Characterization of CD81 associated proteins on T cells

As a member of the tetraspanin superfamily of proteins, CD81 has been linked to multiple biological functions including cellular proliferation, differentiation, activation and degranulation. On T cells CD81 exerts effects on early thymocyte differentiation, LFA-1 activation and costimulation. As a co-receptor for Hepatitis C virus CD81 may play a vital role in pathology. Despite the importance of CD81 in multiple cellular functions, its mechanism of action in these processes remains elusive.; To better understand the mechanism(s) of CD81 functions we evaluated CD81 molecular complexes on murine T cells. The most prominent CD81 associated cell surface protein on thymocytes, and T and B cell lines has an approximate molecular mass of 75 kDa. The 75 kDa protein was purified and analyzed by MALDI-TOF mass spectrometry followed by post-source-decay profiling. p75, here named PGRL, is a novel type I transmembrane protein of the Ig-superfamily which is most similar to FPRP, a prostaglandin regulatory protein. We cloned and sequenced both human and mouse PGRL and characterized mouse PGRL expression in both lymphocytes and non-lymphoid tissues. The discovery of PGRL brings to light a new family of related proteins including PGRL, FPRP, V7/CD101 and IGSF3. Epitope tagged deletion mutants of PGRL were used to determine that the interaction of CD81 with PGRL requires the membrane distal Ig3–Ig4 domains of PGRL. Transwell chamber experiments show that prostaglandins and CD81 coordinately regulate T cell motility. A soluble recombinant PGRL protein shows binding to splenic B cells and suggests the presence of a cellular ligand.; Here we also report an association between CD81 and the intracellular signaling protein 14-3-3 epsilon. We provide evidence that this association is influenced by the palmitoylation state of the CD81 cytoplasmic tails. We have generated a series of CD81 cysteine mutants to identify palmitoylated intracellular motifs of CD81. One of these mutants lacks all five of its intracellular cysteines, and therefore cannot be palmitoylated. This unpalmitoylated version of CD81 shows constitutive association with 14-3-3. Interestingly, we find that under oxidative conditions CD81 palmitoylation is inhibited and that condition correlates with the association of CD81 and 14-3-3. These findings suggest that CD81 signaling events could be mediated by 14-3-3 adapter proteins and these signals may be dependent on cellular redox.