Marcel Wehrli - US grants

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand how Wnt signals regulate normal development and cancer. The immediate goal is to refine our understanding of how Wnt signals are transduced by studying Arrow, a single pass transmembrane protein recently identified by the PI. Arrow, together with members of the Frizzled (Fz) family, is a co-receptor for the fly Wnt, Wingless (Wg). A fusion of the Arrow cytoplasmic tail to the Fz2 carboxy-terminus (Fz2-Arr[i]) recapitulates the proximity normally generated during receptor activation by the Wnt ligand, demonstrating that Arrow and Fz2 function as co-receptors. Fz2-Arr[i] functions as an activated receptor that is necessary and sufficient to generate ligand- independent signaling. In the first aim, structure/function analyses of this chimeric receptor, which is locked in the signaling conformation, will be performed to identify domains that may be required for assembly of intracellular complexes that transduce Wnt signals from the membrane to the nucleus. The second aim will identify Arrow-interacting proteins by (a) Immunoprecipitating and micro sequencing Arrow-associated proteins from fly embryos, and (b) a novel and highly sensitive yeast two-hybrid system that allows the isolation of components interacting with a membrane protein, the activated receptor. Fly genetics will then be used to identify the in vivo function of Arrow- interacting proteins. The third aim is based on our finding that Axin, a far downstream component of the Wnt cascade, directly interacts with the Arrow cytoplasmic domain (and therefore the activated receptor). This interaction was unexpected since Axin was previously shown to bind and negatively regulate Armadillo (fly B-catenin), the final cytoplasmic Wg/Wnt pathway component. This raises the possibility that Arrow not only functions as a receptor subunit but also regulates the final step in signaling by binding Axin, thereby freeing Armadillo to enter the nucleus and signal. This hypothesis will be tested by analyzing Wnt signaling in transgenic flies made to express mutant Axin proteins that discriminate between binding to Arrow or Armadillo. Collectively, these studies will provide insights into a signaling pathway that is required for normal development of all species and insights into cancers due to deregulation of these processes.

Throughout development and in the adult, cells receive specific signals that they must interpret precisely according to the signal's strength. Secreted proteins of the Wnt family represent one such signal. Wnts elicit precise changes in gene expression mediated by increased levels of the transcription factor Beta-catenin. Too much, or too little, of this signal leads to patterning defects and/or disease.

Wnts induce a Beta-catenin signal by blocking the function of the central pathway regulator, a protein complex assembled around the scaffold protein Axin. Multiple, potentially conflicting, inputs interact within the Axin complex. How these different inputs are integrated and dynamically controlled during Wnt signal transduction remains unclear. The focus of this project is to establish a dynamic model of Wnt/B-catenin signaling by identifying the location of the Axin complex and its sub-complexes during signal transduction. Genetic manipulations will then be used to determine the functional significance of the findings.

The current model of the Wnt/B-catenin pathway is based largely on studies using overexpression in tissue culture, and protein-protein interactions in vitro. The functional significance of these findings for normal signaling in vivo remains unclear. Significantly, in the proposed studies, the Wnt/B-catenin pathway will be analyzed in the model organism Drosophila, allowing its analysis in the normal cellular context and under physiological conditions. Additionally, visualization of the Axin protein complexes in real-time will establish a dynamic picture of Wnt signaling not achieved in previous studies. These studies are expected to identify novel regulatory interactions and may open the Wnt pathway to pharmacological intervention.
This project will provide an opportunity for one graduate student, and two undergraduate students to engage in cutting edge research. In addition, it will allow the PI to introduce K-12 students to basic development and genetics and to promote a science-based curriculum in local schools.

DESCRIPTION (provided by applicant): Wnt/??catenin signaling is critically important to development and disease. How this signaling pathway functions normally in vivo remains unclear. At least a dozen different models have been postulated to explain ?-catenin regulation; most are based on studies requiring protein overexpression in highly artificial in vitro assays. Many contradictions may be resolved by a unifying model built on the hypothesis that Wnt signaling in different contexts always uses the same 'core' Wnt pathway, which is fine-tuned by context-specific 'accessory modules'. A new paradigm for Wnt signaling will be developed by determining how components of the core pathway are assembled and regulated by different accessory modules, and what constitutes an accessory module. The goal is to identify these mechanisms by generating a dynamic model of the pathway (Aim 1). The hypothesis is that the core pathway responds to Wnt signal with faster kinetics than accessory mechanisms. An innovative assay has been established in the Drosophila model system to detect interacting Wnt pathway components at molecular resolution and in real time. This has already revealed several highly significant findings, namely the detection, for the first time in cells or in vivo, of the ky regulator in the pathway, termed the 'destruction complex'. This complex is deemed part of the core pathway in contrast to an accessory mechanism represented by the interaction between the receptor Arrow and the destruction complex scaffold Axin, based work by the PI. The molecular mechanism of this accessory module will be probed by functional tests of model-based predictions (Aim 2). In addition, the hypothesis that a set of mutations in Arrow selectively affects Arrow's function in the same accessory module will be tested. Similarities to defects associated with osteoporosis caused by mutations in a human homologue of Arrow, LRP5, raise the possibility that this disorder may be caused by defects in a Wnt accessory mechanism, rather than in the core mechanism, as assumed. Aim 3 tests the hypothesis that the Wnt receptor may initiate signaling by dissociation of the receptor subunits Arrow and Frizzled, contrary to current models for the assembly and activation of this receptor complex. Whether the strong negative regulation of the Frizzled2-Arrow interaction is part of the core pathway or represents a previously unrecognized mechanism for modulating Wnt-dependent processes in a context- specific manner will also be determined. Taken together, the analyses proposed here will significantly advance the understanding of Wnt signaling by generating a dynamic model of normal signaling in vivo at unprecedented resolution. The emerging model is expected to provide a clear distinction between universal regulation by the core pathway and tissue-specific modulation of the Wnt signal by accessory mechanisms. Such a paradigm shift will reveal, in many instances, that Wnt-associated diseases are defective in tissue-specific accessory mechanisms and such a distinction will greatly facilitate the development of targeted therapies.

NSF Proposal IOS-1353799
Marcel Wehrli, Ph.D.

Dynamics of Wnt receptor activation, signal initiation and signal termination in vivo

Cells of developing embryos and adult tissues must receive and precisely interpret specific signals for proper growth. Secreted proteins of the Wnt family represent one such signal. Wnts elicit precise changes in gene expression mediated by increased levels of the protein ?beta-catenin. Too much or too little signal leads to tissue defects and disease.

While it is known that Wnts activate cell surface receptors, it remains unclear where on the cell membrane or inside the cell the ligand-activated receptor functions, how it activates the intracellular signaling cascade via the protein Dishevelled and how the receptor is turned off. This proposal addresses these questions directly with a novel in vivo approach. In addition, it will identify how Dishevelled blocks the central regulator in the pathway, a protein complex assembled around the scaffold protein Axin, and is expected to identify in vivo how the receptor-initiated signal leads to the inhibition of the Axin complex and induces beta-catenin signaling, a process that remains controversial.

Broader Impact
Our understanding of the Wnt/beta-catenin pathway has increased dramatically in recent years. However, many findings are derived from in vitro experiments and their functional significance is limited. The signaling events of the Wnt/beta-catenin pathway are amenable to investigation in their normal cellular context using the Drosophila model system, underscoring the significance of the studies proposed here. These investigations are expected to identify dynamically changing interactions as the signal is transduced, thereby greatly increasing our understanding of this important signaling mechanism.

This proposal will provide an opportunity for one graduate student and three undergraduates to engage in cutting edge research. In addition, it will allow the PI to introduce K-12 students to basic development and genetics and to promote a science-based curriculum in local schools.