Adriano Marchese - US grants

DESCRIPTION (provided by applicant): The primary goal of this proposal is to elucidate the cellular and molecular mechanisms by which the chemokine receptor CXCR4 is regulated. CXCR4 dysregulation has been associated with several pathologies including breast cancer, HIV, WHIM syndrome and cardiovascular disease sates such as chronic ischemic heart disease, angina and human end-stage heart failure. Understanding the mechanisms regulating CXCR4 may provide new strategies for the prevention and treatment of the pathologies associated with CXCR4 dysregulation. CXCR4 undergoes agonist-dependent ubiquitination at the plasma membrane mediated by the E3 ubiquitin ligase AIP4, which targets the receptor for degradation in lysosomes by serving as an endosomal sorting signal. However, insight into the cellular and molecular mechanisms by which AIP4 recognizes and ubiquitinates activated CXCR4 and its role on CXCR4 signaling is lacking. We provide initial evidence demonstrating that AIP4 interacts with non-visual arrestins and that phosphorylation may play a role in targeting CXCR4 to lysososmes. We hypothesize that AIP4-mediated ubiquitination of CXCR4 is preceded by phosphorylation leading to arrestin recruitment, which serves as an adaptor to in turn recruit AIP4 to CXCR4. We also provide evidence that suggests that the ubiquitin moiety on CXCR4 not only serves as an endosomal sorting signal, but may also serve as a rapid and immediate terminator of signaling at the plasma membrane. We propose a comprehensive series of studies aimed at examining the interaction between arrestins and AIP4 in cells and in vitro using GST pull-down assays, co- immunoprecipitation studies, siRNA analysis and immunofluorescence microscopy experiments. Our objective is to define the cellular and molecular mechanisms that determine how AIP4 recognizes and ubiquitinates activated CXCR4 to regulate its signaling. The following Specific Aims are proposed: 1. To define how the ubiquitin ligase AIP4 recognizes and ubiquitinates activated CXCR4. 2. To determine the role of phosphorylation in mediating AIP4-dependent ubiquitination and degradation of CXCR4. 3. To determine the role of AIP4-dependent CXCR4 ubiquitination on regulation of CXCR4 mediated signaling.

DESCRIPTION (provided by applicant): GPCR signaling is essential for a wide variety of physiological processes. GPCR signaling can also play an active role in the pathogenesis of multiple diseases. GPCRs are normally tightly regulated so that signals are of the appropriate magnitude and duration and any perturbations in these regulatory processes can be deleterious. Typically, GPCR signaling is tightly regulated by G protein-coupled receptor kinases (GRKs) and beta arrestins. beta arrestins bind to GRK-phosphorylated GPCRs at the plasma membrane to terminate signaling by promoting G protein uncoupling and receptor endocytosis. The molecular mechanisms by which GRKs and beta arrestins regulate GPCR signaling remain poorly understood. The goal of this proposal is to elucidate the molecular mechanisms by which GRKs and beta-arrestins govern GPCR signaling. Recently, we described a novel function for beta arrestins, as endosomal sorting molecules, whereby they function on endosomes to mediate sorting of GPCRs from endosomes to lysosomes, leading to receptor degradation and long-term attenuation of signaling. However, mechanistic insight into this new function is lacking. beta arrestin-1 mediates endosomal sorting of the chemokine receptor CXCR4, which also requires the action of the E3 ubiquitin ligase AIP4 and the ESCRT (endosomal sorting complex required for transport) machinery. How beta arrestin-1 functionally integrates with AIP4 and ESCRTs to mediate CXCR4 endosomal sorting remains to be determined. Based on our strong preliminary data we hypothesize that beta arrestin-1 regulates the ESCRT machinery to control CXCR4 endosomal trafficking and signaling. We propose the following two specific aims: Aim 1: To determine the molecular mechanisms by which beta-arrestin-1 integrates with AIP4 and ESCRTs to control CXCR4 endosomal sorting. Aim 2: To determine the molecular mechanisms by which ESCRTs regulate CXCR4 signaling and function. We will utilize a comprehensive series of state-of-the-art biochemical, molecular and cellular biology and imaging approaches to complete these aims. We anticipate that new knowledge gained from this proposal will lead to the identification of new and innovative approaches to manipulate GPCR signaling to prevent and treat a variety of diseases.

PROJECT SUMMARY G protein-coupled receptor (GPCR) signaling plays a critical role in many physiological processes and is also involved in many human diseases, yet the mechanisms governing GPCR signaling remain poorly understood. The goal of the proposed research is to fill in gaps in knowledge regarding these mechanisms because we believe that elucidating novel aspects of GPCR signaling could identify new targets for drug development. The focus here will be on the therapeutically relevant GPCR called chemokine receptor C-X-C-receptor 4 (CXCR4). CXCR4 signaling is important for embryogenesis, immune function and stem cell regulation, among others. In addition, CXCR4 signaling is involved in several human diseases, including cancer. CXCR4 is aberrantly expressed in many cancers and its expression correlates with poor prognosis. This is mainly because CXCR4 signaling contributes to metastatic disease, the reason for most cancer related deaths. Yet the mechanisms governing CXCR4 signaling remain poorly understood. The present proposal seeks to fill in these knowledge gaps. Metastasis mediated by CXCL12/CXCR4 occurs via migration and/or survival of tumor cells. Several signaling pathways have been implicated in these processes, including the Akt signaling pathway. Akt is a serine/threonine kinase belonging to the AGC family of kinases. It is fully activated by PDK1 (PtdIns(3,4,5)P3- dependent protein kinase 1) phosphorylation at Thr-308, which is located in the kinase domain, and mTORC2 (mammalian or mechanistic target of rapamycin complex 2) phosphorylation at Ser-473, which is located in the C-terminal hydrophobic motif. One mTORC2 subunit, called DEPTOR (DEP-domain containing mTOR- interacting protein), is an inhibitor of its kinase activity. We recently showed that CXCR4 signaling promotes DEPTOR dissociation from mTORC2 and its subsequent lysosomal degradation, which is linked to mTORC2 activation and phosphorylation of Akt at Ser-473 for subsequent signaling. Yet the spatial and temporal regulation of DEPTOR by CXCR4 signaling leading to mTORC2 activation and the functional relevance of Akt signaling remain unknown. The proposed studies seek to fill in this knowledge gap. To address this we will test the hypothesis that ubiquitin-dependent DEPTOR degradation in lysosomes regulates GPCR promoted mTORC2 activity and Akt survival signaling from the surface of endosomes. To test this hypothesis three specific aims are proposed: Aim 1. To elucidate the role of AMSH in Akt signaling by CXCR4; Aim 2. To identify the mechanism by which mTORC2 is recruited to and activated at the surface of endosomes by CXCR4; and Aim 3. To elucidate the functional role of Akt signaling from the surface of endosomes. Elucidating the mechanisms by which CXCR4 promotes Akt-dependent survival signaling is highly significant because it will provide a novel conceptual understanding of CXCR4 and GPCR promoted Akt signaling, as well as provide the exciting potential to translate this knowledge into innovative drug discovery efforts to selectively target Akt signaling downstream of CXCR4 in cancer.

PROJECT SUMMARY The heterotrimeric G protein-coupled receptor (GPCR) C-X-C motif receptor 4 (CXCR4) and its cognate ligand CXCL12 play important roles in health and disease. A large body of evidence indicates that CXCR4 signaling is linked to cancer progression. CXCR4 expression and signaling in cancer correlates with poor prognosis2-5, mainly because cancer cells expressing CXCR4 colonize distant anatomical sites where CXCL12 is located, resulting in metastatic disease, the cause of most cancer related deaths. CXCR4 signaling regulates several aspects of cell physiology linked to cancer progression. This includes directed cell migration and cell survival, which occur via several discrete signaling pathways. Yet the mechanisms remain poorly understood. The focus of this proposal is on the signal transduction mechanisms that regulate CXCR4-mediated chemotaxis towards CXCL12. We recently reported that CXCR4-mediated chemotaxis occurs via a novel mechanism involving a complex formed between endocytic adaptor proteins b-arrestin1 (barr1) and STAM1 (barr1:STAM1). The barr1:STAM1 complex does not act on Akt or ERK-1/2 signaling pathways, but instead is necessary for activating focal adhesion kinase (FAK), which is also necessary for CXCL12 driven chemotaxis. FAK is typically linked to integrin signaling and focal adhesion dynamics, but these aspects of FAK function are not regulated by the barr1:STAM1 complex. Despite our contribution how barr1:STAM1 activates FAK downstream of CXCR4 to promote chemotaxis remains poorly understood. The overall objective of this proposal is to fill in knowledge gaps. Based on our published and preliminary studies we hypothesize that G protein-dependent barr1:STAM1 signaling spatially and temporally controls FAK activity required for CXCR4-dependent chemotaxis. To test this hypothesis we will pursue the following specific aims: Aim 1. To elucidate the role of CXCR4 site-specific phosphorylation on FAK activation; Aim 2. To identify the structural and biophysical properties of the barr1 interaction with STAM1; Aim 3. To elucidate the functional role of the barr1:STAM1 complex in chemotaxis. Because of the mechanistic focus of our proposal we will use cell culture models and other in vitro approaches spanning techniques in cell and molecular biology, genetics, biochemistry and biophysics plus advanced live cell imaging strategies and mass spectrometry approaches. At the conclusion of this project we will have learned novel signal transduction mechanisms by which barr1:STAM1 collaborate to activate FAK to promote chemotaxis. This is significant because it will reveal novel aspects of CXCR4 signaling that could be targeted therapeutically.

PROJECT SUMMARY Cervical cancer remains one of the leading causes of cancer-related deaths world-wide. A large body of evidence indicates that a sub-family of G protein-coupled receptors (GPCRs), known as chemokine receptors, are linked to progression of several cancers, including cervical cancer. Dysregulated signaling of certain chemokine receptors correlates with poor prognosis in cervical cancer, yet the mechanisms remain poorly understood. The overall objective of this proposal is to determine the mechanisms governing chemokine receptor regulation in the context of cervical cancer growth and metastasis. This is significant because these studies will define new targets for clinical potential. Specifically, we will focus on a novel paradigm that governs chemokine receptor bi-directional regulation by A kinase anchoring proteins (AKAPs). AKAPs are scaffolding proteins that bind and nucleate multiple proteins, usually belonging to a common signaling pathway, to spatially and temporally control signaling to drive physiological responses. We performed a siRNA screen of AKAPs expressed in HeLa cells, a cervical cancer cell line, and provide evidence that AKAPs are involved in cross- regulation of chemokine receptor trafficking. Further, using biochemical and biophysical approaches we provide evidence that chemokine receptors reside in a compartment with AKAPs and protein kinas C (PKC), but that other GPCRs are excluded. It is possible that chemokine receptors might co-reside in a sub- compartment at the plasma membrane that enables their cross-regulation without input from other GPCRs, likely to exclusively fine tune their signaling. Based on published and new preliminary data we hypothesize that chemokine receptors are part of a multimeric protein complex compartmentalized by AKAPs that governs their bi-directional regulation. To test this hypothesis we will pursue three specific aims. Aim 1: To determine the role of PKC in bi-directional chemokine receptor regulation; Aim 2: To determine the role of AKAPs in chemokine receptor bi-directional trafficking and signaling; and Aim 3: To determine the role of bi-directional chemokine receptor regulation in cervical cancer in vitro and in vivo. At the conclusion of the proposed studies we expect to determine the mechanism of bi-directional regulation of chemokine receptor signaling. Importantly, we expect to establish that novel aspects of chemokine receptor regulation and signaling could be targeted to treat cervical cancer.