Detection of homodimer formation of CD99 through extracelluar domain using bimolecular fluorescence complementation analysis.
Although various functions of CD99 have been reported, such as apoptosis and homotypic aggregation of thymocyte and transendothelial migration of immune cells, biochemical/molecular natures of CD99 are still elusive. Using mouse CD99 gene, we show that CD99 forms homodimer through its extracellular domain. Expression of mouse CD99 is up-regulated on T cells after CD3-mediated activation, like the case for human CD99. The potential of CD99 to form homodimer was tested with a recently developed bimoleular fluorescence complementation analysis (BiFC). In BiFC analysis, the dimerization-induced fluorescence was strong near the perinuclear region and was faded at the cell membrane. However, surface expression of CD99 was still detected by flow cytometry, suggesting that CD99 either in monomer form or in association with other molecules exists on the cell surface. In BiFC analysis using CD99 mutants with its extracellular, transmembrane, or cytosolic domains changed to corresponding human CD4 domains, the mutant replaced with human CD4-extracellular domain did not produce fluorescence. Purified soluble CD99-Fc fusion proteins bound to CD99-Fc immobilized onto the gold sensor chip in surface plasmon resonance analysis, confirming that the extracellular domain was responsible for dimer formation. Intracytoplasmic staining for CD99 expression in the thymocytes and mature T cells showed that most of the cells, even the cells with low surface level of CD99, contained the molecule inside the cell. Our results suggest that majority of CD99 homodimers may exit in the cell and be exported to the cell surface, dissociating from each other, after a certain regulatory signal is delivered.
Choi
Gowoon
G
Interdisciplinary Graduate Program in Genetic Engineering, Seoul National University, Seoul 110-799, Korea.
Lee
Sang Wook
SW
Jung
Kyoung Cheon
KC
Choi
Eun Young
EY
D000071443:12E7 Antigen; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry / Q000302:isolation & purification*; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000302:isolation & purification*; D005434:Flow Cytometry; D005453:Fluorescence; D008163:Luminescent Measurements / Q000379:methods*; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D013601:T-Lymphocytes / Q000276:immunology
D000071443:12E7 Antigen; D015703:Antigens, CD; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules