CD99’s highly conserved region III facilitates the homodimerization and function in cell-cell adhesion

Abstract CD99 is a type I transmembrane protein and its function has been known to be implicated in various cellular processes including cell-cell adhesion and leukocyte diapedesis. These functions were all related to actin polymerization and/or reorganization. Nonetheless, the molecular mechanism has remained unclear. In previous studies, we showed that CD99 is capable of forming a homodimer and the homodimerization requires the extracellular domain. This trait was confirmed to be conserved in mouse CD99. In this study, we investigated the molecular basis and functional role of homodimer formation. In sequence comparison analysis, CD99 amino acid sequences from different species have three conserved subdomains in the extracellular domain, named highly conserved region (HCR) I, II, and III. Based on this information, we generated various subdomain-deleted mutants. In the bimolecular fluorescence complementation assay, the HCRIII-deleted mutant (named HCRIIIΔ ext-mut), significantly reduced the ability to form a homodimer, compared with other mutants. Additionally, HCRI and II-deleted mutant, that is, a mutant expressing only HCRIII-subdomain on the cell surface (named HCR3ext-mut), was enough to form the homodimer. Overexpression of HCRIIIΔext-mut in T cells prevented T cells from forming stable interaction with the antigen-presenting cells (APCs), and polarization of polymerized actin to the T-APC interface. However, overexpressing HCRIIIext-mut enhanced the T-APC stable interaction. These results identify the HCRIII of CD99 as the key subdomain required for dimer formation and indicate that the homodimer would contribute to the formation of stable interaction with APCs of T cells via actin cytoskeleton reorganization.