Klipsun Magazine, 1999, Volume 29, Issue 04 - April

https://cedar.wwu.edu/klipsun_magazine/1195/thumbnail.jp

Isoform-specific insulin receptor signaling involves different plasma membrane domains

In pancreatic β-cells, insulin selectively up-regulates the transcription of its own gene and that of the glucokinase gene by signaling through the two isoforms of the insulin receptor, i.e., A-type (Ex11−) and B-type (Ex11+), using different signaling pathways. However, the molecular mechanism(s) that allows the discrete activation of signaling cascades via the two receptor isoforms remains unclear. Here we show that activation of the insulin promoter via A-type and of the glucokinase promoter via B-type insulin receptor is not dependent on receptor isoform–specific differences in internalization but on the different localization of the receptor types in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12–amino acid string encoded by exon 11, which acts as a sorting signal rather than as a physical spacer. Moreover, our data suggest that selective activation of the insulin and glucokinase promoters occurs by signaling from noncaveolae lipid rafts that are differently sensitive toward cholesterol depletion

Spatial Segregation of BMP/Smad Signaling Affects Osteoblast Differentiation in C2C12 Cells

BACKGROUND: Bone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway. METHODOLOGY/PRINCIPAL FINDINGS: Here we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis

The beta secretase BACE1 regulates the expression of insulin receptor in the liver

Insulin receptor (IR) plays a key role in the control of glucose homeostasis; however, the regulation of its cellular expression remains poorly understood. Here we show that the amount of biologically active IR is regulated by the cleavage of its ectodomain, by the β-site amyloid precursor protein cleaving enzyme 1 (BACE1), in a glucose concentration-dependent manner. In vivo studies demonstrate that BACE1 regulates the amount of IR and insulin signaling in the liver. During diabetes, BACE1-dependent cleavage of IR is increased and the amount of IR in the liver is reduced, whereas infusion of a BACE1 inhibitor partially restores liver IR. We suggest the potential use of BACE1 inhibitors to enhance insulin signaling during diabetes. Additionally, we show that plasma levels of cleaved IR reflect IR isoform A expression levels in liver tumors, which prompts us to propose that the measurement of circulating cleaved IR may assist hepatic cancer detection and management

Selective insulin signaling in the pancreatic beta-cell via the two insulin receptor isoforms

Insulin exhibits pleiotropic effects that are tissue- as well as development-dependent. However, the mechanisms by which insulin gains selective effects are poorly understood. Selectivity in insulin signaling is currently discussed as the result of the activation of specific signal transduction pathways. This may be gained by activating specific adapter proteins, such as IRS proteins and Shc, that 'channel' the insulin signal in a more defined way by specifically interacting with downstream located effector proteins. The insulin receptor (IR), the first step in these cascades, exists in two isoforms as a result of alternative mRNA splicing of the11th exon of the pro-receptor transcript. IR-A lacks whereas IR-B contains the respective sequence coding for 12 amino acids in the C-terminus of the a-chain of the receptor. Studies on general and tissue-specific IR knockout models have demonstrated that a defect IR-mediated insulin signaling leads to a type 2 diabetes-like phenotype. However, these knockouts do not discriminate between the two IR isoforms. Besides their different affinity for insulin, differences in kinase activity as well as internalization and recycling for IR-A and IR-B have been described. These data implied differences in the function of either IR isoform. Although all cell types express both isoforms to a various degree, little is known about the mechanisms that underlie IR isoform-specific signaling and their biological importance remains obscure. Besides the classical insulin target tissues liver, muscle and fat, recent research disclosed the pancreatic P-cell as an important target for pleiotropic insulin action, here involving signal transduction through IR and IGF-I receptors. The overall objective of the present thesis work was to test the hypothesis that the two IR isoforms contribute to selective insulin signaling. Specifically, we aimed to investigate the molecular mechanisms that allow simultaneous and selective transcriptional activation of three model genes encoding insulin, beta-cell glucokinase (betaGK) and c-fos by insulin signal transduction via the two IR isoforms in the pancreatic P-cell. We show here that insulin activates the transcription of these three genes by different mechanisms. Insulin activates transcription of its own gene by signaling via IR-A and IRS/P13K la/mTOR/p70s6k. In contrast, betaGK and c-fos genes are activated by insulin signaling via IR-B but employing different signaling cascades. While insulin-stimulated betaGK promoter up-regulation requires the integrity of the IR-B NPEY-motif and signaling via PI3K-C2alphaPDK1/PKB, c-fos gene activation needs the intact YTHM-motif and signaling via P13K la/p52-Shc/MEK1/ERK1/2. Studying the molecular mechanisms that underlie the selective signaling via IR-A versus IR-B, we found that both IR-A-mediated insulin and IRB-mediated betaGK promoter activation are not dependent on IR isoform-specific differences in internalization but on their spatial segregation in the plasma membrane. Our data demonstrate that localization and function of the two receptor types depend on the 12 amino acids encoded by exon 11. Moreover, our data suggest that selective activation of the insulin and betaGK promoters occurs by signaling from non-caveolae plasma membrane micro-domains that are differently sensitive towards cholesterol depletion. Analyzing the mechanisms that allow activation of selective signaling cascades downstream of IR-B, we found that insulin activates the betaGK promoter from membrane-standing IR-B, while c-fos promoter activation is dependent on clathrin-mediated IR-B endocytosis. In conclusion, the results of the present thesis work clearly demonstrate that spatial segregation of selective signaling pathways originating from IR-A and IR-B allows the simultaneous activation of discrete signaling cascades that lead to specific insulin effects