Uncovering the role of the small intracellular loop of tetraspanins by using CD9 as example

• Applicant: Professor Dr. Thorsten Lang
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 523941852

Tetraspanins are small membrane proteins playing an evolutionary role in the unicell-to-multicell transition, and are expressed in all metazoans. They are involved in a large variety of cellular processes and diseases, through forming complexes with immunoglobulin superfamily proteins, proteases, integrins, and several other receptors, that they organize in tetraspanin enriched microdomains (TEMs). How the complexes regulate function is unclear. Possibly, they stabilize tetraspanins and their interaction partners, assist in their folding, direct them to their destination organelle, regulate their function and arrangement in TEMs, or mediate membrane curvature. Obtaining an understanding of how they work in general is complicated, because of the many types of involvements, and the observation that tetraspanins can exhibit opposite effects in different cell types. A prerequisite for a coherent picture could be novel, yet to be discovered properties. The proposal is about the uncovering of the function of a so far barely studied protein segment, the small intracellular loop. One study suggests that it mediates interactions, possibly via functional interactions with the intracellular membrane leaflet. We identified in the loop a conserved sequence of five amino acids harboring a glutamate that in the crystal structure of the tetraspanin CD9 forms a salt-bridge with a lysine of the N-terminus. Without salt-bridge, the TEM-association of CD9 and its interaction partner EWI-2 increases. The salt-bridge plays a role in other tetraspanins as well. We aim for addressing two basic questions: 1. Why does the mutant without salt-bridge associate stronger with EWI-2? Because of a higher affinity to EWI-2, a stronger cross-linking between tetraspanins, or palmitoylation? How does the stronger CD9-EWI-2-association, detected by biochemical methods, relate to the lateral organization of the proteins? How do TEMs actually look like, what parameters define their nano-architecture? 2. Three positive charges at the N-terminus of CD9, from which one forms the salt-bridge, are predestined for an interaction with phosphoinositide signaling lipids. For the CD9 interaction partner EWI-2 such an interaction has been already demonstrated. Therefore, the association between CD9 and EWI-2 in TEMs possibly involves phosphoinositides, and perhaps is even regulated by the salt-bridge and Ca2+. A direct link between tetraspanins and second messengers would be very exciting and strongly contribute to the understanding of the general functionality of this protein family. As the loop does not only determine the behavior of CD9, we expect that our findings will provide generally important insights into this yet enigmatic protein family.

• DFG Programme: Research Grants