Cloning from the retina of two cDNAs encoding novel small GTP-binding (G) proteins which define a new subfamily of Ras-related proteins

Purpose. To clone new members of the Ras family of small G proteins that are expressed in the retina. We were interested in identifying such proteins because small G proteins are involved in regulation of a wide variety of cellular processes and because Drosophila Ras1 has been implicated in photoreceptor cell fate determination. Methods. RT-PCR was performed using mouse retinal cDNA as template and degenerate primers corresponding to conserved Ras domains. The resulting PCR products were used to probe retinal cDNA libraries. Northern blots, PCR, in situ hybridization, in vitro expression cell transfection, immunocytochemistry, and confocal microscopy were performed by standard methods. Results. Full-length cDNAs corresponding to Cras and Cran, novel Ras-related proteins, were obtained. The predicted amino acid sequences of Cras and Cran are more similar to each other than to any known Ras-like protein. Unlike most Ras family members, they lack a recognition signal for carboxyl (C)-terminal lipidation, a modification that is generally necessary for plasma membrane association among this class of proteins. Nonetheless, transiently expressed Cras and Cran are plasma membrane-associated. In vitro expressed Cran and Cras both bind GTP and GDP, but not ATP. Like most Ras family members, Cras is ubiquitously expressed. Cran, however, is expressed specifically in neurons, and within the retina it is expressed only by subsets of cells within the inner nuclear and ganglion cell layers. Expression studies with constituitively active and dominant negative forms of Cras and Cran to define their functions, as well as yeast two-hybrid studies to identify interacting proteins, are currently underway. Conclusions. These results suggest that Cras and Cran may define a new subfamily of Ras-relaled proteins, and that Cran may be specifically involved in transmemhrane signaling by retinal and other neurons.