Philip B. Wedegaertner - US grants

Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. This research grant will address this question in the context of heterotrimeric (alphabetagamma) G proteins. G proteins act as molecular switches to relay information from activated receptors to appropriate effector proteins (e.g. adenylyl cyclase, phospholipase C, cGMP phosphodiesterase, and ion channels). Molecular mechanisms underlying the GTPase cycle of G proteins and its regulation by receptors and effectors are becoming increasingly well understood. Much less well understood are mechanisms responsible for targeting G proteins to their appropriate cellular location, and how a unique cellular environment - and proteins that interact with G proteins to target or retain them there - affect a G protein's function. The major objectives of this proposal are to elucidate mechanisms of cellular palmitoylation, covalent attachment to cysteines of a 16 carbon fatty acid, of G protein alpha subunits (Galpha) and mechanisms of specific targeting of Galpha to intracellular or plasma membranes. The role of G protein betagamma subunits in the palmitoylation of Galpha will be tested by employing strategies to disrupt the alphaybetagamma interaction in vivo. The subcellular site of palmitoylation of Galpha will be addressed by examining the palmitoylation of differentially localized Galpha; alphai2 resides at the plasma membrane, whereas alphai3 and a splice variant of alphai2 (salphai2) are found at Golgi membranes. Inhibitors of membrane vesicle transport will be used to further define the cellular pathway for palmitoylation. To understand mechanisms of specific Golgi localization of Galpha, the role of lipid modifications, both myristoylation and palmitoylation, and betagamma interaction will be tested by expression of mutant Galpha.

DESCRIPTION (provided by applicant): Rho guanine-nucleotide exchange factors (RhoGEFs) comprise a large family of intracellular signaling proteins that couple diverse inputs to the activation of the small GTPase Rho and ultimately to dynamic cell architecture changes brought about by Rho's role in modifying a cell's actin cytoskeleton. Cellular processes under the control of Rho GTPases include smooth muscle cell contractility, cell adhesion, cell migration, cell proliferation, neurite extension and retraction, gene expression and cell division. A sub-family of three RhoGEFs, termed regulator of G protein signaling domain-containing RhoGEFs (RGS-RhoGEFs), is specifically activated by heterotrimeric G proteins at the plasma membrane. The RGS-RhoGEFs thus mediate signaling from several important G protein-coupled receptors (GPCR) to activation of Rho and changes in a cell's actin cytoskeleton. The research proposed in this application will focus on one RGS-RhoGEF, termed leukemia-associated RhoGEF (LARG). Critical physiological pathways mediated by LARG include contraction of vascular smooth muscle cells in response to vasoconstrictors angiotensin II and endothelin, and genetic deletion of LARG prevents salt-induced hypertension in the mouse. Thus, LARG has potential as a therapeutic target in the treatment of cardiovascular disease. Work in this laboratory has recently uncovered a novel and unexpected role for LARG in cell division. In cultured cells, LARG is localized at specific mitotic structures, including centrosomes and mitotic spindles in early mitosis and the cytokinesis cleavage furrow and midbody in late mitosis/cytokinesis. Moreover, LARG undergoes mitotic-dependent phosphorylation, and depletion of LARG causes a strong late cytokinesis defect. This application will focus on defining and understanding this new role for LARG. To address this question, the major objectives of this proposal are 1) Define the role of LARG in mitosis; 2) Characterize mitotic-dependent phosphorylation of LARG; and 3) Investigate mitotic localization of LARG and the role LARG in recruiting critical proteins to the midbody. These objectives will be pursued by a variety of experimental approaches, including cultured cells, time-lapse microscopy, immunofluorescence microscopy, pharmacological inhibitors, mutational analysis, and biochemical assays. PUBLIC HEALTH RELEVANCE: Rho guanine-nucleotide exchange factors (RhoGEFs) are key intracellular signaling proteins, connecting activated cell-surface receptors to dynamic changes in a cell's interior cytoskeleton. The RhoGEFs mediate a number of physiological responses that involve changes in a cell's shape, including smooth muscle cell contractility, cell migration, and developmental processes, and represent potential and novel therapeutic targets in disease states such as hypertension and cancer. The research in this application will provide new knowledge about the functions of RhoGEFs and thus better define how the RhoGEFs can be therapeutically inhibited in disease without affecting their critical normal functions.

DESCRIPTION (provided by applicant): Heterotrimeric G proteins (???) are well known for their function in linking G protein-coupled receptors (GPCRs) to a variety of intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological process. In the textbook view, G proteins carry out their function while associated with the cytoplasmic surface of a cell's plasma membrane. In contrast to the classical view of plasma membrane limited G protein signaling, it is becoming increasingly recognized that G protein localization is dynamic and regulated, such that they can reversibly traffic from the plasma membrane to intracellular locations, and that G proteins can have important cellular functions at intracellular sites. The research in this proposal focuses on one such non-canonical G protein function: Golgi-localized G??-mediated regulation of a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the Golgi exit of select protein cargo destined for the plasma membrane. This research will not only provide new insight into heterotrimeric G protein signaling functions, but will also further our understanding f mechanisms that regulate protein transport, a complex process for which a great deal remains to be understood. This essential cellular process involves protein synthesis at and translocation into the endoplasmic reticulum, transit through the endoplasmic reticulum and Golgi, sorting into distinct cargo-containing domains at the trans Golgi network, and finally exit from the Golgi in vesicles or transport carriers and movement to the plasma membrane or intracellular organelle destinations. This application will focus on the role of G?? in regulating the step of Golgi exit o select cargo proteins. To address this non-canonical function of G??, the major objectives are 1) Investigate the role of Golgi-localized G?? in the Golgi-to-PM transport of select cargo; 2) Investigate the role of RKTG in G??-dependent regulation of Golgi-to-PM traffic; and 3) Investigate the role of GPCRs and the trafficking of G?? in the regulation of Golgi-to-PM transport. These objectives will be pursued by a variety of experimental approaches, including cultured cells, immunofluorescence microscopy, fluorescence microscopy of live cells, subcellular fractionation, pharmacological inhibitors, mutational analysis, biochemical assays, and the development of novel approaches for spatial and temporal inhibition of GPCRs and G proteins.

? DESCRIPTION (provided by applicant): Heterotrimeric G proteins (???) are well known for their function in linking G protein- coupled receptors (GPCRs) to intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological process. G proteins function as molecular switches whereby a GPCR promotes GDP release from the ? subunit (G?), followed by subsequent binding of GTP by G? and dissociation of G? and G??. G?-GTP and G?? can then regulate a variety of signaling proteins until the GTP hydrolysis activity of G? turns off signaling by generating the inactive G?-GDP, which then re- associates with G?? to complete the cycle of activation and inactivation. However, in uveal melanoma this tightly controlled G protein cycle is corrupted by a mutation in the closely related G?, ?q or ?1. Such mutations, occurring in over 90% of uveal melanoma, generate a constitutively active ?q or ?11 in which GTP hydrolysis activity is abrogated, thereby locking the G? in an active GTP bound state and turning ?q or ?11 into an oncogenic driver of this cancer. Uveal melanoma, the most common cancer of the eye in adults, metastasizes in up to 50% of uveal melanoma patients, and, once metastasis occurs, it is invariably fatal with an average survival of less than six months. There are currently no effective therapies for metastatic uveal melanoma, and thus there exists an urgent need to better understand the molecular mechanisms that promote the development of uveal melanoma. Based on numerous basic research studies on G protein function, it is clear that mutationally activated G? exhibit key cell biology properties, such as changes in subcellular localization, trafficking, lipid modification, degradation and activation of unique signaling pathways, that set them apart from the wild type G? counterpart. In uveal melanoma, it has been difficult to compare mutationally activated ?q or ?11 with wild type ?q or ?11 due to a lack of tools to differentiate between and effectively study the endogenous mutant versus wild type protein. Therefore, the objective of this project is to develop such a tool by using gene-editing approaches with uveal melanoma cell lines to generate cells in which the endogenous ?q or ?11, either the wild type or mutationally activated form, is fused in-frame to GFP. These genome-edited uveal melanoma cell lines will be a novel and much-needed tool to better understand how mutationally activated ?q or ?11 functions in uveal melanoma and to identify key aspects of regulation of mutationally activated ?q or ?11 that differ from that of wld type ?q or ?11.

Abstract Heterotrimeric G proteins (???) are well known for their function in linking G protein-coupled receptors (GPCRs) to a variety of intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological processes. In the textbook view, G proteins carry out their function while associated with the cytoplasmic surface of a cell?s plasma membrane. In contrast to the classical view of plasma membrane-limited G protein signaling, it is becoming increasingly recognized that G protein localization is dynamic and regulated, such that they can reversibly traffic from the plasma membrane to intracellular locations, and that G proteins can have important cellular functions at intracellular sites. The research in this proposal focuses on understanding non-canonical functions of G?? subunits, and specifically roles for G?? in regulating signaling at the Golgi. Our previous work revealed an important role for Golgi-localized G?? in regulating a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the Golgi exit of select protein cargo destined for the plasma membrane. The research in this current proposal will examine the hypothesis that G?? regulates signaling pathways that control Golgi integrity by regulating the fragmentation of the Golgi under physiological and pathophysiological conditions. Reversible Golgi fragmentation is a cellular phenomenon that occurs under normal conditions, such as during mitosis, and that occurs in disease states, such as infection, cancer and neurodegenerative disease. This application will focus on defining a novel role for G?? in regulating the mitotic Golgi fragmentation checkpoint and by interrogating a novel role for G?? in regulating microtubule- dependent Golgi fragmentation. In addition, this application will define upstream mechanisms that directly promote signaling by G?? at the Golgi. These objectives will be pursued by a variety of experimental approaches, including cultured cells, immunofluorescence microscopy, fluorescence microscopy of live cells, biosensors, pharmacological inhibitors, mutational analysis, and biochemical assays.

Abstract Heterotrimeric G proteins (???) are well known for their function in linking G protein-coupled receptors (GPCRs) to a variety of intracellular responses, and thereby playing essential roles in transmitting a wide variety of extracellular signals into regulation of countless physiological processes. In the textbook view, G proteins carry out their function while associated with the cytoplasmic surface of a cell?s plasma membrane. In contrast to the classical view of plasma membrane-limited G protein signaling, it is becoming increasingly recognized that G protein localization is dynamic and regulated, such that they can reversibly traffic from the plasma membrane to intracellular locations, and that G proteins can have important cellular functions at intracellular sites. The research in this proposal focuses on understanding non-canonical functions of G?? subunits, and specifically roles for G?? in regulating signaling at the Golgi. Our previous work revealed an important role for Golgi-localized G?? in regulating a signaling pathway on the cytoplasmic surface of Golgi membranes that controls the Golgi exit of select protein cargo destined for the plasma membrane. The research in this current proposal will examine the hypothesis that G?? regulates signaling pathways that control Golgi integrity by regulating the fragmentation of the Golgi under physiological and pathophysiological conditions. Reversible Golgi fragmentation is a cellular phenomenon that occurs under normal conditions, such as during mitosis, and that occurs in disease states, such as infection, cancer and neurodegenerative disease. This application will focus on defining a novel role for G?? in regulating the mitotic Golgi fragmentation checkpoint and by interrogating a novel role for G?? in regulating microtubule- dependent Golgi fragmentation. In addition, this application will define upstream mechanisms that directly promote signaling by G?? at the Golgi. These objectives will be pursued by a variety of experimental approaches, including cultured cells, immunofluorescence microscopy, fluorescence microscopy of live cells, biosensors, pharmacological inhibitors, mutational analysis, and biochemical assays.

! BISR Summary The mission of the Sidney Kimmel Cancer Center Bioimaging Shared Resource (BISR) is to advance the scientific research programs of the Cancer Center by providing powerful, reliable, and readily accessible light microscopic image acquisition and analysis capabilities to SKCC investigators. The Bioimaging Shared Resource provides laser point-scanning and spinning disk confocal, TIRF (total internal refection fluorescence), and widefield epifluorescence microscopy capabilities allowing for multi-wavelength visualization and analysis of fixed and living specimens, Z series, single molecule events at surfaces and interfaces, as well as image analysis and processing expertise. The BISR, founded in 1991 by its long-term director James Keen, PhD and continuously funded by the NCI since 1996, is operated under the leadership of Director James Keen, PhD. During 2016, Philip Wedegaertner, PhD, who has long-term expertise using fluorescence microscopy to understand mechanisms of subcellular localization and trafficking of signaling proteins, was appointed as Co- Director in response to the expanded capabilities realized in BISR over the last funding period. Experienced manager, Yolanda Covarrubias, PhD, provides operational consultation, and training and assistance for all instruments. The BISR is centrally located in the Bluemle Life Sciences Building and after training, is accessible for experienced, trained investigators 24 hours a day, seven days a week through the iLab scheduling system. Current BISR goals include: 1) Provide state-of-the-art visualization capabilities; 2) Train users to properly and independently use microscopes and data analysis software; 3) Provide ready access (24/7) to reliable light microscopic image acquisition and analysis tools