Mast Cells in Airway Hyporesponsive C3H/HeJ Mice Express a Unique Isoform of the Signaling Protein Ras Guanine Nucleotide Releasing Protein 4 That Is Unresponsive to Diacylglycerol and Phorbol Esters

Abstract

cDNAs were recently isolated from BALB/c mouse mast cells (MCs) that encode the new signaling protein mouse Ras guanine nucleotide releasing protein 4 (mRasGRP4). The present study evaluates the expression pattern and biological activity of mRasGRP4 in a variety of mouse strains. As assessed immunohistochemically and by RNA analysis, mRasGRP4 is not coordinately expressed with any of its family members. Normally, mRasGRP4 is an MC-restricted protein in tissues, and kinetic studies revealed that mRasGRP4 is expressed relatively early in developing MCs. The expression of mRasGRP4 in the fetus before granulated MCs become abundant supports the conclusion that RasGRP4 participates in MC-specific differentiation pathways. Functional studies conducted with recombinant material revealed that mRasGRP4 is a cation-dependent, diacylglycerol (DAG)-regulated, guanine nucleotide exchange factor. Immunoelectron microscopic studies revealed that mRasGRP4 resides in either the cytosol or inner leaflet of the plasma membrane of the MC, implying that DAG controls the intracellular movement of this signaling protein in c-kit-stimulated MCs. The mRasGRP4 gene resides on chromosome 7B1 within a site that is prominently linked to baseline airway reactivity in backcrossed C3H/HeJ and A/J mice. A truncated isoform of mRasGRP4 that lacks its DAG-regulatory domain was isolated from C3H/HeJ mouse MCs. Sequence analysis showed that this isoform is the result of defective splicing of the precursor transcript. MCs play a central role in allergic inflammation. The discovery of a novel isoform of mRasGRP4 in hyporesponsive mice suggests that airway reactivity is influenced by RasGRP4-dependent signaling events in pulmonary MCs.

Ras Guanine Nucleotide-releasing Protein-4 (RasGRP4) Involvement in Experimental Arthritis and Colitis

“…Immunogold ultrastructural studies of the in vivo differentiated MCs in the V3 mastocytosis mouse revealed that RasGRP4 tends to reside either in the cytoplasm or on the cytosolic side of the plasma membrane (4). The latter data imply that RasGRP4 participates in early signaling events at the surface of the MC with an undefined receptor.…”

“…The ϳ19-kb mouse RasGRP4 gene was isolated and mapped to chromosome 7B1 (4). Its human (3) and rat (5) orthologs were then isolated using homology-based cloning approaches.…”

“…RasGRP4 is a guanine nucleotide exchange factor that can activate H-Ras efficiently (3,4,6). Immunogold ultrastructural studies of the in vivo differentiated MCs in the V3 mastocytosis mouse revealed that RasGRP4 tends to reside either in the cytoplasm or on the cytosolic side of the plasma membrane (4).…”

The Role of Ras Guanine Nucleotide Releasing Protein 4 in FcϵRI-mediated Signaling, Mast Cell Function, and T Cell Development

“…These mice express a dysfunctional RasGRP4 protein that lacks a DAG-binding domain. Consequently, they are hyporesponsive to methacholine stimulation (14). We further analyzed why these RasGRP4 Ϫ/Ϫ mast cells are hyporesponsive to stimulation and found that Fc⑀RI-mediated Erk and p38 activation was reduced, whereas activation of other signaling events was not largely affected.…”

“…Its function in mast cells was suggested from studies with C3H/HeJ mice, which are hyporesponsive to methacholine stimulation via the airway (14). These mice express a dysfunctional RasGRP4 protein that lacks a DAG-binding domain (C1 domain) because of aberrant splicing.…”