ANALYSIS OF SMALL GTP-BINDING PROTEINS OF THE LENS BY GTP OVERLAY ASSAY REVEALS THE PRESENCE OF UNIQUE GTP-BINDING PROTEINS ASSOCIATED WITHFIBER CELLS

Low molecular weight GTP-binding proteins are molecular switches which are thought to play pivotal roles in cell growth, differentiation, cy toskeletal organization and vesicular trafficking. In this study, memb ers of this family of proteins have been identified and characterized in the eye lens, for the first time. [alpha-P-32]GTP blot overlay assa ys of monkey and human lens water soluble and membranous insoluble fra ctions revealed the presence of specific GTP-binding proteins in the r ange of 20-30 kDa (small GTPases) in both fractions, with much higher amounts in the membranous insoluble fraction. In the insoluble fractio n, in addition to 20-30 kDa GTPases, there are three distinct GTP-bind ing proteins, ranging from 33-45 kDa, The small GTPases (20-30 kDa) we re present throughout the lens in epithelium, cortex and nucleus, whil e the 33-45 kDA GTP-binding protein bands were exclusively associated with the cortex and nucleus (fiber cells). Analysis of lens fractions by two-dimensional electrophoresis, immunoprecipitation using monoclon al and sequence specific polyclonal antibodies and C3 exoenzyme mediat ed ADP-ribosylation demonstrated the presence of Ras, Rap, Rho, Rac, R ab and several other small GTPases. The 33-45 kDa GTP-binding proteins that are associated with lens fiber cells appear to be distinct from the small GTPases and from heterotrimeric GTPases, and were not detect ed in brain or heart tissue. The presence of different complements of GTP-binding proteins in lens fibers and epithelial cells suggests thei r involvement in important regulatory functions, possibly related to c ell growth, differentiation and organization of the cytoskeleton. (C) 1997 Academic Press Limited.