Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins

A critical step in the activation of cellular Ras is the release of bound GDP. Oligonucleotide primers derived from a mouse cDNA sequence homologous to the Saccharomyces cerevisiae CDC25 gene product were used to screen a human brain cDNA library. The cloning led to the isolation of a 2.8-kb cDNA predicted to encode a protein of 488 amino acids. This protein was produced in Escherichia coli as a glutathione S-transferase fusion protein and functioned in vitro as a specific guanine nucleotide-releasing factor. Polyclonal antibodies raised against the last 281 amino acids of the protein allowed a protein in the molecular weight range of 55 kDa to be identified in human cortex homogenates. Analysis by Northern blotting led to the identification of a 5.5-kb mRNA in brain poly(A)+ RNA. The functionality of the encoded protein was evaluated after expression in different cells: (i) in Saccharomyces cerevisiae the effects of the cdc25.5 and RAS2 Ala-22 mutations were reversed; (ii) in chinese hamster ovary cells, a RAS-responsive element was transactivated as demonstrated by the expression of a CAT reporter gene under the control of the polyomavirus enhancer. Finally, in situ hybridization on of human chromosomes revealed a localization on band 15q2.4.