Lipopolysaccharide Is in Close Proximity to Each of the Proteins in Its Membrane Receptor Complex
The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation ofDrosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group (125I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex. The structural features of some proteins of the innate immune system involved in mediating responses to microbial pathogens are highly conserved throughout evolution. Examples include members of the Drosophila Toll (dToll) and the mammalian Toll-like receptor (TLR) protein families. Activation ofDrosophila Toll is believed to occur via an endogenous peptide rather than through direct binding of microbial products to the Toll protein. In mammals there is a growing consensus that lipopolysaccharide (LPS) initiates its biological activities through a heteromeric receptor complex containing CD14, TLR4, and at least one other protein, MD-2. LPS binds directly to CD14 but whether LPS then binds to TLR4 and/or MD-2 is not known. We have used transient transfection to express human TLRs, MD-2, or CD14 alone or in different combinations in HEK 293 cells. Interactions between LPS and these proteins were studied using a chemically modified, radioiodinated LPS containing a covalently linked, UV light-activated cross-linking group (125I-ASD-Re595 LPS). Here we show that LPS is cross-linked specifically to TLR4 and MD-2 only when co-expressed with CD14. These data support the contention that LPS is in close proximity to the three known proteins of its membrane receptor complex. Thus, LPS binds directly to each of the members of the tripartite LPS receptor complex. lipopolysaccharide LPS-binding protein radioiodinated sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3-dithiopropionate derivative of Salmonella minnesota Re595 LPS Toll-like receptor Drosophila Toll soluble CD14 membrane CD14 polyacrylamide gel electrophoresis fluorescence-activated cell sorter Bacterial endotoxin (lipopolysaccharide, LPS)1 okonp61 is a complex glycolipid composed of a hydrophilic polysaccharide moiety and a hydrophobic domain known as lipid A (1Seydel U. Schromm A.B. Blunck R. Brandenburg K. Chem. Immunol. 2000; 74: 5-24Crossref PubMed Scopus (73) Google Scholar). LPS is an outer membrane constituent of all Gram-negative bacteria where it has indispensable barrier functions. LPS is also a potent activator of innate immune responses that result in the production of pro- and anti-inflammatory mediators from myeloid lineage and other cell types (2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar). LPS-induced cell activation depends on the presence of three proteins comprising a multiprotein cell surface receptor complex here termed the LPS receptor complex. One essential protein of the LPS receptor complex is CD14 (3Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3406) Google Scholar), a 55-kDa glycoprotein present in soluble form (sCD14) in blood or as a membrane-bound form (mCD14) in myeloid lineage cells. This latter form is attached to the outer leaflet of the cell membrane via a glycosylphosphatidylinositol anchor. Multiple lines of biochemical and genetic evidence support the contention that CD14 principally acts to bind LPS and does not participate in signaling directly. Thus, others and we postulated that there must be at least one transmembrane protein that acts in concert with CD14 (2Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1323) Google Scholar). This putative transmembrane protein is now identified as a member of the mammalian Toll-like receptor (TLR) family and is TLR4 (4Medzhitov R. Preston-Hurlburt P. Janeway Jr., C. Nature. 1997; 388: 394-397Crossref PubMed Scopus (4439) Google Scholar, 5Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar). Genetic and biochemical studies suggest that TLR4 plays an important role in LPS signaling under physiological conditions. Positional cloning and sequencing of the lps d locus localized the defect to the tlr4 gene (6Poltorak A. He X. Smirnova I. Liu M.-Y. Van Huffel C. Birdwell X.D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar). The importance of TLR4 in LPS signaling is further supported by the fact that TLR4-deficient mice are LPS hyporesponsive but respond normally to products of Gram-positive organisms (7Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2790) Google Scholar). MD-2 is another protein that appears to be important in LPS signaling (8Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1749) Google Scholar). However, its function in the LPS receptor complex is currently unknown. TLR2 is a signaling receptor for a variety of microbial products that in some cases require CD14 for maximum activation (9Aderem A. Ulevitch R.J. Nature. 2000; 406: 7-782Crossref Scopus (2624) Google Scholar). Although TLR2 was initially thought to function as an LPS receptor current evidence suggests that it does not play a major role in the physiological response to LPS (9Aderem A. Ulevitch R.J. Nature. 2000; 406: 7-782Crossref Scopus (2624) Google Scholar, 10Hirschfeld M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar, 11Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar). Nonetheless a recent publication provides data showing that when TLR2 is overexpressed in 293 cells it mediates cell activation induced by a variety of purified LPS isolates (12Dziarski R. Wang Q. Miyake K. Kirschning C.J. Gupta D. J. Immunol. 2001; 166: 1938-1944Crossref PubMed Scopus (181) Google Scholar). It thus appears that TLR2 is more promiscuous with respect to ligand recognition and that LPS must be added to the list of TLR2 ligands. Numerous reports describe the direct binding of LPS to CD14 (3Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3406) Google Scholar,13Frey E.A. Miller D.S. Jahr T.G. Sundan A. Bazil V. Espevik T. Finlay B.B. Wright S.D. J. Exp. Med. 1992; 176: 1665-1671Crossref PubMed Scopus (606) Google Scholar, 14Pugin J. Schurer-Maly C.C. Leturcq D. Moriarty A. Ulevitch R.J. Tobias P.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2744-2748Crossref PubMed Scopus (731) Google Scholar, 15Pugin J. Ulevitch R.J. Tobias P.S. J. Exp. Med. 1993; 178: 2193-2200Crossref PubMed Scopus (136) Google Scholar). The question of whether LPS binds to, or even is in close proximity to the other proteins of the LPS receptor remains unanswered. Here we have used radioiodinated Re595 LPS with a covalently attached UV-activated phenylazide that is capable of cross-linking to nearby proteins in order to characterize interactions between LPS and the LPS receptor complex at the cell surface. Our results show that LPS is brought into close proximity to TLR4 only when it is present as an LPS·CD14 complex and when CD14 and TLR4 are co-expressed with MD-2. Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10‥ fetal bovine serum, 2 mm glutamine, 100 units/ml penicillin, and 10 μg/ml streptomycin. 3.5 × 106 cells in 10-cm Petri dishes were transfected using LipofectAMINE Reagent Plus (Life Technologies, Inc., Gaithersburg, MD). The total amount of DNA was kept constant by supplementing pFLAG-CMV1 vector DNA (Eastman Kodak Co., Rochester, NY). Murine anti-human TLR4 monoclonal antibodies were from Dr. K. Miyake. The following reagents were prepared as described in the references: Re595 LPS (16Mathison J.C. Wolfson E. Ulevitch R.J. J. Clin. Invest. 1988; 81: 1925-1937Crossref PubMed Scopus (461) Google Scholar), rabbit LBP (17Theofan G. Horwitz A.H. Williams R.E. Liu P.-S. Chan I. Birr C. Carroll S.F. Meszaros K. Parent J.B. Kasler H. Aberle S. Trown P.W. Gazzano-Santoro H. J. Immunol. Google Scholar), and human soluble CD14 V. J. Moriarty A. Leturcq D. Tobias P.S. Ulevitch R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). monoclonal antibodies CD14 and were a from A. Moriarty and D. Leturcq monoclonal was from LPS and LPS were from E. LPS was from was from In order to protein the Re595 LPS was also to the of M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar). This was also used to results with derivative of the Re595 LPS were not from the Human CD14 was in vector as described V. J. Moriarty A. Leturcq D. Tobias P.S. Ulevitch R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). The for human TLR2 and TLR4 were from P. and C. The for Drosophila Toll and human were from Dr. was in pFLAG-CMV1 using for was by DNA into pFLAG-CMV1 in the an 293 cells in 10-cm Petri dishes were transfected and 2 were 2 with mm mm and 2 with containing mm were and in Dulbecco's modified Eagle's medium containing mm and human bovine Inc., were and as described P.S. K. K. Ulevitch R.J. J. Immunol. 1993; Google Scholar). μg/ml and LPS were in a containing mm and mm 10 at were added to 10 at and with a UV for on A a of Re595 LPS and μg/ml was cells were for at in binding containing 10 mm 2 mm and mm to of were and in of containing mm 100 mm 2 mm 10‥ 100 mm mm 10 mm 100 units/ml 100 mm for on cell were 10 and the were were for at with of protein and with of monoclonal antibodies for at under constant were to bind to of protein were with and the in 100 of and for 10 from of the were on and to were with in mm mm and with for 2 and with for at were using the Plus system of of were on The were and the with protein was with a described here was at least three Here we show 293 cells were at a of × in and transfected with the with vector Inc., for 2 cells were in containing fetal and and with or μg/ml for at 2 cells were for with were then and with a HEK 293 cells were transfected with combinations of DNA human CD14 and of TLR4 and MD-2. transfection the cells were and to a complex of LBP and LPS as described P.S. K. K. Ulevitch R.J. J. Immunol. 1993; Google Scholar). cells were and the with monoclonal The were to to by and then to TLR4 and MD-2 protein by with for LPS cross-linking to TLR4 and MD-2 was by the protein only when CD14 was We to binding of LPS to TLR4 or MD-2 when alone 2 and or in with CD14 and TLR4 and MD-2 were a to MD-2 was In not were even when we the LPS to as as 10 all were with of MD-2 appears as by and to Silva and R. J. in Although CD14 does not an we were to it from a and lines of evidence suggest a amount of in the of the amount of LPS that LPS to TLR4 and MD-2 than of total LPS to the complex. The is to CD14 or other This is not the major function of CD14 to bind and LPS in a of cell only the presence of LPS to CD14 was to LPS to TLR4 and MD-2 not and function to responses to LPS (3Wright S.D. Ramos R.A. Tobias P.S. Ulevitch R.J. Mathison J.C. Science. 1990; 249: 1431-1433Crossref PubMed Scopus (3406) Google E. T. M. Wright S.D. J. Immunol. Google Scholar, Mathison J.C. Tobias P.S. Leturcq Moriarty R.J. Ulevitch R.J. J. Clin. Invest. 1992; 90: PubMed Scopus Google Scholar, P.S. K. D. Ulevitch R.J. J. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). We whether for in LPS binding to TLR4 and MD-2. 293 cells were transfected with TLR4 and MD-2 alone or in The cells were then and to the complex LPS binding to TLR4 and MD-2 only when proteins were co-expressed of TLR4 or MD-2 was when these proteins are alone 2 and These data suggest that LPS was from an complex to a complex in a whether other member of the family bind LPS in a CD14 and MD-2 were with cells TLR4, or 293 cells were transfected with MD-2 and CD14 in the presence of TLR4, or A a binding of LPS only to binding to or be under conditions. of each of the proteins was by 2 and 2 TLR4 and MD-2 were at cell surface of 293 cells when alone using a monoclonal or with The of TLR4 with MD-2 the of This was with or it was on MD-2, the of CD14 in the of MD-2 on the of TLR4 not and were at cell surface as in 2 with TLR4, with MD-2 not cell surface of or not TLR2 to be a receptor for Gram-negative LPS C.J. H. M. J. Exp. Med. 1998; PubMed Scopus Google Scholar, A. A. M. A. Nature. 1998; PubMed Scopus Google Scholar). However, these in from present in the LPS isolates M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar, Akashi S. Miyake K. Tobias P.S. J. Immunol. 2000; 165: PubMed Scopus Google Scholar). the other recent of (12Dziarski R. Wang Q. Miyake K. Kirschning C.J. Gupta D. J. Immunol. 2001; 166: 1938-1944Crossref PubMed Scopus (181) Google suggests that TLR2 some of LPS that of Re595 as of the of the we whether Re595 LPS also be brought into close proximity with TLR2 and whether CD14 and MD-2 are We cross-linking of Re595 LPS to TLR2 in the of CD14 and MD-2 We that of CD14 the amount of LPS to TLR2 In to with TLR4, of MD-2 on Re595 LPS cross-linking to TLR2 whether or not CD14 was present and we to binding of Re595 LPS to MD-2 of whether TLR2 and/or CD14 were TLR2 protein was constant in each of the in and by and in to with TLR4, of MD-2 to cell surface of TLR2 that of TLR2 and MD-2 in total of each protein in cell when with the with each (12Dziarski R. Wang Q. Miyake K. Kirschning C.J. Gupta D. J. Immunol. 2001; 166: 1938-1944Crossref PubMed Scopus (181) Google Scholar). However, the latter not the surface we directly In order to be of the of the LPS used in studies we Re595 LPS to the described in M. Ma Y. Weis J.H. Vogel S.N. Weis J.J. J. Immunol. 2000; 165: 618-622Crossref PubMed Scopus (971) Google Scholar). We used the LPS to the radioiodinated derivative for cross-linking The Re595 LPS in all the of cross-linking to TLR2 as not it is that the results we describe here with the Re595 LPS interactions of LPS and not protein with cell surface of LPS to CD14 has to be of Leturcq D. Moriarty A. Ulevitch R.J. Tobias P.S. J. Chem. 1993; Full Text PDF PubMed Google Scholar). We for LPS binding to MD-2 and we binding of Re595 LPS to TLR4 and MD-2 in 293 LPS was added to the cells in the presence and of of and to Re595 LPS cross-linking to of LPS from CD14 to the complex does not require results were with The lipid A moiety has identified as the LPS for LPS-induced biological through direct with CD14 (1Seydel U. Schromm A.B. Blunck R. Brandenburg K. Chem. Immunol. 2000; 74: 5-24Crossref PubMed Scopus (73) Google Scholar). whether LPS binding to TLR4 and MD-2 is a lipid were in 293 cells transfected with CD14, TLR4, and MD-2. were with an of LPS a lipid A known as lipid Immunol. 1992; 13: Full Text PDF PubMed Scopus Google and for to the of LPS LPS from E. and binding of Re595 The different LPS isolates as as lipid LPS binding to TLR4 and MD-2 and In not Re595 LPS binding to TLR4 and MD-2. have that is a ligand for TLR2 R. R. H. M. Kirschning C.J. J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. B. P. A. Science. 1999; PubMed Scopus Google Scholar, A. E. E. R. D. J. Immunol. 1999; PubMed Google Scholar). These data support the contention that the interactions between Re595 LPS and are lipid further the importance of CD14 in of LPS to the we LPS binding to TLR4 and MD-2 in the or presence of monoclonal antibodies monoclonal antibodies were known to LPS binding to and Ulevitch R.J. Tobias P.S. J. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), an LPS-induced cell activation but does not LPS binding to CD14 Leturcq D. Moriarty A. Ulevitch R.J. Tobias P.S. J. Chem. 1993; Full Text PDF PubMed Google and an that has of cell activation and on LPS binding to CD14 K. J. Tobias P.S. Ulevitch R.J. J. 1997; PubMed Scopus Google Scholar). with LPS binding to TLR4 and MD-2. with not LPS binding to TLR4 and binding to MD-2. The cross-linking to TLR4 and MD-2. In these results suggest that binding of LPS to CD14 is essential for the that interactions of LPS with TLR4 and MD-2. an anti-human CD14 monoclonal that cell activation LPS binding to CD14 not LPS interactions with other members of the receptor complex. This suggests that to LPS binding to TLR4 and MD-2 are for cell support for from studies with monoclonal the monoclonal antibodies to and all LPS-induced and from blood cells Akashi S. Miyake K. Tobias P.S. J. Immunol. 2000; 165: PubMed Scopus Google Scholar). to show the in the system not we used these antibodies to whether LPS binding to TLR4 and MD-2. of the antibodies of TLR4 or MD-2. One of the three the by not it at or an LPS-binding of Here we have used a radioiodinated derivative of Re595 LPS with a UV-activated phenylazide to binding to CD14, we cross-linking of Re595 LPS to other proteins in the putative LPS receptor complex. we that binding to CD14, Re595 LPS is in close proximity to TLR4 and MD-2 that cross-linking These interactions not require but are by CD14, as in monoclonal antibodies that LPS binding to CD14 the interactions with TLR4 and MD-2. monoclonal antibodies that LPS-induced cell activation not LPS binding to CD14, TLR4, or MD-2. these data suggest that LPS binds directly to proteins in the LPS membrane receptor complex but that binding to TLR4 and/or MD-2 is not to cell as protein or of another member of the LPS receptor complex be to transmembrane In we used HEK 293 cells not express endogenous TLR4, MD-2, or CD14. transient transfection we were to of each of the three of LPS of TLR4 that was to be at in cells B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar, A. Ricciardi-Castagnoli P. S. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). cell system to TLR4 and MD-2 as proteins in and by the proteins are essential for of studies to binding to proteins other than CD14 at the cell surface as a result of the of surface of TLR4 and MD-2 P.S. K. K. Ulevitch R.J. J. Immunol. 1993; Google Scholar, S. Ogata H. T. K. M. R. Nagai Y. Fukudome K. Kimoto M. Miyake K. 2000; PubMed Scopus Google Scholar). LPS binding to TLR4 and MD-2 only in the presence of the complex composed of CD14, TLR4, and MD-2. CD14 the major binding on the cell surface it more than of total LPS to the Our data suggest that only a of the LPS to CD14 is to This is of the results of Ulevitch R.J. Tobias P.S. J. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google that of the LPS not to be involved in the present system only provides binding data and of require the of more The by TLR4 LPS signaling the membrane is not known. Our suggest a complex for activation of cells by LPS is from a complex to a complex at cell surface. lines of evidence support LPS binds to TLR4 and MD-2 only in the presence of CD14. Although MD-2 and TLR4 were at cell surface of cells when co-expressed as by only binding of LPS to protein be in the of CD14. CD14 be present on the cell surface as a membrane-bound protein, or added to the cells as a soluble an that has to the LPS-binding on CD14, LPS binding not only to CD14, but also to TLR4 and MD-2. there is a role of CD14 in LPS to TLR4 and MD-2. LPS binding to TLR4 and MD-2 was not in the presence of a of that of LPS at the The role of LPS binding to TLR4 and MD-2 with respect to is not the monoclonal known to LPS-induced cell activation binding of LPS to CD14 Leturcq D. Moriarty A. Ulevitch R.J. Tobias P.S. J. Chem. 1993; Full Text PDF PubMed Google Scholar), to LPS binding to TLR4 and MD-2. monoclonal antibodies a or of LPS binding to TLR4 or MD-2. One of these data is that TLR4 has different in the an LPS-binding an activation and involved in with TLR4 MD-2 and/or CD14. It is that of TLR4 following interactions with A. Ricciardi-Castagnoli P. S. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google have also that LPS binds to TLR4 and initiates for cell biochemical and genetic studies a role of TLR2 as a receptor for and from Gram-positive bacteria R. R. H. M. Kirschning C.J. J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. E. E. R. D. J. Immunol. 1999; PubMed Google Scholar, E. A. G. Carroll Espevik T. J. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, O. E. G. Sundan A. Espevik T. J. Immunol. 2000; PubMed Scopus Google its role as a receptor for Gram-negative LPS under physiological is in (7Takeuchi O. Hoshino K. Kawai T. Sanjo H. Takada H. Ogawa T. Takeda K. Akira S. Immunity. 1999; 11: 443-451Abstract Full Text Full Text PDF PubMed Scopus (2790) Google Scholar, 11Beutler B. Curr. Opin. Immunol. 2000; 12: 20-26Crossref PubMed Scopus (648) Google Scholar). It does that purified LPS is capable of cells TLR2 in the presence of MD-2 (12Dziarski R. Wang Q. Miyake K. Kirschning C.J. Gupta D. J. Immunol. 2001; 166: 1938-1944Crossref PubMed Scopus (181) Google Scholar). it has that cells as as from mice respond to LPS from E. and lipid A specifically through TLR2 Y. T. M. H. J. Ogawa T. O. Akira S. Y. Y. J. Immunol. 2000; 165: PubMed Scopus Google Scholar, M. Weis J.J. V. Vogel S.N. 2001; PubMed Scopus Google Scholar). Here we show that LPS and TLR2 are capable of as by the of TLR2 following to the Re595 However, in to with TLR4, of MD-2 to these It is important to that we not cross-linked products when other members of the Toll or were at the surface of 293 cells even when co-expressed with CD14 and MD-2. we are that the cross-linked products in cells TLR4 or TLR2 LPS interactions with proteins as studies an essential role for MD-2 in responses to LPS H. J.C. J. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). data were to suggest a for MD-2 Here we show that MD-2 is essential to LPS binding to TLR4, a that be to be essential in the activation a protein some into MD-2 functions. is a protein that binds to a protein known as a member of Toll receptor family that is specifically on K. R. J. T. Akashi S. Ogata H. Y. Y. Kimoto M. J. Immunol. 1998; Google Y. R. Miyake K. Akashi S. Ogata H. Y. Y. Kimoto M. 1998; PubMed Google Scholar). is thought to of at the cell surface. and (8Shimazu R. Akashi S. Ogata H. Nagai Y. Fukudome K. Miyake K. Kimoto M. J. Exp. Med. 1999; 189: 1777-1782Crossref PubMed Scopus (1749) Google have that MD-2 is not on the cell surface of the cell in the of In that MD-2 be at the cell surface of 293 cells in the of TLR4 was also at cell surface when However, the presence of MD-2 cell MD-2 complex with TLR4 and with the However, an was with and the monoclonal we the latter is studies in have that MD-2 with of the of studies are to these interactions through of that the of TLR4, containing play a role in binding its LPS binding but not with MD-2 not The biochemical data we have with results of others A. Ricciardi-Castagnoli P. S. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google a where LPS binds directly to the known proteins of the putative LPS receptor complex. This with is believed to occur in Drosophila where products of microbial pathogens not directly with Toll-like essential question that remains is binding to TLR4 and MD-2 is to the of a transmembrane We Dr. for of the