Identification of fusion regulatory protein (FRP)-1/4F2 related molecules: cytoskeletal proteins are associated with FRP-1 molecules that regulate multinucleated giant cell formation of monocytes and HIV-induced cell fusion.

Abstract

Fusion regulatory proteins (FRPs) regulate virus-mediated cell fusion and multinucleated giant cell formation of monocytes. Anti-FRP-1 mAbs immunoprecipitated 80 kDa and 38 kDa proteins from HeLa cells. After long exposure other bands were detected, suggesting the presence of molecule(s) associated with FRP-1. To identify the molecule(s), we prepared monoclonal antibodies against immunoaffinity-purified FRP-1 complex derived from membrane fractions of HeLa cells. Immunofluorescence microscopy revealed that these monoclonal antibodies recognized the intracytoplasmic molecules in HeLa cells. Using immunoblotting, the antibodies reacted with 200 kDa, 70 kDa, 55 kDa and 35 kDa molecules, so we designated these molecules as FRP-related molecules (FRMs). Subsequently, we performed gene cloning from a HeLa lambda gt11 cDNA library using anti-FRM mAbs and immunoblotting analysis with either purified cytoskeletal proteins or specific antibodies against various cytoskeletal proteins. Three kinds of positive clone were obtained, which encoded partial sequences of vimentin, tropomyosin, and heat shock cognate protein 70 (hsc70). The 200 kDa molecule was expected to be a myosin heavy chain, judging from the immunoblotting pattern. Immunoblotting confirmed that these purified proteins were readily recognized by anti-FRM mAbs. Furthermore, anti-vimentin and anti-myosin mAbs reacted with the precipitates by anti-FRP-1 mAb, indicating a physical association between FRP-1 molecules and these cytoskeletal proteins. When anti-FRP-1 mAb was added to culture fluids of HeLa cells, the cell-shape and immunofluorescence-pattern stained with anti-FRM mAbs changed. Taken together, the fusion regulatory molecular complex is suggested to consist of at least FRP-1, hsc70, actomyosin and vimentin systems.