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E.E. Geisert, X. Wang, G. Geisert, G. Kang, B.K. Song; CD81–Molecular Interactions and RPE Proliferation. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4606.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:We have identified a small membrane protein CD81 that appears to be directly involved in the regulation of RPE cell number. Antibodies directed against CD81 suppress proliferation in culture and mice with CD81 –/– mutations have an increased number of glia within the brain. In this study, we define the molecular interactions of CD81 that may in part control proliferation of RPE .Methods: For this study we used two human cell lines: AREP19 cells and D407 cells that were stability transfected with rat CD81. Confluent cultures of cultures were rinsed and lysed in ice–cold 1% Brij 97 in 0.05 M Tris, 0.05 M NaCl, pH 7.3 containing protease inhibitors. CD81 and the associated proteins were co–isolated by immunoprecipitation with species–specific, anti–CD81 affinity column. Associated proteins were identified by surface biotinalation and cross blotting with antibodies directed against known proteins Results: The immunoprecipitated proteins from D407–ratCD81, demonstrate that CD81 can associate with itself as well as other tetraspanins CD9 and CD151. CD81 was not associated with CD63, an additional tetraspanin in human RPE cell lines. We examined the precipitates and found several integrins were present: alpha 3, alpha 5, alpha v, Beta1 and Beta 5. In addition we observed the presence of two recently described members of the Ig superfamily, EWI–F and EWI–2. Surface biotinalation and immunoprecipitations demonstrate that these associated proteins precipitate in three specific CD81 complexes. The first has CD81 primarily associating with integrins (Fig. 3). In the second complex, the primary component is EWI–F. In the final complex there are approximately equal levels of EWI–F and EWI–2. In this experiment living cells were surface labeled with biotin, and proteins made soluble with a mild detergent (1% Brij) and affinity isolated with one of three antibodies: anti–rat–CD81, anti–EWI–F or anti–EWI–2. Within each of the immunoprecipitations multiple biotin labeled bands were observed. Many of the protein bands are similar in each of the immunoprecipitation samples. However, the intensity of each band and the specific pattern of banding differ between each three samples. Conclusions:CD81 forms a series of distinct molecular complexes in RPE. One or a combination of these complexes may be linked into the halt of the cell–cycle progression observed following antibody perturbation studies.
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