by Gram-negative bacterial endotoxin requires sequential protein-endotoxin and protein-protein interactions involving lipopolysaccharide-binding protein, CD14, MD-2, and Toll-like receptor 4 (TLR4). TLR4 activation requires simultaneous binding of MD-2 to endotoxin (E) and the ectodomain of TLR4. We now describe mutants of recombinant human MD-2 that bind TLR4 and react with E·CD14 but do not support cellular responsiveness to endotoxin. The mutants F121A/K122A MD-2 and Y131A/K132A MD-2 react with E·CD14 only when co-expressed with TLR4. Single mutants K122A and K132A each react with E·CD14 ± TLR4 and promote TLR4-dependent cell activation by endotoxin suggesting that Phe¹²¹ and Tyr¹³¹ are needed for TLR4-independent transfer of endotoxin from CD14 to MD-2 and also needed for TLR4 activation by bound E·MD-2. The mutant F126A MD-2 reacts as well as wild-type MD-2 with E·CD14 ± TLR4. E·MD-2^F126A binds TLR4 with high affinity (K_d 200 pM) but does not activate TLR4 and instead acts as a potent TLR4 antagonist, inhibiting activation of HEK/TLR4 cells by wild-type E·MD-2. These findings reveal roles of Phe¹²¹ and Tyr¹³¹ in TLR4-independent interactions of human MD-2 with E·CD14 and, together with Phe¹²⁶, in activation of TLR4 by bound E·MD-2. These findings strongly suggest that the structural properties of E·MD-2, not E alone, determine agonist or antagonist effects on TLR4.

Received for publication, July 23, 2007 , and in revised form, October 30, 2007.

^* This work was supported by United States Public Health Service Grants R21 AI 54665 (to F. R.), P0144642 and AI59372 (to J. P. W.), and a Veterans' Administration Merit Review grant (to T. L. G.). 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: Roy J. and Lucille A. Carver College of Medicine, University of Iowa and Veterans Administration Medical Center, Inflammation Program, 2501 Crosspark Rd., Coralville, IA 52241. Tel.: 319-335-4268; Fax: 319-335-4191; E-mail: jerrold-weiss{at}uiowa.edu.

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