CD9-association with PIP₂ areas is regulated by a CD9 salt bridge

Tetraspanins are membrane proteins involved in multiple cellular functions, which they regulate by means of tetraspanin-enriched microdomains. While many tetraspanin-associated processes are regulated by intracellular signaling, possibly through the crosstalk between intracellular tetraspanin segments and second messengers like PIP₂, this molecular crosstalk has remained largely unknown. To improve our understanding of this crosstalk, we investigate the possible relationship between an intracellular salt bridge of the tetraspanin CD9 and PIP₂. We find that CD9 readily associates with PIP₂-rich areas, in contrast to its interaction partner EWI-2. The opening of the CD9 salt bridge lowers the abundance of CD9 in these PIP₂ areas. Instead, open-CD9 can be located in regions that are more strongly populated with EWI-2, promoting CD9-EWI-2 association. This study uncovers the process of an intracellular salt bridge regulating the association of CD9 with PIP₂-enriched areas. This points toward a possible link between intracellular tetraspanin segments and signaling.