Domain analysis of the tetraspanins: Studies of CD9/CD63 chimeric molecules on subcellular localization and upregulation activity for diphtheria toxin binding

CD9 and CD63 belong to a tetramembrane-spanning glycoprotein family called tetraspanin, and are involved in a wide variety of cellular processes, but the structure-function relationship of this family of proteins has yet to b e clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activ ity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activit y in spite of the fact that this protein also associates with DTR/proHB-EGF on the cell surface, CD9 molecules localize on the cell surface, while tho se of CD63 localize predominantly at lysosomes and intracellular compartmen ts, We made CD9/CD63 chimeric molecules and then studied their intracellula r localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effe ct on the expression of the chimeric proteins at the cell surface, while mu tants lacking the lysosome sorting motif delivered more efficiently on the cell surface, indicating that the lysosome sorting motif contributes to the inhibitory effect of the C-terminal region. However, the N-terminal half o f this family of proteins containing the 1st to 3rd transmembrane domains a lso seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essentia l, while the remaining regions influenced the upregulation activity by chan ging the efficiency of cell surface expression, From these results we discu ssed the structure-function relationship of this family of proteins.