The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.
Insulin controls target cells by binding to its cell surface receptor. The further intracellular transmission of the insulin signal involves phosphorylation of the receptor as well as other proteins, in particular the insulin receptor substrate (IRS), 1 on specific tyrosine residues. After tyrosine phosphorylation IRS is recognized by Src homology 2 domain-containing proteins for metabolic and glucose transport control, or activation of the mitogen-activated protein kinase (MAP kinase) pathway and mitogenic control (1-4). In type 2 diabetes target cells of the hormone are not fully responsive, which is compensated temporarily by enhanced insulin secretion. The pathogenic mechanisms for this insulin resistance are not known, but an important common feature appears to be a reduced activation/ tyrosine phosphorylation of IRS-1 (5).The insulin receptors are sequestered in the caveolae microdomains of the plasma membrane in adipocytes, and caveolae appear to be critical for insulin control (6). By thin-section electron microscopy, caveolae appear as omega-shaped invaginations of 50 -100 nm diameter in the plasma membrane (7). Caveolae invaginations are found in the plasma membrane of many cell types, but are particularly abundant in adipocytes where they increase in number in conjunction with the differentiation of 3T3-L1 fibroblasts to mature adipocytes (8 -10). Caveolae are involved in receptor-mediated uptake of solutes into the cytosol (11) and in transcytosis (12). A number of proteins, in addition to the insulin receptor, involved in signal transduction are localized to caveolae, which suggests that they may be involved in cellular signaling and control (reviewed in Refs. 13-16).Caveolae are rich in cholesterol and sphingolipids. Caveolae may indeed form from cholesterol-and sphingolipid-rich rafts in the membrane in a process requiring the caveolae-specific structural protein caveolin. Caveolin is found in the plasma membrane and intracellularly, but in the plasma membrane is confined to caveolae; it is therefore used as a marker for these structures. The function of caveolae is dependent on a sufficient level of cholesterol in the plasma membrane and caveolae (12,17). We have also demonstrated a critical dependence of the insulin receptor signal transduction on cholesterol; depletion of cholesterol from the plasma membrane of rat adipocytes reversibly inhibited insulin stimulation of glucose transport and metabolic protein phosphorylation control (6). The importance of caveolae for insulin receptor signaling is further indicated by a consensus binding site for interaction with caveolin (18), and coprecipitation of the receptor with caveolin (4) indicates that the interaction may be physiological. Moreover, the insulin receptor appears to phosphorylate caveolin (19), whereas caveolin was shown to activate the isolated receptor, although the physiological relevance of this is not known (20).Herein we examine in detail the dependence of the insulin receptor on caveolae for signal transduction: the effects of cho...
One of the major challenges limiting the efficacy of anti–PD-1/PD-L1 therapy in nonresponding patients is the failure of T cells to penetrate the tumor microenvironment. We showed that genetic or pharmacological inhibition of Vps34 kinase activity using SB02024 or SAR405 (Vps34i) decreased the tumor growth and improved mice survival in multiple tumor models by inducing an infiltration of NK, CD8+, and CD4+ T effector cells in melanoma and CRC tumors. Such infiltration resulted in the establishment of a T cell−inflamed tumor microenvironment, characterized by the up-regulation of pro-inflammatory chemokines and cytokines, CCL5, CXCL10, and IFNγ. Vps34i treatment induced STAT1 and IRF7, involved in the up-regulation of CCL5 and CXCL10. Combining Vps34i improved the therapeutic benefit of anti–PD-L1/PD-1 in melanoma and CRC and prolonged mice survival. Our study revealed that targeting Vps34 turns cold into hot inflamed tumors, thus enhancing the efficacy of anti–PD-L1/PD-1 blockade.
Background: ␥-Secretase modulators (GSMs) hold potential as disease modifiers in Alzheimer disease; however, their mechanism of action is not completely understood. Results: Second generation in vivo active GSMs were described and shown to modulate A production via a non-APP targeting mechanism, different from the NSAIDs class of GSMs. Conclusion: A growing class of second generation GSMs appears to target ␥-secretase and displays a different mechanism of action compared with first generation GSMs. Significance: The identification of in vivo active non-APP targeting second generation GSMs may facilitate the development of novel therapeutics against AD.
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