RESUMEN
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.
Asunto(s)
Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/metabolismo , Pruebas de Provocación Bronquial , Diglicéridos/farmacología , Mastocitos/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Factores de Intercambio de Guanina Nucleótido ras/biosíntesis , Secuencia de Aminoácidos , Animales , Hiperreactividad Bronquial/fisiopatología , Pruebas de Provocación Bronquial/métodos , Mapeo Cromosómico , Cruzamientos Genéticos , Factores de Intercambio de Guanina Nucleótido , Mastocitos/efectos de los fármacos , Mastocitos/fisiología , Ratones , Ratones Endogámicos A , Ratones Endogámicos C3H , Datos de Secuencia Molecular , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/fisiología , ARN Mensajero/biosíntesis , Factores de Intercambio de Guanina Nucleótido ras/genética , Factores de Intercambio de Guanina Nucleótido ras/aislamiento & purificación , Factores de Intercambio de Guanina Nucleótido ras/fisiologíaRESUMEN
Erythroid dematin is a major component of red blood cell junctional complexes that link the spectrin-actin cytoskeleton to the overlying plasma membrane. Transcripts of dematin are widely distributed including human brain, heart, lung, skeletal muscle, and kidney. In vitro, dematin binds and bundles actin filaments in a phosphorylation-dependent manner. The primary structure of dematin consists of a C-terminal domain homologous to the 'headpiece' domain of villin, an actin-binding protein of the brush border cytoskeleton. Except filamentous actin, no other binding partners of dematin have been identified. To investigate the physiological function of dematin, we employed the yeast two-hybrid assay to identify dematin-interacting proteins in the adult human brain. Here, we show that dematin interacts with the guanine nucleotide exchange factor Ras-GRF2 by yeast two-hybrid assay, and this interaction is further confirmed by blot overlay, surface plasmon resonance, co-transfection, and co-immunoprecipitation assays. Human Ras-GRF2 is expressed in a variety of tissues and, similar to other guanine nucleotide exchange factors (GEFs), displays anchorage independent growth in soft agar. Co-transfection and immunoblotting experiments revealed that dematin blocks transcriptional activation of Jun by Ras-GRF2 and activates ERK1 via a Ras-GRF2 independent pathway. Because much of the present evidence has centered on the identification of the Rho family of GTPases as key regulators of the actin cytoskeleton, the direct association between dematin and Ras-GRF2 may provide an alternate mechanism for regulating the activation of Rac and Ras GTPases via the actin cytoskeleton.