Domain Analysis of the Tetraspanins. Studies of CD9/CD63 Chimeric Molecules on Subcellular Localization and Upregulation Activity for Diphtheria Toxin Binding.

Abstract

ABSTRACT. 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 be clarified. CD9 associates with diphtheria toxin receptor (DTR), which is identical to the membraneanchored form of heparin-binding EGF-like growth factor (proHB-EGF). CD9 upregulates the diphtheria toxin (DT) binding activity of DTR/proHB-EGF, while CD63 does not upregulate the DT binding activity 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 those of CD63 localize predominantly at lysosomes and intracellular compartments. We made CD9/ CD63 chimeric molecules and then studied their intracellular localization and upregulation activities. The C-terminal regions of CD63, which includes the lysosome sorting motif, showed a strong inhibitory effect on the expression of the chimeric proteins at the cell surface, while mutants 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 of this family of proteins containing the 1st to 3rd transmembrane domains also seems to influence the cell surface expression. For the upregulation of DT binding activity the large extracellular loop (EC2) of CD9 was essential, while the remaining regions influenced the upregulation activity by changing the efficiency of cell surface expression. From these results we discussed the structure-function relationship of this family of proteins.

Identification of CD9 extracellular domains important in regulation of CHO cell adhesion to fibronectin and fibronectin pericellular matrix assembly

“…[27][28][29] Our studies extended these findings by identifying a region within this CD9 segment that was functionally important. In our investigation, EC2 mutants ⌬173-192, ⌬152-192, and ⌬133-192 had an adhesive and FN matrix assembly phenotype comparable to that of CHO MOCK cells.…”

“…Additionally, the interaction between CD9 EC2 and the EC domain of proHB-EGF was important for up-regulation of both mitogenic and DT-binding activities of proHB-EGF. 28,29 Our present investigation examined the importance of the CD9 EC2 region on the adhesion and pericellular FN matrix assembly of CD9 CHO cells. Using peptides that correspond to amino acid sequences of EC2 and CD9 mutants lacking specific regions of EC2, we identified EC2 domains important in the regulation of CHO cell adhesive functions.…”

The C-terminal tail of tetraspanin protein CD9 contributes to its function and molecular organization

“…This particular conservative strategy was chosen because: the C-terminal EMV motif in CD9 partly resembles the functionally important PDZdomain-interacting C-terminal EVM site in CD63 (Latysheva et al, 2006); more radical CD9 C-tail mutations lead to loss of cellsurface expression (e.g. Ryu et al, 2000); CD9 and CD82 are the two tetraspanins most known for having tumor suppressor functions 2703 CD9 C-terminal tail functions (Cajot et al, 1997;Huang et al, 1998;Miranti, 2009;Miyake et al, 1995); and various other CD9-CD82 chimeras have been effectively utilized in prior studies (Charrin et al, 2003; GutierrezLopez et al, 2003). Several CD9-deficient cell lines (MOLT-4, K562, RD, HT1080 and U937) were stably transfected with wildtype and mutant CD9, to yield similar total expression levels, as seen by immunoblotting of CD9 (supplementary material Fig.…”

Structure of the Tetraspanin Main Extracellular Domain

“…As for "true" tetraspanins, the functions of many tetraspanin-like proteins such as RDS/ROM proteins or uroplakins appear to involve self-association and/or association with other proteins (21,22). Some data indicate that part of the specific activity of tetraspanins is determined by the large extracellular EC2 region (23)(24)(25). Unlike the TM regions that are significantly conserved, this region is highly variable in size and sequence composition.…”