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    Signalling and Endocytic Trafficking

    Embryonic Stem Cells and Human Induced Pluripotent Stem Cells: signalling and trafficking. The role of endocytic trafficking in differentiation

    We are interested in the role of the endocytic compartment in GFR signalling. We have chosen to investigate the TGF-β/Activin A and VEGFR families due to their role in angiogenesis, cancer and development, and our approach is:

    • To define the trafficking routes in cultured primary cells with high precision, combining conventional confocal microscopy, super-resolution STED CW microscopy and automated image analysis

    • To define the molecular machinery responsible for this transport, using proteomics and functional genomic approaches

    • To define the signalling networks downstream of receptor activation by high throughput siRNA approaches

    • To integrate the information from cultured cells with animal models

    • To test the relevance of modulation of endocytic trafficking on signalling in disease models

    • To investigate the trafficking circuitries and pathways in human stem cells and induced pluripotent stem cells and address trafficking pathway alterations during differentiation from stem cells to mature endothelial cells

    Cell Signaling and Membrane Trafficking

    No cell lives in isolation. In multicellular organisms, an enormous variety of chemicals, including small molecules, peptides, and proteins are used in intercellular communication. The signaling molecule acts as a ligand, which binds to a structurally complementary site on the extracellular or membrane-spanning domains of the receptor. Binding of a ligand to its receptor causes a conformational change in the cytosolic domain or domains of the receptor that ultimately induces specific cellular responses. The output of a signaling process depends not only on activation of a particular set of signaling molecules but also on the endocytic route of the ligand/receptor complex, which determines where and for how long the signal is emitted. Traditionally, endocytosis has been recognized as a means to terminate signaling via degradation of activated receptor complexes after their internalization from the cell surface. However it is now accepted that the signaling machinery exploits the compartmentalization and the functional specialization of the endocytic pathway, beyond its conventional role in cargo degradation. Following ligand binding, ligand/receptor complexes can be internalised via clathrin mediated endocytosis, caveolae, macropinocytosis, the APPL, the non-clathrin and non-caveolar pathway, the FEME pathway and others (see figure for some of the pathways). Cargo entering via various endocytic routes is transported to a series of intracellular compartments, such as early endosomes, from where it signals or is recycled to the cell surface or is directed to degradative compartments thereby effecting the signaling outcome. Therefore, the endocytic route chosen by a receptor/ligand complex will ultimately determine the final response of the ligand/receptor complex, and this is our area of interest. Most of the work in this field has been carried out in differentiated cells. Indeed, to date we have focused our work on endocytic trafficking of members of the TGFβ/Activin A family and VEGF, due to their involvement in angiogenesis, carcinogenesis and development, in mature differentiated endothelial cells. In the last few years we initiated a new line of research investigating the role of endocytic trafficking in the differentiation of hESCs/hiPSCs to mature endothelial cells. We are especially interested in understanding the role of endocytic trafficking during pluripotency and differentiation.

    Some of the main endocytic pathways (Conner & Schmid, Nature 422(2003)37-44).

    TGFβ family signaling

    The transforming growth factor β (TGFβ) family of ligands consists of evolutionary conserved pleiotropic secreted cytokines, which include TGFβ1, Activins and bone morphogenetic proteins (BMPs). Individual members of this family play crucial roles in multiple processes throughout development and in the maintenance of tissue homeostasis in adult life. As a consequence, deranged signaling by TGFβ family members has been implicated in many human diseases, including cancer, fibrosis, autoimmune and vascular diseases.

    Overview of TGF Signalling Components
    TGFβs, Inhibins, Activins, Nodals and GDF1 exert their signalling by binding specific receptors on the plasma membrane that will activate SMAD2/3 proteins to affect transcription. BMPs, most GDFs and AMH bind their own receptors that activate SMAD1/5/9 to affect separate pathways. Interestingly, aided by the accessory receptor Endoglin, TGFβ can activate the BMP-related R-SMADs and propagate signal via their cascade. Betaglycan facilitates Inhibin binding to Activin receptors therefore attenuating Activin signalling. Cripto has been found to mediate the interaction of Nodal and GDF1 to Activin receptors, enabling signal propagation. Ligand binding proteins such as Follistatin and LAP and Noggin serve as traps, sequestering TGF and BMP ligands away from receptors and attenuating signalling.

    TGFβ ligands trigger heteromeric complex formation between specific transmembrane type I and type II Ser/Thr kinase receptors, in which the type II receptor transphosphorylates and activates the type I receptor. R-SMADs are phosphorylated by type I receptor, and in turn can form heteromeric complexes with SMAD4. These activated SMAD complexes accumulate in the nucleus, where they directly or indirectly bind to specific promoter region on target genes together with transcription factors and/or co-activators/repressors.
    TGFβ receptors localize to both raft and non-raft membrane domains and the internalization route dictates whether signaling or degradation will ensue. Internalization of TGFβ receptors, via the clathrin-coated pathway into an EEA1 and SARA positive endocytic compartment, where SARA recruits non-phosphorylated SMAD2/3 to the activated receptors for phosphorylation, promotes downstream signaling. However, internalization via the raft-caveolar pathway, where SMAD7 and SMURF2 are localized, promotes ubiquitin-dependent receptor degradation. Indeed, inhibition of this pathway leads to receptor stabilization, suggesting that trafficking of receptors to the SARA positive early endosomes functions to sequester receptors from the rafts and caveolae, thereby stabilizing the receptors. Thus, partitioning between these two internalization pathways appears to be a dynamic and balanced process influencing the signaling outcome of the activated TGFβ family receptors. There is sparse information about the role of internalization of TGFβ family receptors via the other routes or the internalization route of Activin A.
    Most of our work to date on trafficking and signalling has been performed in mature primary endothelial cells. Some highlights are documented below:

    ERBIN is a new SARA-interacting protein: competition between SARA and SMAD2 and SMAD3 for binding to ERBIN

    A model illustrating the interactions between SARA, ERBIN and SMAD2/3. ERBIN interacts with SARA using a domain that interacts also with SMAD2 and SMAD3 (amino acids 1208–1265, SSID). As a consequence, SARA competes with SMAD2/3 for binding to ERBIN. As ERBIN binds and segregates phosphorylated SMAD2/3 in the cytoplasm, SARA not only ensures proper presentation of SMAD2/3 for phosphorylation by TGFβ and activin A receptors but also facilitates the nuclear transfer of phosphorylated SMAD2/3 by competing for their cytoplasmic segregation by ERBIN. Thus, the cellular response to TGFβ/activin A might be regulated by a complex interplay between the relative concentrations of SARA, ERBIN and SMAD2/3, as well as their binding affinities.

    SARA and RNF11 interact with each other and ESCRT-0 core proteins and regulate degradative EGFR trafficking

    Smad anchor for receptor activation (SARA) is highly enriched on endocytic membranes via binding to phosphatidylinositol 3-phosphates through its FYVE (Fab1p-YOTB-Vps27p-EEA1) domain. SARA was originally identified as a protein that recruits non-phosphorylated SMAD2/3 to the activated TGFb receptors for phosphorylation, but later reports suggested a regulatory role in endocytic trafficking. Here we demonstrate that the ubiquitin ligase RNF11 is a SARA-interacting protein residing on early and late endosomes, as well as the fast recycling compartment. RNF11 and SARA interact with the ESCRT-0 subunits STAM2 and Eps15b, but only RNF11 associates with the core subunit Hrs. Both gain- and loss-of-function perturbation of RNF11 and SARA levels result in delayed degradation of epidermal growth factor (EGF)-activated EGF receptor (EGFR), while loss-of-function sustained/enhanced EGF-induced ERK1/2 phosphorylation. These findings suggest that RNF11 and SARA are functional components of the ESCRT-0 complexes. Moreover, SARA interacts with clathrin, the ESCRT-I subunit Tsg101 and ubiquitinated cargo exhibiting all the properties of Hrs concerning ESCRT-0 function, indicating that it could substitute Hrs in some ESCRT-0 complexes. These results suggest that RNF11 and SARA participate structurally and functionally in the ESCRT-dependent lysosomal degradation of receptors. As a consequence, the negative influence that perturbation of RNF11 and SARA levels exerts on the lysosomal degradation of EGFRs could underscore the reported overexpression of RNF11 in several cancers. In these cancers, deficient termination of the oncogenic signaling of mutated receptors, such as the EGFRs, through suboptimal lysosomal degradation could contribute to the process of malignant transformation.

    Ongoing projects include: