Identification and characterization of members of the Ras super family GTPases in the eye lens

Purpose: Small GTPases of the Ras super family (Ras, Rho, Arf, Rab and Ran) are molecular switches that regulate aspects of cellular proliferation and differentiation, cytoskeletal organization and vesicular trafficking. In this study, these proteins have been identified and characterized in the eye lens Methods: Using monkey and human lenses, the distribution and identification of low molecular weight GTP-binding proteins was investigated by employing the [α-P³²] GTP overlay assay, immunoprecipitation and ADP ribosylation. Results: GTP overlay assays of lens soluble and insoluble fractions revealed the presence of specific GTP-binding proteins in the range of 20-30 kDa in both fractions, with much higher amounts in the membranous insoluble fraction. In the insoluble fraction, in addition to 20-30 kDa GTPases, there are three distinct GTP-binding proteins, with molecular weights of 35-45 kDa. The small GTPases (20-30 kDa) were present in epithelium, cortex and nucleus, while the 35-45 kDa GTP- proteins were exclusively associated with the cortex and nucleus. Analysis of lens fractions by 2D electrophoresis and immunoprecipitation using monoclonal/ polyclonal antibodies directed against specific GTPases, revealed the presence of Ras, Rap, Rho, Rac, Rab and several other small GTPases. Conclusions: This study demonstrates the presence of a variety of regulatory small GTPases in the various regions of the lens. In addition to these GTPases, the lens cortex and nucleus contain 35-45 kDa GTP- proteins which appear to be distinct from heterotrimeric GTPases. The presence of these GTP- binding proteins suggests their involvement in important regulatory function(s) in the lens.