ABSTRACT
BACKGROUND: Mammalian Shc (Src homology and collagen) proteins comprise a family of four phosphotyrosine adaptor molecules which exhibit varied spatiotemporal expression and signaling functions. ShcD is the most recently discovered homologue and it is highly expressed in the developing central nervous system (CNS) and adult brain. Presently however, its localization within specific cell types of mature neural structures has yet to be characterized. RESULTS: In the current study, we examine the expression profile of ShcD in the adult rat CNS using immunohistochemistry, and compare with those of the neuronally enriched ShcB and ShcC proteins. ShcD shows relatively widespread distribution in the adult brain and spinal cord, with prominent levels of staining throughout the olfactory bulb, as well as in sub-structures of the cerebellum and hippocampus, including the subgranular zone. Co-localization studies confirm the expression of ShcD in mature neurons and progenitor cells. ShcD immunoreactivity is primarily localized to axons and somata, consistent with the function of ShcD as a cytoplasmic adaptor. Regional differences in expression are observed among neural Shc proteins, with ShcC predominating in the hippocampus, cerebellum, and some fiber tracts. Interestingly, ShcD is uniquely expressed in the olfactory nerve layer and in glomeruli of the main olfactory bulb. CONCLUSIONS: Together our findings suggest that ShcD may provide a distinct signaling contribution within the olfactory system, and that overlapping expression of ShcD with other Shc proteins may allow compensatory functions in the brain.
Subject(s)
Central Nervous System/metabolism , Shc Signaling Adaptor Proteins/metabolism , Animals , Central Nervous System/cytology , Immunohistochemistry , Male , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Neurons/cytology , Neurons/metabolism , Rats, Sprague-Dawley , Src Homology 2 Domain-Containing, Transforming Protein 2/metabolism , Src Homology 2 Domain-Containing, Transforming Protein 3/metabolismABSTRACT
The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT1 receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET2 technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.
