Insulin signaling in microdomains of the plasma membrane

Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.     

Ras signaling from plasma membrane and endomembrane microdomains

Ras proteins are compartmentalized by dynamic interactions with both plasma membrane microdomains and intracellular membranes. The mechanisms underlying Ras compartmentalization involve a series of protein/lipid, lipid/lipid and cytoskeleton interactions, resulting in the generation of discrete microdomains from which Ras operates. Segregation of Ras proteins to these different platforms regulates the formation of Ras signaling complexes and the generation of discrete signal outputs. This temporal and spatial modulation of Ras signal transduction provides a mechanism for the generation of different biological outcomes from different Ras isoforms, as well as flexibility in the signal output from a single activated isoform.     

EGFR-RAS-MAPK signaling is confined to the plasma membrane and associated endorecycling protrusions

The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS–containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.     

Designer bugs: structural engineering to build a better mouse model

Many animal models of bacterial diseases are hampered by differences in tissue tropism and the course of pathogenesis. In a recent issue of Cell, by rationally mutating a surface invasion protein (InlA) to have higher binding affinity for its cognate host receptor (E-cadherin), Wollert et al. were able to "murinize"Listeria monocytogenes, creating a strain capable of invading intestinal epithelial cells in mice, mimicking the route of infection in humans.     

Proximal events in signaling by plasma membrane estrogen receptors

Estradiol (E2) rapidly stimulates signal transduction from plasma membrane estrogen receptors (ER) that are G protein-coupled. This is reported to occur through the transactivation of the epidermal growth factor receptor (EGFR) or insulin-like growth factor-1 receptor, similar to other G protein-coupled receptors. Here, we define the signaling events that result in EGFR and ERK activation. E2-stimulated ERK required ER in breast cancer and endothelial cells and was substantially prevented by expression of a dominant negative EGFR or by tyrphostin AG1478, a specific inhibitor for EGFR tyrosine kinase activity. Transactivation/phosphorylation of EGFR by E2 was dependent on the rapid liberation of heparin-binding EGF (HB-EGF) from cultured MCF-7 cells and was blocked by antibodies to this ligand for EGFR. Expression of dominant negative mini-genes for Galpha(q) and Galpha(i) blocked E2-induced, EGFR-dependent ERK activation, and Gbetagamma also contributed. G protein activation led to activation of matrix metalloproteinases (MMP)-2 and -9. This resulted from Src-induced MMP activation, implicated using PP2 (Src family kinase inhibitor) or the expression of a dominant negative Src protein. Antisense oligonucleotides to MMP-2 and MMP-9 or ICI 182780 (ER antagonist) each prevented E2-induced HB-EGF liberation and ERK activation. E2 also induced AKT up-regulation in MCF-7 cells and p38beta MAP kinase activity in endothelial cells, blocked by an MMP inhibitor, GM6001, and tyrphostin AG1478. Targeting of only the E domain of ERalpha to the plasma membrane resulted in MMP activation and EGFR transactivation. Thus, specific G proteins mediate the ability of E2 to activate MMP-2 and MMP-9 via Src. This leads to HB-EGF transactivation of EGFR and signaling to multiple kinase cascades in several target cells for E2. The E domain is sufficient to enact these events, defining additional details of the important cross-talk between membrane ER and EGFR in breast cancer.     

Seven transmembrane receptor core signaling complexes are assembled prior to plasma membrane trafficking

Much is known about beta2-adrenergic receptor trafficking and internalization following prolonged agonist stimulation. However, less is known about outward trafficking of the beta2-adrenergic receptor to the plasma membrane or the role that trafficking might play in the assembly of receptor signaling complexes, important for targeting, specificity, and rapidity of subsequent signaling events. Here, by using a combination of bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and confocal microscopy, we evaluated the steps in the formation of the core receptor-G protein heterotrimer complex. By using dominant negative Rab and Sar GTPase constructs, we demonstrate that receptor dimers and receptor-G betagamma complexes initially associate in the endoplasmic reticulum, whereas G alpha subunits are added to the complex during endoplasmic reticulum-Golgi transit. We also observed that G protein heterotrimers adopt different trafficking itineraries when expressed alone or with stoichiometric co-expression with receptor. Furthermore, deliberate mistargeting of specific components of these complexes leads to diversion of other members from their normal subcellular localization, confirming the role of these early interactions in targeting and formation of specific signaling complexes.     

Fluorescent castasterone reveals BRI1 signaling from the plasma membrane

Receptor-mediated endocytosis is an integral part of signal transduction as it mediates signal attenuation and provides spatial and temporal dimensions to signaling events. One of the best-studied leucine-rich repeat receptor-like kinases in plants, BRASSINOSTEROID INSENSITIVE 1 (BRI1), perceives its ligand, the brassinosteroid (BR) hormone, at the cell surface and is constitutively endocytosed. However, the importance of endocytosis for BR signaling remains unclear. Here we developed a bioactive, fluorescent BR analog, Alexa Fluor 647-castasterone (AFCS), and visualized the endocytosis of BRI1-AFCS complexes in living Arabidopsis thaliana cells. Impairment of endocytosis dependent on clathrin and the guanine nucleotide exchange factor for ARF GTPases (ARF-GEF) GNOM enhanced BR signaling by retaining active BRI1-ligand complexes at the plasma membrane. Increasing the trans-Golgi network/early endosome pool of BRI1-BR complexes did not affect BR signaling. Our findings provide what is to our knowledge the first visualization of receptor-ligand complexes in plants and reveal clathrin- and ARF-GEF-dependent endocytic regulation of BR signaling from the plasma membrane.     

Integrin bi‐directional signaling across the plasma membrane

Integrins are heterodimeric cell adhesion molecules that are important in many biological functions, such as cell migration, proliferation, differentiation, and survival. They can transmit bi‐directional signals across the plasma membrane. Inside‐out activating signal from some cell surface receptors bound with soluble agonists triggers integrins conformational change leading to high affinity for extracellular ligands. Then binding of ligands to integrins results in outside‐in signaling, leading to formation of focal adhesion complex at the integrin cytoplasmic tail and activation of downstream signal pathways. This bi‐directional signaling is essential for rapid response of cell to surrounding environmental changes. During this process, the conformational change of integrin extracellular and transmembrane/cytoplasmic domains is particularly important. In this review, we will summarize recent progress in both inside‐out and outside‐in signaling with specific focus on the mechanism how integrins transmit bi‐directional signals through transmembrane/cytoplasmic domains. J. Cell. Physiol. 228: 306–312, 2013. © 2012 Wiley Periodicals, Inc.     

Using topographic networks to build a representation of consciousness

The subject of consciousness has intrigued both psychologists and neuroscientists for many years. Recently, following many recent advances in the emerging field of cognitive neuroscience, there is the possibility that this fundamental process may soon be explained. In particular, there have been dramatic insights gained into the mechanisms of attention, cognition and perception in recent decades. Here, simple network models are proposed which are used to create a representation of consciousness. The models are inspired by the structure of the thalamus and all incorporate topographic layers in their structure. Operation of the models allows filtering of the information reaching the representation according to (1) modality and/or (2) sub-modality, in addition several of the models allowing filtering at the topographic level. The models presented have different structures and employ different integrative mechanisms to produce gating or amplification at different levels; the resultant representations of consciousness are discussed.     

Giant plasma membrane vesicles to study plasma membrane structure and dynamics

The plasma membrane (PM) is a highly heterogenous structure intertwined with the cortical actin cytoskeleton and extracellular matrix. This complex architecture makes it difficult to study the processes taking place at the PM. Model membrane systems that are simple mimics of the PM overcome this bottleneck and allow us to study the biophysical principles underlying the processes at the PM. Among them, cell-derived giant plasma membrane vesicles (GPMVs) are considered the most physiologically relevant system, retaining the compositional complexity of the PM to a large extent. GPMVs have become a key tool in membrane research in the last few years. In this review, I will provide a brief overview of this system, summarize recent applications and discuss the limitations.     

Dose-response aligned circuits in signaling systems

Cells use biological signal transduction pathways to respond to environmental stimuli and the behavior of many cell types depends on precise sensing and transmission of external information. A notable property of signal transduction that was characterized in the Saccharomyces cerevisiae yeast cell and many mammalian cells is the alignment of dose-response curves. It was found that the dose response of the receptor matches closely the dose responses of the downstream. This dose-response alignment (DoRA) renders equal sensitivities and concordant responses in different parts of signaling system and guarantees a faithful information transmission. The experimental observations raise interesting questions about the nature of the information transmission through DoRA signaling networks and design principles of signaling systems with this function. Here, we performed an exhaustive computational analysis on network architectures that underlie the DoRA function in simple regulatory networks composed of two and three enzymes. The minimal circuits capable of DoRA were examined with Michaelis-Menten kinetics. Several motifs that are essential for the dynamical function of DoRA were identified. Systematic analysis of the topology space of robust DoRA circuits revealed that, rather than fine-tuning the network's parameters, the function is primarily realized by enzymatic regulations on the controlled node that are constrained in limiting regions of saturation or linearity.     

Nerve growth factor signaling in caveolae-like domains at the plasma membrane

Nerve growth factor (NGF) binding to its receptors TrkA and p75(NTR) enhances the survival, differentiation, and maintenance of neurons. Recent studies have suggested that NGF receptor activation may occur in caveolae or caveolae-like membranes (CLM). This is an intriguing possibility because caveolae have been shown to contain many of the signaling intermediates in the TrkA signaling cascade. To examine the membrane localization of TrkA and p75(NTR), we isolated caveolae from 3T3-TrkA-p75 cells and CLM from PC12 cells. Immunoblot analysis showed that TrkA and p75(NTR) were enriched about 13- and 25-fold, respectively, in caveolae and CLM. Binding and cross-linking studies demonstrated that the NGF binding to both TrkA and p75(NTR) was considerably enriched in CLM and that about 90% of high affinity binding to TrkA was present in CLM. When PC12 cells were treated with NGF, virtually all activated (i.e. tyrosine phosphorylated) TrkA was found in the CLM. Remarkably, in NGF-treated cells, it was only in CLM that activated TrkA was coimmunoprecipitated with phosphorylated Shc and PLCgamma. These results document a signaling role for TrkA in CLM and suggest that both TrkA and p75(NTR) signaling are initiated from these membranes.