CD96 Interaction with CD155 via Its First Ig-like Domain Is Modulated by Alternative Splicing or Mutations in Distal Ig-like Domains

The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes. The adhesion receptor CD96 (TACTILE) is a transmembrane glycoprotein possessing three extracellular immunoglobulin-like domains. Among peripheral blood cells, CD96 is expressed on T cells as well as NK cells and a subpopulation of B cells. A possible function of this receptor in NK cell-mediated killing activities was suggested recently. Moreover, CD96 was described as a tumor marker for T-cell acute lymphoblastic leukemia and acute myeloid leukemia. CD96 binds to CD155 (poliovirus receptor) and nectin-1, an adhesion receptor related to CD155. Here we report that human but not mouse CD96 is expressed in two splice variants possessing either an I-like (variant 1) or V-like (variant 2) second domain. With the notable exception of an AML tumor sample, variant 2 predominates in all the CD96-expressing cell types and tissues examined. Using chimeric human/murine CD96 receptors, we show that the interaction with its ligands is mediated via the outermost V-like domain. In contrast to mouse, however, the binding of human CD96 to CD155 is sensitive to the characteristics of the two downstream domains. This is illustrated by a significantly weaker CD96/CD155 interaction mediated by variant 1 when compared with variant 2. Moreover, recent evidence suggested that mutations in human CD96 correlate with the occurrence of a rare form of trigonocephaly. One such mutation causing a single amino acid exchange in the third domain of human CD96 decreased the capacity of both variants to bind to CD155 considerably, suggesting that a CD96-driven adhesion to CD155 may be crucial in developmental processes. CD96 is a single pass transmembrane glycoprotein belonging to the Ig superfamily (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). In its extracellular part, CD96 consists of three Ig-like domains in the order VN-term-V-CC-term and a membrane proximal region rich in proline/serine/threonine. It is assumed that extensive O-linked glycomodification of this region equips CD96 with a semi-flexible stalk. Therefore, the Ig-like domains may protrude among other membrane-bound macromolecules comprising the outer layer of the cell (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). Even though such exposed presentation predestines CD96 for roles in adhesion, interaction partners of CD96 as well as its biological function(s) remained largely obscure until recently. However, CD96 was described as tumor marker for T-ALL and AML characteristic for the AML stem cell in particular (2Gramatzki M. Ludwig W.D. Burger R. Moos P. Rohwer P. Grunert C. Sendler A. Kalden J.R. Andreesen R. Henschke F. Moldenhauer G. Exp. Hematol.. 1998; 26: 1209-1214Google Scholar, 3Hosen N. Park C.Y. Tatsumi N. Oji Y. Sugiyama H. Gramatzki M. Krensky A.M. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 11008-11013Google Scholar). Among human peripheral blood cells, CD96 expression was observed on T and NK cells but not on the majority of B cells, monocytes, and granulocytes (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar). The murine CD96, characterized recently, is widely expressed on T cells of all developmental and differentiation stages tested albeit to varying extents (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). This is in line with the original observation of elevated CD96 expression in activated human T cells (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). In humans, CD96 promotes adhesion of NK cells to tumor cells expressing CD155, an interaction that may assist in NK-mediated destruction of the targets (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar); yet direct evidence proving CD96 as NK receptor is missing. Although CD155 remained the only identified interaction partner for CD96 in human (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar), nectin-1 also binds to CD96 in mouse (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). Although the significance of the CD96/nectin-1 interaction remains unclear, it is interesting to note that nectin-1 is expressed prominently in brain and, in particular, localizes to the synaptic junctions. Nectin-1-deficient mice suffer from microphthalmia (small eyes), an eye disorder caused by malformation of cell layers of the ciliary epithelia (6Inagaki M. Irie K. Ishizaki H. Tanaka-Okamoto M. Morimoto K. Inoue E. Ohtsuka T. Miyoshi J. Takai Y. Development.. 2005; 132: 1525-1537Google Scholar). In humans, mutations in nectin-1 were associated with the occurrence of cleft lip/palate-ectodermal dysplasia syndrome (7Suzuki K. Hu D. Bustos T. Zlotogora J. Richieri-Costa A. Helms J.A. Spritz R.A. Nat. Genet.. 2000; 25: 427-430Google Scholar), whereas a mutation in CD96 (T280M) or disruption of the genetic locus of CD96 by chromosomal translocation link to a distinct manifestation of the C syndrome (Opitz Trigonocephaly) (8Kaname T. Yanagi K. Chinen Y. Makita Y. Okamoto N. Maehara H. Owan I. Kanaya F. Kubota Y. Oike Y. Yamamoto T. Kurosawa K. Fukushima Y. Bohring A. Opitz J.M. Yoshiura K. Niikawa N. Naritomi K. Am. J. Hum. Genet.. 2007; 81: 835-841Google Scholar). Nectin-1 as well as CD155 belong to a subfamily of Ig-like adhesive receptors (9Takai Y. Irie K. Shimizu K. Sakisaka T. Ikeda W. Cancer Sci.. 2003; 94: 655-667Google Scholar). An evolutionary analysis revealed that the members of this family share a common progenitor with CD96 and CD226 (10Du Pasquier L. Zucchetti I. De Santis R. Immunol. Rev.. 2004; 198: 233-248Google Scholar), another binding partner for CD155 (11Bottino C. Castriconi R. Pende D. Rivera P. Nanni M. Carnemolla B. Cantoni C. Grassi J. Marcenaro S. Reymond N. Vitale M. Moretta L. Lopez M. Moretta A. J. Exp. Med.. 2003; 198: 557-567Google Scholar). Mice deficient for CD155 produce a to the via the whereas or not M.K. S. N. Qiu Q. Ravens I. Kremmer E. M. W. Forster R. Bernhardt G. J. Immunol.. 2007; Scholar). the of CD96 and members of the family in as well as developmental processes. In this we were to the interaction of CD96 with its binding partners in this chimeric receptors were of distinct mouse and human Ig-like domains. It be that the domain of CD96 the for CD155 However, the of binding is by the second domain in of human but not mouse to murine CD96, human CD96 is to two in the Ig of the second domain. analysis by revealed that the variant is expressed in all and cell types CD155 binds to both splice variants yet with Moreover, the mutation in Ig-like domain three of CD96 in a significantly binding to CD155. of CD96 human murine T-cell acute lymphoblastic acute myeloid deficient in and was of blood cell by The 1 of 2 and an of 2 with an of The and the were types of were also 1 the whereas in variant 2 this is causing the of amino in such that not amino acid of by the the expression of murine or were cells 2 the cells were The AML cell line expressing as a cells were with for and with and in 1 of 2 of was in 1 of and to the cells. The was for and with of The was in 1 of for on and to cell the cell were either with of (2Gramatzki M. Ludwig W.D. Burger R. Moos P. Rohwer P. Grunert C. Sendler A. Kalden J.R. Andreesen R. Henschke F. Moldenhauer G. Exp. Hematol.. 1998; 26: 1209-1214Google or of with of The were with and with The were in of on a the were and the was revealed with in and with and of and for and were to to or to the Although the of the was that of the caused amino acid were The mutation was both of the were to The and C. and of or of the human/murine CD96 were cells with 2 of a the The cells were and with either or a of or S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google for or mouse were for CD155 and nectin-1 were expressed and as as described (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). of the of CD155 or to domains of cells were with 2 of on and and was by mouse of on cells expressing or the cells were with the of in of for 1 on three was In the cells were with of and and revealed by was to cells from The cells were on a or and the were The binding of and in was to a from to was to the binding to the receptors in of and for and the 1) were in the the of the and for The binding to variants were by The were for the 2) from binding were to the expression of CD96 variant by by the of The were to a from to the binding by binding to the of to were in the the of the and for of and cells were in and 2 in of cells were in a with of expression of and of 1 was and The cells were for 2 in cell adhesion receptors were the of the for the was to the of either variant were by for in the of were as described with the as the and G. The of the and was by of the cells that with the of a for expression were with 1 of of or murine The were with 2 cells in of expressing and of receptors were in cells expressing of as The were and for cells were by two of The cells were the cell to the of the of and blood cells from were a The cells from of blood were the and the cell was with mouse to NK cells on a The and were from human blood of cells with and with and and B cells were as and The of all was by was the was other human and were human and human The of the and AML and T-ALL G. S. A. P. J. A. M. A. L. Ludwig W.D. K. B. M. M. J. 2007; 25: were from J. and M. The expression of and was a and the or the and of expression was as described N. Bernhardt G. F. H. Forster R. J. Immunol.. 2005; Scholar). were to and for were and the were by were as and also to that the not on the and The and Ig and the Ig-like domains of CD96 were to the of the and to the of the of for CD96 in the Ig of the human CD96, we a that a of CD96 A and This is in the when and to the second Ig-like domain A and A analysis two that this Ig-like domain to the V-like when amino whereas the domain by an or a a V-like we to the splice variants in that murine CD96 is to human the to a variant 1 by on murine were and This observation is with the of the mouse that not the of the human in this region of the revealed that CD96 is expressed by the human AML cell line Therefore, cells were and to to by the as well as a with an of This (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google and extensive as for murine CD96 (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). of the and the of the two variants were expression in cells, of A in was not though to a This also the of as a single in the that both variants expressed by cells the expression of the two human CD96 in human with a on revealed expression of CD96 in peripheral as well as and in and An not be in and Among was to be expressed in and with the and to a in and we to in this may that is expressed distinct developmental was to be expressed on T cells, a subpopulation of B cells, and NK cells but not on and Therefore, blood cells from were by and the the two variants in B cells, T cells, T cells, monocytes, and NK cells In all of the a of the blood was T cell in was identified as tumor marker expressed in T-ALL and AML (2Gramatzki M. Ludwig W.D. Burger R. Moos P. Rohwer P. Grunert C. Sendler A. Kalden J.R. Andreesen R. Henschke F. Moldenhauer G. Exp. Hematol.. 1998; 26: 1209-1214Google Scholar, 3Hosen N. Park C.Y. Tatsumi N. Oji Y. Sugiyama H. Gramatzki M. Krensky A.M. Weissman I.L. Proc. Natl. Acad. Sci. U. S. A.. 2007; 104: 11008-11013Google Scholar), it was of to the expression in a of types of C and we observed a of expression among both AML and T-ALL However, in all of the T-ALL the to an as for T cells. In AML cells, the expression of CD96 when compared with though also the AML is by a the CD96 expression In contrast to AML a in the expression observed with variant 1 It that either or the original of cell (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). the of is T cell peripheral T cells with or were expression was the but a was observed (1Wang P.L. O'Farrell S. Clayberger C. Krensky A.M. J. Immunol.. 1992; 148: 2600-2608Google Scholar). This particular of was by the and to the variant remained with by a of all The Ig-like of CD96 for to to the of the binding on CD96 for CD155 and The that the interaction is not the (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google was to chimeric CD96 receptors on and were the domain and domain by receptor chimeric receptors were by domain exchange and to receptor a CD96 the human domain was by its murine In all of the the of the third domain also that of the the transmembrane and the The receptors were expressed in cells, and binding of mouse or human to was and In the binding of to the chimeric receptors was tested C and the not bind all to the murine binding to chimeric receptors that in the two domains of the second human domain or binding whereas that of the domain caused binding of the third human domain only on The binding to the chimeric receptors that the of the human domain is for the interaction with the second and third the of binding of the of an or albeit to a whereas of the was was to all receptors a murine domain with and to bind possessing a domain of human murine CD96 and nectin-1 binding were and both were to downstream that the for both CD96 ligands in domain nectin-1 was also tested for but this receptor not to binding to another This that the interaction is not the two of the to the binding of the and and also a of binding were to the of and murine nectin-1, to domain in a that was largely of the of the downstream domains. that of binding is to possessing murine domain three the exchange of this domain in all In in both domain 1 and to binding of as by the to bind either or The binding of and the chimeric CD96 receptors whereas that of binding to and This observation is in with the capacity of the to CD155 binding (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar), but not with CD155 of CD96 to the binding the chimeric CD96 receptors revealed that the second domain an on CD155 it is possible that the of this domain the of the CD96/CD155 Therefore, cells expressing either were with of or that binding not the two variants In of the CD155 in Although binding a when were in the of of that of was by a that binding Therefore, the in binding were for an of of binding partner in the in also and the of both and tested binding to CD155 expressed by cells. both it was that the interaction is when compared with that and as observed for the to binding of to CD155 P. R. G. J. G. G. J. also that of CD96 that CD155 the only binding for on the of the cells and the domain of CD155 is in CD96 via in the outermost V-like domain of CD155 G. A. M. E. Scholar). the adhesive of the variants in a cells, a human B cell tumor line were with expression for In this the receptor not in yet for a direct of expression by cells expressing of the variants were to to in the adhesive were observed as for the ligands The of to the was to binding capacity a to not of cells. a binding of cell expressed to a of not a weaker capacity of this variant when compared with the binding by variant 2. The adhesion mediated by the may also observation that cells expressing not variant 1) a to A in of to was that genetic in the human CD96 locus may to a form of the C syndrome (Opitz Trigonocephaly) (8Kaname T. Yanagi K. Chinen Y. Makita Y. Okamoto N. Maehara H. Owan I. Kanaya F. Kubota Y. Oike Y. Yamamoto T. Kurosawa K. Fukushima Y. Bohring A. Opitz J.M. Yoshiura K. Niikawa N. Naritomi K. Am. J. Hum. Genet.. 2007; 81: 835-841Google Scholar). A chromosomal was to the CD96 in whereas in a mutation domain three of (T280M) was expressing or the in adhesive the stages of with the of the Therefore, the mutation was both variants and and binding capacity as well as was tested as revealed that binding to both CD96 is by the mutation In the of both binding to is only be the of that this particular domain three mutation an on the adhesive capacity of with the that variant 1 a of the in the mutation may adhesive for cells on CD96 a adhesion to and from extracellular or whereas cells may be to in adhesion receptor It be interesting to the cell expressing CD96 in brain or to a mouse to the of this receptor in developmental processes. CD96 function(s) evidence that it an adhesion receptor in brain as well as in The is by the expression of CD96 on T as well as on NK cells A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google and S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google and this from T and NK cells (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar), is expression in other cell types or in the brain (8Kaname T. Yanagi K. Chinen Y. Makita Y. Okamoto N. Maehara H. Owan I. Kanaya F. Kubota Y. Oike Y. Yamamoto T. Kurosawa K. Fukushima Y. Bohring A. Opitz J.M. Yoshiura K. Niikawa N. Naritomi K. Am. J. Hum. Genet.. 2007; 81: 835-841Google Scholar). Therefore, we the expression of in tissues and in all and In only a expression was observed in tissues such as and with the of expression not in and to an only in In is in but it that this in expression is caused by an elevated of was observed in brain with that observed in but was in the Moreover, the that variant 1 to the CD96 mediated in and brain when compared with other A analysis of blood cell revealed that the of expressed by both and T cells on the blood However, a of variant that for variant 1 a of the expression of its expression in all This was from the of and to the when tumor from from AML or T-ALL were However, in particular the AML a was observed with the of variant that of variant 2. both the expression or the variant 1 and 2 expression be as but not mouse CD96 be expressed in two on The assumed function of as a NK receptor to its binding to CD155 (4Fuchs A. Cella M. Giurisato E. Shaw A.S. Colonna M. J. Immunol.. 2004; 172: 3994-3998Google Scholar). is also to in splice whereas only of for I. S. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2003; Scholar, E. Scholar, S. H. I. A. M. N. K. T. A. Scholar). However, of not the Ig-like whereas the capacity of the receptor to with its ligands is for all In of the for the of receptors in the of the second Ig-like domain. The function of adhesion receptors is by the of ligands Therefore, we the binding of human as well as murine CD96 in with to the domains the interaction with CD155 and the three Ig-like domains by chimeric human/murine CD96 receptors revealed that binding the in domain the domain of the that is to be for The for the domain in is illustrated by the that all receptors it CD155 binding capacity of the However, human and murine CD96 in other the human receptor not with and CD96 binding to CD155 and is of domain two in In binding is sensitive to the of the downstream domains also It is not the of human and murine domains with of the chimeric receptors to a binding of However, such also binding its binding is largely to the domain of In the that a splice variant binding an of the downstream domains in the of It is that both in human but not mouse of binding on downstream domains and the of the domain binding is direct or in remains unclear, but it is interesting to note that the not only of the but also of the Ig-like It is to that the Ig an presentation of the domain with to the other domains of the to amino for in of variant is also to in two splice variants to either a V-like or a J. Immunol.. Scholar). Even though the of this a NK and the CD96 and to the observation that both receptors expressed in brain M. B. 2005; Scholar). Although we only a expression in in the of expression the correlate with a function of this receptor in the of the Therefore, it was of to the on the of a mutation of that to the third Ig-like domain (T280M) and that in a from C syndrome (Opitz In contrast to that of is not by This is in line with the that by in domain 1 and 2 with domain only on This is illustrated by the observation that with characteristics to receptor whereas that of CD155 remained a In binding to was whereas this a capacity to with CD155. The that also the third human CD96 domain is in (8Kaname T. Yanagi K. Chinen Y. Makita Y. Okamoto N. Maehara H. Owan I. Kanaya F. Kubota Y. Oike Y. Yamamoto T. Kurosawa K. Fukushima Y. Bohring A. Opitz J.M. Yoshiura K. Niikawa N. Naritomi K. Am. J. Hum. Genet.. 2007; 81: 835-841Google that the exchange to suggesting that this mutation a of the domains with to other or to receptors, the as for the splice The by the mutation to the of the of the third domain in to the rich from the of the domain. The human The murine is and that the human region is with a whereas the murine only such Although remains the mutation from a to a may the characteristics of the of the domain. Ig domains in with other or with as yet ligands show in this R. F. M. M. D. 2005; for in and N. 2004; for in and in this region be to in of binding However, in the of CD96 the binding to on the domain an in by the either the of the or the interaction with a the in the human is by a in mouse, the mutation the of the human a with the observation that the murine CD155 binding to CD96 is to the whereas the human interaction on downstream we that the of the third Ig-like domain the of the domains in human for also the murine CD155 only with murine CD96 but not with human CD96 and the amino for binding be or In this we that CD96 in via to the Ig-like domain. However, the binding and mouse as by the interaction of CD96 with CD155. In contrast to the adhesive of human CD96 by characteristics of downstream domains that not by but by This also that the interaction evolutionary in and a binding to CD155 in mouse and a interaction with CD155 that is to by in The of was also by the evolutionary of both CD96 and CD155, as by the of amino acid (5Seth S. Maier M.K. Qiu Q. Ravens I. Kremmer E. Forster R. Bernhardt G. Biochem. Biophys. Res. Commun.. 2007; 364: 959-965Google Scholar). the interaction the receptors was its biological the CD96 function(s) the but may be by the murine CD96/nectin-1 interaction that is in only the human a a for the of the in of the Ig superfamily and in Immunol.. Scholar). It be to the mediated by human and murine CD96 in transmembrane and share other such as an A in the membrane domain with amino the as a binding for Immunol. Scholar). In a region in the possessing domains to CD96 M. 2000; Scholar). M. and for cell M. for in and M. and J. for tumor T-ALL and