Analysis of low-molecular-weight GTP-binding proteins in two functionally different intestinal epithelial cell lines

Low molecular weight GTP-binding proteins are molecular switches that are believed to play pivotal roles in cell growth, differentiation, cytoskeletal organization, and vesicular trafficking. In this study, for the first time, members of this family of proteins in two functionally different intestinal epithelial cell lines are identified and characterized. [alpha-P-32]GTP blot overlay assays of cytosolic and membranous fractions revealed the presence of specific GTP-binding proteins in the range of 20-30 kDa (small GTPases) in both fractions, with considerably higher amounts in the membranous insoluble fraction. Analysis by two-dimensional electrophoresis, immunoprecipitation using monoclonal and sequence-specific polyclonal antibodies, and C3 exoenzyme-mediated ADP ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, Rab, and several other small GTPases. The pattern of small GTP-binding proteins corresponded to the characteristics of the cell lines. Caco-2 cells showed a Rab5 protein that is known to be involved in endocytosis but was not found in T-84 cells. On the contrary Rab3 has been shown to participate in secretory processes. It is highly expressed in T-84 cells (sixfold compared to Caco-2 cells).