Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing.     

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.     

Src kinase modulates the activation, transport and signalling dynamics of fibroblast growth factor receptors

The non-receptor tyrosine kinase Src is recruited to activated fibroblast growth factor receptor (FGFR) complexes through the adaptor protein factor receptor substrate 2 (FRS2). Here, we show that Src kinase activity has a crucial role in the regulation of FGFR1 signalling dynamics. Following receptor activation by ligand binding, activated Src is colocalized with activated FGFR1 at the plasma membrane. This localization requires both active Src and FGFR1 kinases, which are inter-dependent. Internalization of activated FGFR1 is associated with release from complexes containing activated Src. Src-mediated transport and subsequent activation of FGFR1 require both RhoB endosomes and an intact actin cytoskeleton. Chemical and genetic inhibition studies showed strikingly different requirements for Src family kinases in FGFR1-mediated signalling; activation of the phosphoinositide-3 kinase-Akt pathway is severely attenuated, whereas activation of the extracellular signal-regulated kinase pathway is delayed in its initial phase and fails to attenuate.     

Substrates and signalling complexes: the tortured path to insulin action.

In the last few years several potential substrates of the insulin receptor tyrosine kinase have been identified, purified, and their cDNAs isolated. These putative substrates include: 1) pp15, a fatty acid-binding protein; 2) pp120, a plasma membrane ecto-ATPase; 3) pp42, a MAP serine/threonine kinase; 4) pp85, a subunit of the Type 1 phosphatidylinositol kinase; and 5) pp185, a phosphatidylinositol kinase binding protein. Although the tyrosine phosphorylation of several of these substrates correlates with the signalling capabilities of various mutant receptors, the role of these substrates in mediating any one of insulin's many biological responses is still unknown. In addition, recent data indicate that the tyrosine phosphorylation of pp42 may in fact be due to autophosphorylation, thereby removing it from the list of putative substrates of the insulin receptor kinase. Finally, the present review discusses the question of whether signalling occurs as a result of the tyrosine phosphorylation of substrates or via the formation of signalling complexes.     

Insulin receptor kinase following internalization in isolated rat adipocytes

We have studied how insulin-mediated internalization of insulin receptors and insulin activation of the insulin receptor kinase might be inter-related. Isolated rat adipocytes were exposed to 0, 6, or 500 ng/ml insulin for 40 min at 37 degrees C. Subsequently, plasma membrane, low-density microsomal membrane and high-density microsomal membrane subcellular fractions were prepared. Measurement of insulin binding to insulin receptors isolated from the membrane fractions revealed that exposure of cells to insulin resulted in a loss of binding activity (13% at 6 ng/ml, 27% at 500 ng/ml insulin) from the plasma membranes which was completely accounted for by the appearance of receptors in the low-density and high-density microsomal membrane fractions, indicating that insulin had induced translocation of insulin receptors from the surface to the cell interior. Measurement of kinase activity of the isolated receptors revealed that exposure of intact cells to 500 ng/ml insulin resulted in as much as a 35-fold increase in the intrinsic kinase activity of receptors from subcellular fractions. The kinase activity per receptor was equal in all fractions at 3-4 min but by 20 min the activity of the internalized receptors fell approximately 40% to a steady state; plasma membrane receptors, on the other hand, remained fully active over time. This indicates that newly internalized receptors retain their kinase activity but undergo subsequent deactivation. Following exposure of cells to 6 ng/ml insulin, the degree of activation of the insulin receptor kinase was lower in the plasma membrane fraction (24% of the insulin effect at 500 ng/ml) than in the low-density and high-density microsomal membrane fractions (54 and 77%, respectively, of the insulin effect at 500 ng/ml). These results suggest that receptors with an activated kinase are preferentially internalized. We conclude that exposure of adipocytes to insulin causes endocytosis of insulin receptors and activation of insulin receptor kinase, newly internalized receptors are fully active tyrosine kinases but are deactivated as they traverse the intracellular organelles represented by low-density and high-density microsomal membranes, and insulin receptor occupancy, possibly by stimulating phosphorylation and activating the insulin receptor kinase, is important for targeting insulin receptors for internalization.     

Membrane receptors with protein-tyrosine kinase activity

Protein-tyrosine kinase activities have appeared so far to be intrinsic for two classes of proteins: the transforming proteins of certain retroviral oncogenes and the membrane receptors for certain cellular growth factors. In this latter family, the protein-tyrosine kinase is activated upon binding of the growth factor to its receptor and phosphorylates both the receptor itself and other cell target proteins. Growth factor receptors are transmembrane glycoproteins able to undergo not only autophosphorylation but also phosphorylation by other protein kinases (e.g., protein kinase C). Both autophosphorylation and heterologous phosphorylation of the receptor are regulatory events for the ligand binding and protein-tyrosine kinase intrinsic activities of the growth factor receptors.     

Structure and function of the receptor-like protein kinases of higher plants

Cell surface receptors located in the plasma membrane have a prominent role in the initiation of cellular signalling. Recent evidence strongly suggests that plant cells carry cell surface receptors with intrinsic protein kinase activity. The plant receptor-like protein kinases (RLKs) are structurally related to the polypeptide growth factor receptors of animals which consist of a large extracytoplasmic domain, a single membrane spanning segment and a cytoplasmic domain of the protein kinase gene family. Most of the animal growth factor receptor protein kinases are tyrosine kinases; however, the plant RLKs all appear to be serine/threonine protein kinases. Based on structural similarities in their extracellular domains the RLKs fall into three categories: the S-domain class, related to the self-incompatibility locus glycoproteins of Brassica; the leucine-rich repeat class, containing a tandemly repeated motif that has been found in numerous proteins from a variety of eukaryotes; and a third class that has epidermal growth factor-like repeats. Distinct members of these putative receptors have been found in both monocytyledonous plants such as maize and in members of the dicotyledonous Brassicaceae. The diversity among plant RLKs, reflected in their structural and functional properties, has opened up a broad new area of investigation into cellular signalling in plants with far-reaching implications for the mechanisms by which plant cells perceive and respond to extracellular signals.     

Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1.

The Drosophila melanogaster protein sprouty is induced upon fibroblast growth factor (FGF)- and epidermal growth factor (EGF)-receptor tyrosine kinase activation and acts as an inhibitor of the ras/MAP kinase pathway downstream of these receptors. By differential display RT-PCR of activated vs. resting umbilical artery smooth muscle cells (SMCs) we detected a new human sprouty gene, which we designated human sprouty 4 (hspry4) based on its homology with murine sprouty 4. Hspry4 is widely expressed and Northern blots indicate that different isoforms of hspry4 are induced upon cellular activation. The hspry4 gene maps to 5q31.3. It encodes a protein of 322 amino acids, which, in support of a modulating role in signal transduction, contains a prototypic cysteine-rich region, three, potentially Src homology 3 (SH3) binding, proline-rich regions and a PEST sequence. This new sprouty orthologue can suppress the insulin- and EGF-receptor transduced MAP kinase signaling pathway, but fails to inhibit MAP kinase activation by constitutively active V12 ras. Hspry4 appears to impair the formation of active GTP-ras and exert its activity at the level of wild-type ras or upstream thereof. In a yeast two-hybrid screen, using hspry4 as bait, testicular protein kinase 1 (TESK1) was identified from a human fetal liver cDNA library as a partner of hspry4. The hspry4-TESK1 interaction was confirmed by coimmunoprecipitation experiments and increases by growth factor stimulation. The two proteins colocalize in apparent cytoplasmic vesicles and do not show substantial translocation to the plasma membrane upon receptor tyrosine kinase stimulation.     

A 180,000 molecular weight glycoprotein substrate of the insulin receptor tyrosine kinase is present in human placenta and in rat liver, muscle, heart and brain plasma membrane preparations

Cell signalling for insulin may include insulin receptor tyrosine kinase catalysing the phosphorylation of one or more cell proteins. Since temporally the insulin receptor will encounter plasma membrane protein first, we have studied the in vitro phosphorylation of purified plasma membrane preparations. Two proteins were immunoprecipitated with anti-phosphotyrosine antibody from rat liver, muscle, heart and brain membranes and from human placenta membranes: the insulin receptor (detected as a phosphorylated-β-subunit) and a 180,000 molecular weight protein (pp180). pp180 is a monomeric glycoprotein that in the absence of dithiothreitol migrated in denaturing gels like a 150,000 molecular weight protein. pp180 was a substrate for the insulin receptor: (i) receptor and pp180 phosphorylation followed a similar insulin dose-response, although fold-stimulation of autophosphorylation was greater; and (ii) removal of insulin receptors with monoclonal antibodies prevented subsequent pp180 phosphorylation. Insulin-activated receptors increased the extent, but not the rate, of pp180 phosphorylation; the increased phosphate was incorporated into tyrosine and appeared to do so in three or four of pp180's 12 tryptic phosphopeptides. Some data suggest that pp180 is the same protein in each of the tested tissues. The occurrence of pp180, an insulin receptor substrate, in plasma membranes of several insulin responsive tissues suggests that it has a role in insulin signalling.     

Modulation of the insulin growth factor II/mannose 6-phosphate receptor in microvascular endothelial cells by phorbol ester via protein kinase C

Phosphorylation of hormone receptors by protein kinase C (PKC) may be involved in the regulation of receptor recycling. We have studied the recycling and the phosphorylation state of the insulin growth factor (IGF) II/mannose 6-phosphate (Man-6-P) receptor in microvascular endothelial cells from rat adipose tissue. Scatchard analysis showed these cells have over 2 x 10(6) receptors/cell with an affinity constant of 1 x 10(9) M-1. In the presence of phorbol myristate acetate (PMA), an activator of PKC and analog of diacylglycerol, IGF-II receptor number increased in the plasma membrane by 60% without changes in the binding affinity. This increase in cell surface receptor number was confirmed by affinity cross-linking and 125I-surface labeling studies, occurred with a half-time of 20 min, and was reversible upon withdrawal of PMA. The redistribution of IGF-II/Man-6-P receptors was not due to an inhibition of internalization which was in fact stimulated by PMA. The effect of PMA on IGF-II receptor recycling correlated with its stimulation of PKC activity. Furthermore, after down-regulation of cellular PKC levels by preincubation with PMA, PMA was unable to activate residual PKC activity in the membranous pool or increase IGF-II receptor number at the cell surface. The phosphorylation state of the IGF-II/Man-6-P receptor was determined by 32P labeling of intact cells and immunoprecipitation with anti-receptor antibodies. In the basal state, the receptor was phosphorylated only on serine residues which was increased by 75% after treatment with PMA. In contrast, IGF-II decreased receptor phosphorylation and plasma membrane binding in a parallel and dose-dependent manner. Thus, PKC-stimulated serine phosphorylation of IGF-II/Man-6-P receptor may promote the translocation of the receptor to the cell surface, whereas IGF-II-stimulated dephosphorylation of the receptor may lead to a decrease in the number of cell surface receptors. These data suggest a role for PKC-mediated serine phosphorylation in the regulation of intracellular trafficking of receptors in endothelial cells.     

Ethanol mimics ligand-mediated activation and endocytosis of IL-1RI/TLR4 receptors via lipid rafts caveolae in astroglial cells

We have recently reported that ethanol-induced inflammatory processes in the brain and glial cells are mediated via the activation of interleukin-1 beta receptor type I (IL-1RI)/toll-like receptor type 4 (TLR4) signalling. The mechanism(s) by which ethanol activates these receptors in astroglial cells remains unknown. Recently, plasma membrane microdomains, lipid rafts, have been identified as platforms for receptor signalling and, in astrocytes, rafts /caveolae constitute an important integrators of signal events and trafficking. Here we show that stimulation of astrocytes with IL-1β, lipopolysaccharide or ethanol (10 and 50 mM), triggers the translocation of IL-1RI and/or TLR4 into lipid rafts caveolae-enriched fractions, promoting the recruitment of signalling molecules (phospho-IL-1R-associated kinase and phospho-extracellular regulated-kinase) into these microdomains. With confocal microscopy, we further demonstrate that IL-1RI is internalized by caveolar endocytosis via enlarged caveosomes organelles upon IL-1β or ethanol treatment, which sorted their IL-1RI cargo into the endoplasmic reticulum–Golgi compartment and into the nucleus of astrocytes. In short, our findings demonstrate that rafts /caveolae are critical for IL-1RI and TLR4 signalling in astrocytes, and reveal a novel mechanism by which ethanol, by interacting with lipid rafts caveolae, promotes IL-1RI and TLR4 receptors recruitment, triggering their endocytosis via caveosomes and downstream signalling stimulation. These results suggest that TLRs receptors are important targets of ethanol-induced inflammatory damage in the brain.     

Effect of oxidative stress on receptors and signal transmission.

Reactive oxygen metabolites affect binding of ligands to membrane receptors and also coupling of receptors to G-proteins and effector enzymes. Peroxidation of membrane lipids may lead to a lowered receptor density and also will alter the viscosity of the plasma membrane, which affects receptor coupling. Reactive oxygen species may also interact with thiol/disulfide moieties on receptor proteins or on other factors in the receptor system, which is responsible for alterations in receptor binding or coupling. Moreover, lipid peroxidation is associated with the phospholipase A2 pathway, which might indirectly affect receptor function. Moreover, oxidative stress may lead to a disturbance in cellular Ca(2+)-homeostasis. This might be related to an effect on Ca(2+)-mobilizing receptors, but there is also evidence for a decreased Ca(2+)-sequestration by ATPases. In addition, peroxidation of membrane lipids increases membrane permeability to Ca2+. Finally, reactive oxygen species interfere with actions of nitric oxide, thus affecting another pharmacological messenger system.     

Possible mechanism for the alpha subunit of the interleukin-2 receptor (CD25) to influence interleukin-2 receptor signal transduction

The receptors for interleukin 2 (IL-2) and interleukin 15 (IL-15) in T cells share the IL-2R beta subunit (CD122) and gamma(C) subunit but have private alpha subunits. Despite utilizing the same receptor chains known to be necessary and sufficient to transduce IL-2 signals the two cytokines manifest different cellular effects. It is commonly held that the alpha subunit of the IL-2R (CD25) is involved solely in the generation of a high affinity receptor complex. This is questioned by the development of autoimmune diseases in instances where the expression of CD25 is absent. The timely expression of CD25 in the thymus has been linked with clonal deletion. Evidence from peripheral T cells indicates that survival signals arising from the intermediate affinity IL-2R (lacking CD25) do not require the activation of Janus kinase 3 (Jak3) but do require the presence of the membrane proximal region of the gamma(C) chain. This particular signalling pathway is not observed in the high affinity receptor complex where Jak3 is activated. Recent data point to CD25 having a surface distribution consistent with it being localized within membrane microdomains. Here we suggest that in the absence of CD25 expression, IL-2R activation occurs within the soluble membrane fraction. This membrane environment and the absence of CD25 promotes Jak3 independent signal transduction and induction of antiapoptotic mechanisms. T cell antigen receptor (TCR) signalling leads to the induction of CD25 expression, which localizes to membrane microdomains. There is a dynamic pre-association of CD25 and CD122 leading to the loose association of the heterodimer with membrane microdomains. High affinity IL-2R signalling in the context of CD25 and the microdomain environment is characterized by Jak3 activation. The relative levels of high to intermediate affinity receptor signalling determines whether a cell proliferates or undergoes activation induced cell death dependent upon cell status.     

The role of the PI3K/AKT signalling pathway in the corneal epithelium: recent updates

Phosphatidylinositol 3 kinase (PI3K)/AKT (also called protein kinase B, PKB) signalling regulates various cellular processes, such as apoptosis, cell proliferation, the cell cycle, protein synthesis, glucose metabolism, and telomere activity. Corneal epithelial cells (CECs) are the outermost cells of the cornea; they maintain good optical performance and act as a physical and immune barrier. Various growth factors, including epidermal growth factor receptor (EGFR) ligands, insulin-like growth factor 1 (IGF1), neurokinin 1 (NK-1), and insulin activate the PI3K/AKT signalling pathway by binding their receptors and promote antiapoptotic, anti-inflammatory, proliferative, and migratory functions and wound healing in the corneal epithelium (CE). Reactive oxygen species (ROS) regulate apoptosis and inflammation in CECs in a concentration-dependent manner. Extreme environments induce excess ROS accumulation, inhibit PI3K/AKT, and cause apoptosis and inflammation in CECs. However, at low or moderate levels, ROS activate PI3K/AKT signalling, inhibiting apoptosis and stimulating proliferation of healthy CECs. Diabetes-associated hyperglycaemia directly inhibit PI3K/AKT signalling by increasing ROS and endoplasmic reticulum (ER) stress levels or suppressing the expression of growth factors receptors and cause diabetic keratopathy (DK) in CECs. Similarly, hyperosmolarity and ROS accumulation suppress PI3K/AKT signalling in dry eye disease (DED). However, significant overactivation of the PI3K/AKT signalling pathway, which mediates inflammation in CECs, is observed in both infectious and noninfectious keratitis. Overall, upon activation by growth factors and NK-1, PI3K/AKT signalling promotes the proliferation, migration, and anti-apoptosis of CECs, and these processes can be regulated by ROS in a concentration-dependent manner. Moreover, PI3K/AKT signalling pathway is inhibited in CECs from individuals with DK and DED, but is overactivated by keratitis.Subject terms: Growth factor signalling, Apoptosis, Extracellular matrix     

Phase separation enhances probability of receptor signalling and drug targeting

The probability of a given receptor tyrosine kinase (RTK) triggering a defined cellular outcome is low because of the promiscuous nature of signalling, the randomness of molecular diffusion through the cell, and the ongoing nonfunctional submembrane signalling activity or noise. Signal transduction is therefore a ‘numbers game’, where enough cell surface receptors and effector proteins must initially be engaged to guarantee formation of a functional signalling complex against a background of redundant events. The presence of intracellular liquid–liquid phase separation (LLPS) at the plasma membrane provides a mechanism through which the probabilistic nature of signalling can be weighted in favour of the required, discrete cellular outcome and mutual exclusivity in signal initiation.     

Properties of an Ezrin Mutant Defective in F-actin Binding

Ezrin, radixin and moesin are a family of proteins that provide a link between the plasma membrane and the cortical actin cytoskeleton. The regulated targeting of ezrin to the plasma membrane and its association with cortical F-actin are more than likely functions necessary for a number of cellular processes, such as cell adhesion, motility, morphogenesis and cell signalling. The interaction with F-actin was originally mapped to the last 34 residues of ezrin, which correspond to the last three helices (αB, αC and αD) of the C-terminal tail. We set out to identify and mutate the ezrin/F-actin binding site in order to pinpoint the role of F-actin interaction in morphological processes as well as signal transduction. We report here the generation of an ezrin mutant defective in F-actin binding. We identified four actin-binding residues, T576, K577, R579 and I580, that form a contiguous patch on the surface of the last helix, αD. Interestingly, mutagenesis of R579 also eliminated the interaction of band four-point one, ezrin, radixin, moesin homology domains (FERM) and the C-terminal tail domain, identifying a hotspot of the FERM/tail interaction. In vivo expression of the ezrin mutant defective in F-actin binding and FERM/tail interaction (R579A) altered the normal cell surface structure dramatically and inhibited cell migration. Further, we showed that ezrin/F-actin binding is required for the receptor tyrosine kinase signal transfer to the Ras/MAP kinase signalling pathway. Taken together, these observations highlight the importance of ezrin/F-actin function in the development of dynamic membrane/actin structures critical for cell shape and motility, as well as signal transduction.     

FAK Acts as a Suppressor of RTK-MAP Kinase Signalling in Drosophila melanogaster Epithelia and Human Cancer Cells

Receptor Tyrosine Kinases (RTKs) and Focal Adhesion Kinase (FAK) regulate multiple signalling pathways, including mitogen-activated protein (MAP) kinase pathway. FAK interacts with several RTKs but little is known about how FAK regulates their downstream signalling. Here we investigated how FAK regulates signalling resulting from the overexpression of the RTKs RET and EGFR. FAK suppressed RTKs signalling in Drosophila melanogaster epithelia by impairing MAPK pathway. This regulation was also observed in MDA-MB-231 human breast cancer cells, suggesting it is a conserved phenomenon in humans. Mechanistically, FAK reduced receptor recycling into the plasma membrane, which resulted in lower MAPK activation. Conversely, increasing the membrane pool of the receptor increased MAPK pathway signalling. FAK is widely considered as a therapeutic target in cancer biology; however, it also has tumour suppressor properties in some contexts. Therefore, the FAK-mediated negative regulation of RTK/MAPK signalling described here may have potential implications in the designing of therapy strategies for RTK-driven tumours.