Identification of G-protein alpha-subunits during distinct stages of lens cell differentiation.

PURPOSE The purpose of this study was to identify alpha-subunits of heterotrimeric guanosine triphosphate-binding proteins in lens cell populations at various stages of terminal differentiation. METHODS Crude cell membranes were isolated from the annular pad, cortical fibers, and nuclear fibers of adult chickens and subjected to cholera and pertussis toxin-mediated ribosylation reactions. Specific labeling of toxin substrates was visualized after SDS-PAGE and radioautography. In complementary experiments, cell membranes were first separated by SDS-PAGE, transferred to a nitrocellulose support membrane, and probed with a panel of commercially available antibodies that recognize various classes of G-protein alpha-subunits. RESULTS A cholera toxin substrate was identified in cortical fibers whose labeling was dependent on a soluble factor. No cholera toxin substrates were labeled in annular pad cells. Two pertussis toxin substrates were seen in the relatively undifferentiated annular pad and the more differentiated cortical fibers. Relative abundance of the pertussis toxin substrates differed between the two cell types. Antibody staining revealed the presence of alpha-subunits belonging to the Gs, G(o), and G(i), families throughout the lens. Molecular weight differences of G alpha 2 polypeptides were noted between annular pad and cortical fiber cells. CONCLUSIONS These results suggest that G-protein-based signal transduction pathways continue to function in lens cells during various stages of terminal differentiation. Differences noted between annular pad and cortical fibers provide additional evidence that dynamic alterations in receptor-mediated processes may be responsible for the accumulation of differentiated characteristics during fiber formation. This also indicates that ocular pharmacologic intervention could affect various aspects of lens physiology throughout the process of fiber formation.