RasGRP4, a New Mast Cell-restricted Ras Guanine Nucleotide-releasing Protein with Calcium- and Diacylglycerol-binding Motifs: IDENTIFICATION OF DEFECTIVE VARIANTS OF THIS SIGNALING PROTEIN IN ASTHMA, MASTOCYTOSIS, AND MAST CELL LEUKEMIA PATIENTS AND DEMONSTRATION OF THE IMPORTANCE OF RasGRP4 IN MAST CELL DEVELOPMENT AND FUNCTION

Abstract

A cDNA was isolated from interleukin 3-developed, mouse bone marrow-derived mast cells (MCs) that contained an insert (designated mRasGRP4) that had not been identified in any species at the gene, mRNA, or protein level. By using a homology-based cloning approach, the approximately 2.6-kb hRasGRP4 transcript was also isolated from the mononuclear progenitors residing in the peripheral blood of normal individuals. This transcript information was then used to locate the RasGRP4 gene in the mouse and human genomes, to deduce its exon/intron organization, and then to identify 10 single nucleotide polymorphisms in the human gene that result in 5 amino acid differences. The >15-kb hRasGRP4 gene consists of 18 exons and resides on a region of chromosome 19q13.1 that had not been sequenced by the Human Genome Project. Human and mouse MCs and their progenitors selectively express RasGRP4, and this new intracellular protein contains all of the domains present in the RasGRP family of guanine nucleotide exchange factors even though it is <50% identical to its closest homolog. Recombinant RasGRP4 can activate H-Ras in a cation-dependent manner. Transfection experiments also suggest that RasGRP4 is a diacylglycerol/phorbol ester receptor. Transcript analysis of an asthma patient, a mastocytosis patient, and the HMC-1 cell line derived from a MC leukemia patient revealed the presence of substantial amounts of non-functional forms of hRasGRP4 due to an inability to remove intron 5 in the precursor transcript. Because only abnormal forms of hRasGRP4 were identified in the HMC-1 cell line, this immature MC progenitor was used to address the function of RasGRP4 in MCs. HMC-1 leukemia cells differentiated and underwent granule maturation when induced to express a normal form of RasGRP4. Thus, RasGRP4 plays an important role in the final stages of MC development.