Abstract
Disabled-2 (Dab2) is a widely expressed endocytic adaptor with a phosphotyrosine-binding domain (PTB) that binds to NPXY motifs found in many transmembrane receptors. Dab2 also contains a proline-rich domain that binds to Grb2 in competition with Sos1, thus Dab2 modulates Ras/MAPK activation. We produced Dab2 conditional knockout mice using Sox2-cre to delete the floxed dab2 allele. Both copies of the dab2 gene were efficiently deleted in the embryos but not in the extraembryonic endoderm. The Dab2 conditional knockout mice were viable and grossly normal. However, our investigation of these mutant mice revealed multiple biological deviations. Firstly, Dab2-deficient mammary glands showed a delay during involution. Dab2 protein was induced during lactation and by estrogen, progesterone, and prolactin in primary mouse mammary gland epithelial cell cultures. Dab2 null mammary epithelial cells were insensitive to growth suppression induced by TGF-beta. Dab2 deletion did not affect Smad2 phosphorylation and activation, but rather TGF-beta-stimulated MAPK activation was enhanced. Secondly, the Dab2-deficient mice were found lean and resistant to a high caloric diet-induced obesity. The impact of Dab2 was only found in young mice; older wild-type and Dab2-deficient mice showed no significant difference in weight gain after being switch to a high caloric diet. In Dab2-deficient mice, the size of adipocytes was enlarged to the same extent as the wild-types, but the number of adipocytes was reduced. Dab2-deficient embryonic fibroblasts and mesenchymal stromal cells showed a reduced ability to differentiate into adipocytes. The mechanism appears related to the regulation of MAPK/Erk1/2 activity by induced Dab2 protein during adipogenic differentiation, which subsequently impacts the phosphorylation and nuclear localization of PPAR-gamma. Thirdly, Dab2 and ARH have homologous functions in LDLR endocytosis. ARH is primarily expressed in the liver and Dab2 is widely distributed throughout the body. We generated compound knockout mice for both Dab2 and ARH. The double knockout mice were viable and grossly normal. When these mice were challenged with high sucrose diet, we found that double knockout mice had a synergistic increase in serum cholesterol and LDL levels comparable to LDL receptor knockout mice. The double knockout mice also developed hepatic steatosis when fed with a high sucrose diet. In the mouse embryonic fibroblasts, we found that in the ARH-/- background, Dab2 depletion greatly reduced LDL uptake, to the level comparable to those of LDLR-/-. We conclude that Dab2 plays a compensatory but not redundant role in LDL endocytosis in the peripheral tissues while ARH is mainly responsible for those in the liver. In summary, mammary glands in Dab2-deficient mice were delayed during involution stage. Dab2 deficient mice were resistant to high caloric diet-induced obesity. Compound knockout mice for both Dab2 and ARH have a synergistic increase in cholesterol and LDL levels. These data suggest that Dab2 functions as a cargo-selective endocytic adaptor as well as a signaling molecule negatively regulating MAPK pathway.