This is an application to study the biological function of the CD44 molecule of activated T lymphocytes, and to define the parameters that determine high affinity binding of this receptor to the extracellular matrix. Hyaluronan is a specific ligand of CD44 and although abundant very few T-cells bind to it despite expressing the receptor, albeit in an inactive form. The goal of this application is to identify the mechanism that conveys a low affinity (non-binding) CD44 molecule to a high affinity one. In cellular terms high affinity-binding appears to be a feature of activated T-cells, and it follows that the expression of this high affinity-receptor is a regulated event. The biological implications are directed migration in inflammation and metastasis. In more specific terms, one can distinguish between an inactive binding state, an inductive one and a constitutive one. The PI has succeeded in deriving mutant cell lines that represent these three phenotypes, allowing the detailed study proposed here of the molecular conditions governing the three binding states. An important lead has been the demonstration that distinct N-glycosylation patterns are correlated with presence or absence of hyaluronan binding. Using transfections with mutants of glycosylation sites it was shown that absence of carbohydrate on one site at position 25 confers constitutive hyaluronan binding activity on an otherwise inducible cell line. These experiments leave little doubt that N glycosylation implies the action of trans acting genes, and since results of a number of biological experiments, including somatic hybridization, also postulate the existence of 2 genes modulating the CD44 function, the search is on to identify these, A transfection strategy free of prejudice regarding the function of such genes is proposed for their isolation. Finally in the third aim, the immunological function of CD44 is approached by studying the role of CD44 in mediating lymphocyte trafficking.
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