A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb

Abstract

We report a novel approach for identifying monomeric GTP-binding proteins that is based on probing cDNA expression libraries with [α-32P]GTP. In short, a nitrocellulose replica from a plated cDNA expression library is treated with 2% SDS to block the GTP-binding activity of various G proteins expressed by E. coli, thus allowing the direct identification of positive clones. Using this procedure we have cloned several small GTP-binding proteins from human keratinocytes including the human homolog of rheb, a novel member of the ras-related GTP-binding proteins. Human rheb cDNA shares 90% identity with the rat counterpart and it is highly upregulated in transformed human cells of various origin. Northern analysis showed that human rheb is ubiquitously expressed, with the highest levels observed in skeletal and cardiac muscle, and not in brain, as it is the case for rat rheb. The human RHEB gene was mapped to chromosome 10q11