Abstract
A novel Ig superfamily protein, EWI-2, was co-purified with tetraspanin protein CD81 under relatively stringent Brij 96 detergent conditions and identified by mass spectrometric protein sequencing. EWI-2 associated specifically with CD9 and CD81 but not with other tetraspanins or with integrins. Immunodepletion experiments indicated that EWI-2-CD9/CD81 interactions are highly stoichiometric, with approximately 70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line. The EWI-2 molecule was covalently cross-linked (in separate complexes) to both CD81 and CD9, suggesting that association is direct. EWI-2 is part of a novel Ig subfamily that includes EWI-F (F2alpha receptor regulatory protein (FPRP), CD9P-1), EWI-3 (IgSF3), and EWI-101 (CD101). All four members of this Ig subfamily contain a Glu-Trp-Ile (EWI) motif not seen in other Ig proteins. As shown previously, the EWI-F molecule likewise forms highly proximal, specific, and stoichiometric complexes with CD9 and CD81. Human and murine EWI-2 protein sequences are 91% identical, and transcripts in the two species are expressed in virtually every tissue tested. Thus, EWI-2 potentially contributes to a variety of CD9 and CD81 functions seen in different cell and tissue types.
Highlights
To observe many other tetraspanin complexes, detergents less disruptive than Triton X-100 are required
Immunodepletion experiments indicated that EWI-2–CD9/CD81 interactions are highly stoichiometric, with ϳ70% of CD9 and CD81 associated with EWI-2 in an embryonic kidney cell line
CD151␣31 integrin complexes are stable in conditions (Triton X-100 plus SDS detergent) that disrupt all other known tetraspanin associations, and at least 90% of ␣31 is associated with CD151 under these conditions [26]
Summary
To observe many other tetraspanin complexes, detergents less disruptive than Triton X-100 are required. CD81 complexes isolated from cell surface-labeled NT2RA cells (Fig. 1, lane A), 293 embryonic kidney cells (Fig. 1, lane B), and A431 epithelial carcinoma cells (Fig. 1, lane C) contained CD9 (ϳ30 kDa) as well as major species migrating above 120 kDa. These were previously identified as a mixture of ␣31 integrin and a novel Ig superfamily protein (FPRP, CD9P-1 [30, 63]).
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