A cysteine residue located in the transmembrane domain of CD44 is important in binding of CD44 to hyaluronic acid.
In the transmembrane domain and cytoplasmic domain of human CD44 protein there are two cysteine residues. These two cysteines are conserved in all known mammalian CD44 proteins. The functions of these cysteine residues are not known. Site-specific mutagenesis was used to create CD44 mutant proteins lacking either one or both of these cysteine residues. Wild-type CD44 and mutant CD44 genes were transfected into CD44- Jurkat cells to establish stable transfectants. These transfectants were used to study whether these two cysteine residues are important in the binding of CD44(H) to fluorescein-conjugated hyaluronic acid (F-HA). Jurkat transfectant bearing wild-type CD44 did not bind F-HA, unless they were stimulated in vitro with immobilized anti-CD3 monoclonal antibody. Anti-CD3 antibody also stimulated the binding of F-HA in Jurkat CD44.C295A transfectant in which the cytoplasmic cysteine residue has been replaced with alanine. In contrast, anti-CD3 antibody failed to stimulate the binding of F-HA in Jurkat transfectant (CD44.C286A), in which the transmembrane domain cysteine 286 has been replaced with an alanine, and in Jurkat transfectant CD44.2C2A, in which both of the cysteine residues have been altered. Binding can also be induced with a monoclonal anti-CD44 antibody (F-44-10-2) in Jurkat wild-type CD44 and Jurkat CD44.C295A transfectants but not in CD44. C286A transfectant. These results provide evidence that the transmembrane domain of CD44, more specifically the cysteine residue in the transmembrane domain, is important for both activation-induced and anti-CD44 antibody-induced binding of soluble HA.
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Department of Dermatology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4943, USA.
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D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine; D003593:Cytoplasm / Q000276:immunology; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry* / Q000378:metabolism*; D006820:Hyaluronic Acid / Q000378:metabolism*; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D015703:Antigens, CD; D018960:Hyaluronan Receptors; D011994:Recombinant Proteins; D006820:Hyaluronic Acid; D003545:Cysteine