John J. Tesmer - US grants

DESCRIPTION (provided by applicant): G protein-coupled receptor kinase 2 (GRK2) regulates heterotrimeric G protein signaling in the heart not only by phosphorylating activated beta-adrenergic receptors, thereby initiating their desensitization, but also by sequestering activated G protein alpha and betagamma subunits. Despite its beneficial role in adaptation, unusually high expression of GRK2 is strongly implicated in the onset of cardiovascular disease. We recently determined the crystal structure of a peripheral membrane complex between GRK2 and Gbetagamma. The structure reveals the core architecture shared by all GRKs, and is the first description of Gbetagamma bound to a bona fide effector target. This proposal seeks to address some of the questions generated by the GRK2:Gbetagamma structure, particularly those pertaining to the mechanism of activation of GRK2 by Gbetagamma, phospholipids and GPCRs. The first aim is to characterize the ligand binding sites of GRK2 by determining co-crystal structures of GRK2:Gbetagamma in complex with phospholipids or phospholipid head groups, and by determining novel structures of GRK2:Gbetagamma in complex with nucleotide analogs and peptide substrates. We will also model peptides corresponding to known GRK2 phosphorylation by docking them to the kinase domain, not only to learn more about the sequence specificity of GRK2, but also potentially to develop better peptide substrates or inhibitors. The second aim is to define the conformational changes induced in GRK2 by the binding of Gbetagamma, primarily by determining the structure of the cytosolic form of GRK2 from existing crystals. In addition, conformational changes in GRK2 induced by ligands or membrane translocation will be evaluated using limited proteolysis and ligand-binding assays. The third aim is to define the receptor-docking site of GRK2. First, site-directed mutants of various residues within the predicted docking site will be tested for their ability to block receptor phosphorylation. Secondly, methods to improve existing crystals of the complex between GRK2:Gbetagamma and the activated beta2-adrenergic receptor will be developed, with the ultimate goal of determining its crystallographic structure. In an alternative approach, structures will be determined of GRK2:Gbetagamma in complex either with peptides that correspond to one or more cytosolic loops of the beta2-adrenergic receptor or with a peptide, mastoparan, that mimics the catalytic activity of GPCRs.

Project Summary. The heterotrimeric G protein Gaq regulates platelet activation, blood pressure and cardiac function. While Gaq is best known for its ability to stimulate phospholipase Cp (PLC|3), it also binds to G protein-coupled receptor kinase 2 (GRK2), which competitively inhibits PLCp activation, and to p63RhoGEF, a Rho guanine nucleotide exchange factor that modulates cytoskeletal structure. We recently demonstrated that a functional Gaq chimera could be produced in amounts sufficient for the structure determination of the Gaq-GRK2-Gpy complex. The surprising arrangement of activated heterotrimeric G proteins in this assembly suggested that RGS proteins and receptors could also associate to form even higher order signaling complexes. Aim 1 examines the ability of RGS proteins such as RGS2 to bind GRK2- bound Gaq using a flow-cytometry binding assay, size-exclusion chromatography and crystallographic studies of Gaq-RGS and RGS-Gaq-GRK2-Gpy complexes. Aim 2 investigates changes in the orientation of activated Gaq at the membrane upon effector binding as well as the interactions of Gaq-GRK2-Gpv and RGS proteins with intact receptors or their cytoplasmic loops in a membrane environment. The Gaq chimera also opens the door to the structural analysis of other Gaq-effector interactions. To initiate these efforts, Aim 3 seeks to define the molecular basis for Gaq-mediated activation of p63RhoGEF through site-directed mutagenesis, fluorescence polarization nucleotide exchange assays and structural studies. Relevance. By focusing our proposal on two unique effectors of Gaq,GRK2 and p63RhoGEF, we seek to define general paradigms for heterotrimeric G protein signaling through Gaq. We have also focused onGaq, GRK2, RGS2 and p63RhoGEF because all are strongly linked to cardiovascular physiology and disease, and it is not unreasonable to expect that these proteins coordinate their activities, either directly or indirectly, in living cells. GRK2 and Gaq/n are essential for proper heart development and function, RGS2 regulates blood pressure via attenuation of Gaq signaling, and p63RhoGEF induces changes in myocytes that are characteristic of cardiachypertrophy.

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from the sensation of light to the contractility of the heart. A family of GPCR kinases (GRKs) modulates the activity of these GPCRs by phosphorylating sites in their cytoplasmic loops and C-terminal tails. Although GRKs allow cells to adapt and can protect them from damage incurred by sustained signaling, aberrant GRK activity has been associated with human disease such as hypertension and heart failure. Inhibition of GRK activity is also expected to enhance the action of the many drugs that target GPCRs. In the last five years, our lab has made significant progress in understanding the structure and function of this kinase family. We have produced high resolution crystal structures that represent all three GRK subfamilies, including that of GRK1 (rhodopsin kinase), GRK2 (2-adrenergic receptor kinase 1), and GRK6, as well as structures of GRK2 in complex with heterotrimeric G1q and G23 subunits. While much has been learned about the modular structure of GRKs, their interactions with G proteins, and their configuration at the membrane, only recently have we determined a crystal structure that permits us to rationally test how GRKs recognize and are allosterically activated by GPCRs. In the first aim of this proposal, we test hypotheses derived from our breakthrough structure of GRK6 in a closed conformation, wherein a conserved N-terminal helix docks with the kinase domain and stabilizes it in a more active state. This helix extends from the kinase domain such that it could interact with a GPCR in a manner analogous to how the C-terminal helix of transducin binds opsin. The second aim is devoted to crystallographic analysis of GRK-receptor complexes. We will pursue structures of the closed conformation of GRK6 in complex with substrate peptides derived from the phosphoacceptor sites of GPCRs. To help define how GRKs dock on the receptor, we will develop peptides and/or peptidomimetics derived from the N-terminal helices of GRKs that bind with high affinity to activated bovine or cephalopod rhodopsin for co-crystallization screens. We will also attempt to determine structures of these prototypical GPCRs in complex with full-length GRKs that we engineer to more readily assume a closed conformation. Our final aim is to use a crystallographic approach to define the molecular basis for how a novel RNA aptamer inhibits GRK2 with high affinity and selectivity. We will develop an assay to screen for selective compounds that target key pockets on the surface of GRK2 bound by the aptamer, and will attempt to engineer new aptamers that are selective for GRK6. Understanding how GPCRs activate GRKs and characterizing the unique and functionally critical sites on these enzymes is key to the development of agents that can selectively regulate GRK function in cells. PUBLIC HEALTH RELEVANCE: G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate and thereby regulate the activity of most of the ~800 GPCRs in the human genome. Some GRKs, such as GRK2, are strongly implicated in the progression of cardiovascular disease and hypertension. This proposal investigates the molecular basis for how GRKs recognize and are regulated by their target GPCRs, and seeks to structurally characterize a novel GRK inhibitor that could lead to the development of therapeutic agents or new molecular tools to dissect GRK function in cells.

DESCRIPTION (provided by applicant): A small family of G protein-coupled receptor (GPCR) kinases (GRKs) negatively regulates heterotrimeric G protein signaling by phosphorylating multiple sites in the cytoplasmic loops and tails of activated GPCRs. Through this process, cells adapt to persistent stimuli that act at GPCRs and protect themselves from damage incurred by sustained signaling. GRKs can also play maladaptive roles in human disease. GRK2 is overexpressed during heart failure, which not only uncouples cardiac receptors from the central nervous system, but also promotes the release of excessive amounts of catecholamines from the adrenal gland. Inhibition of GRK2 by transgenic peptides prevents cardiac failure in mouse models, suggesting that GRK2 is an excellent target for the treatment of heart disease. However, selective small molecule inhibitors of GRKs have not been reported, perhaps due to high homology among the active sites of GRKs and other AGC kinases. Over the last six years, our lab has made significant progress in understanding the structure and function of GRKs, and we are currently investigating the molecular basis for the selective inhibition of GRK2 by a high affinity RNA aptamer. Our preliminary crystallographic studies of this complex demonstrate that the aptamer binds primarily to the large lobe of the kinase domain, where it blocks the entrance to the nucleotide binding site of the kinase domain. We hypothesize that this RNA aptamer can be used in a displacement assay to identify small molecules that bind to regions on GRK2 outside of its active site that are also critical for activity. We have designed improved versions of the original RNA aptamer for use in a robust flow cytometry protein interaction assay to screen for compounds that compete with RNA binding to GRK2. In collaboration with the Center for Chemical Genomics at the University of Michigan, we have conducted a preliminary HTS of ~40,000 compounds with excellent statistics. Using activity-based secondary screens, we will confirm which hits derived from this screen and those from screens conducted at a Molecular Libraries Probe Production Center bind directly to GRK2 and inhibit kinase activity. These compounds will be further characterized to establish membrane permeability, their mode of inhibition, and their selectivity for GRK2. Although all active molecules are of interest, small molecules that do not exhibit competitive inhibition with ATP are of particular importance because they would likely represent novel and selective therapeutic leads for the treatment of heart disease. PUBLIC HEALTH RELEVANCE: GRK2 is strongly linked to cardiovascular physiology and disease. Our flow cytometry protein interaction assay will allow us to rapidly screen large libraries of small molecules with the goal of identifying compounds that interfere with a high-affinity RNA aptamer that selectively binds to the kinase domain of GRK2. These compounds have the potential to interact with novel sites on the surface of the kinase domain and thus serve as selective inhibitors of GRK2.

DESCRIPTION (provided by applicant): The heterotrimeric G protein Gq regulates neuromuscular control as well as platelet activation, blood pressure and cardiac function, and plays a central role in the development of hypertension and cardiac hypertrophy. Activated G?q directly interacts with proteins that have been shown to be important for these processes, including the effector enzymes phospholipase C2 (PLC2) and p63RhoGEF, and the GTPase activating protein regulator of G protein signaling protein 2 (RGS2). We recently determined structures of the autoinhibited catalytic core of invertebrate PLC2 and the G?q -p63RhoGEF-RhoA complex, which led to dramatic new insights into how G?q regulates effector activity, and of the RGS2-G1q complex, the first structure of an RGS protein in complex with a member of the G1q subfamily. In Aim 1, we will confirm our model for how G?q allosterically regulates PLC2 in living cells and in the model organism C. elegans, and investigate how the C-terminal regulatory region of PLC23 enhances catalysis and affinity for G1q using functional studies and X-ray crystallographic and electron microscopic analyses of full-length PLC2 in complex with G1q. In Aim 2, we determine the autoinhibited basal structure of p63RhoGEF by solution NMR and assess how its structure is perturbed upon complex formation with G?q which will help fully describe its mechanism of activation. In Aim 3, we will determine the role of unique interactions formed between RGS2 and the helical domain of G?q and test their contribution to the selective regulation of G1q by RGS2. Collectively, our experiments are expected to reveal the molecular basis for how G?q activates its effectors, and how RGS2 appears to have uniquely evolved to regulate G?q . This knowledge is essential for understanding fundamental processes that underlie cardiac and neuromuscular function, and for developing new approaches to treat cardiovascular disease.

DESCRIPTION (provided by applicant): Lysosomal phospholipase A2 (LPLA2) plays a major role in lipid degradation and is believed to underlie drug-induced phospholipidosis, which commonly occurs in patients taking cationic lipophilic drugs such as the antiarrhythmic amiodarone. Aberrant LPLA2 activity may also be involved in development of autoimmune disease and atherosclerosis. LPLA2 is 50% identical in sequence to lecithin-cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport from arterial plaque macrophages via high density lipoproteins (HDL). Genetic mutations in LCAT are responsible for Familial LCAT Deficiency (FLD), a devastating disease characterized by low serum cholesterol ester levels and renal failure. There are no reported atomic models for either LPLA2 or LCAT, which do not have significant homology to other proteins of known structure. Thus, the molecular bases for their substrate selectivity, regulation, and disease phenotypes remain poorly understood. In this proposal, we address this critical gap in knowledge via functional analysis of our new 1.8 ? crystal structure of LPLA2, determination of the atomic structure of LCAT, imaging LCAT bound to HDL particles by electron microscopy, mapping somatic mutations known to cause genetic disease, and investigating the structural basis for differences in acyl acceptor selectivity. In support of our aims, we provide multiple high resolution structure of LPLA2 in various ligand states, negative stained images of LCAT-HDL complexes, and a low resolution crystal structure of fully glycosylated LCAT. The expected outcome of these studies is a better mechanistic understanding of a structurally uncharacterized family of eukaryotic enzymes that play key roles in lipid metabolism. Our structural and functional studies will help explain the molecular basis for genetic disease and ultimately assist in the design of improved biotherapeutics and small molecule LCAT activators to treat lipid-related disorders such as atherosclerosis and LCAT deficiency.

? DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from the sensation of light to the contractility of the heart. GPCR kinases (GRKs) phosphorylate active GPCRs at sites in their cytoplasmic loops and C-terminal tails, thereby promoting uncoupling of these receptors from heterotrimeric G proteins and ultimately their internalization. Although GRKs allow cells to adapt to changes in their environment and protect against damage incurred by sustained signaling, aberrant GRK activity is strongly associated with diseases such as heart failure and cardiac hypertrophy. Furthermore, inhibition of GRK activity is expected to enhance the action of many drugs that promote GPCR signaling. During and since the last funding cycle, our lab has made substantial advances in the identification and development of GRK selective small molecule inhibitors. We determined the crystal structure of GRK2 in complex with a selective RNA aptamer, and then used this macromolecule as a tool to identify the FDA-approved drug paroxetine as a selective inhibitor of GRK2 activity in vitro and in vivo that improves outcome in myocardial infarcted mice. Furthermore, we conducted several ligand-induced thermal stability screens that identified additional chemical scaffolds that potently and selectivity inhibits GRK2 and GRK5. Subsequent rational design based on crystal structures of these leads in complex with various GRKs led to the development of more potent compounds, one of which assisted us in determining the atomic structure of GRK5. In the first aim, we will further develop and characterize hybrid inhibitors of GRK2 based on the two most promising scaffolds. The second aim is devoted to testing how these compounds affect GRK2 recruitment to membranes and receptors and how they perform in cell-based and whole animal models relevant to human disease. In the third aim, we will use our inhibitors to help investigate how GRK5 interacts with membranes and Ca2+*CaM, which together regulate the entry of GRK5 into the nucleus where it promotes the expression of genes that cause cardiac hypertrophy. Collectively, these studies are designed to create a chemical tool box that can be used to help decipher the function of specific GRKs in living cells and disease states, to take a significant step closer towards development of new therapeutic agents for the treatment of heart disease, and to achieve a better understanding of how GRKs interact with their cellular targets.

Summary G protein-coupled receptors (GPCRs) and their downstream signaling pathways are important for the regulation of many essential cellular processes and are targets of some of the most commonly prescribed drugs, such as those used to treat pain, heart failure, and high blood pressure (e.g. morphine, ? blockers, and angiotensin inhibitors, respectively). GPCR kinases (GRKs) and arrestins work together to regulate GPCR signaling by reducing the ability of receptors to couple with G proteins and by targeting active GPCRs for endocytosis. Although these desensitization mechanisms are important for returning cells to their physiological resting states, GRKs and arrestins are also thought to play prominent roles in addiction and cardiovascular disease, at least in part by instigating other, non-canonical signaling cascades. This proposal seeks funding for a forum that would bring together world-leading researchers who study different aspects of GRK and arrestin biology and their roles in disease called G Protein-Coupled Receptor Kinases and Arrestins: From Structure to Disease. Two major highlights of the meeting will be keynote lectures by 2012 Nobel laureates who are experts in GPCR, GRK, and arrestin structure, function, and cell biology. The meeting is expected to not only stimulate the generation of new hypotheses, collaborations, and methodologies that can be used to study and combat drug abuse and cardiovascular disease, but also provide career advancement and speaking opportunities for junior investigators and underrepresented groups of scientists.

Purdue University Molecular Biophysics Training Program PROJECT SUMMARY In 2015, the Zika virus outbreak emerged as an international public health crisis. In less than a year, scientists at Purdue University published the structure of the mature Zika virus capsid, providing immunologists and drug discovery experts with a sophisticated molecular understanding of how to rationally develop selective anti-viral agents. This remarkably fast transition from the discovery of a novel disease to an atomic model was made possible by a cutting-edge electron microscopy facility, a deep understanding of the theory and application of biophysics, and a diverse team of researchers spearheaded by a well-trained predoctoral student. Successful molecular biophysics training programs will instill upon its trainees many of the same qualities that made this example so effective. To this end, the mission of the Purdue University Molecular Biophysics Training program is to bring together 27 preceptors from six different departments to train an outstanding cohort of graduate students in the underlying theory and practice of cutting-edge biophysical techniques. The program's chief objectives are: (i) to provide enhanced training in the application of molecular biophysics in a rigorous and reproducible way to modern problems in human health and disease, (ii) to foster effective and inclusive teamwork, and (iii) to provide career development opportunities tailored to the goals of individual trainees. To achieve these objectives, selected trainees (6 in each year of the award, typically beginning in their 2nd year of study and supported for up to 2 years) will take a new two-semester gateway class that merges theory with team- based project design and implementation, participate in and help implement an interdepartmental biophysics seminar series that will showcase student-invited external speakers and trainee research on campus, and plan, develop, and implement Purdue's annual biophysics symposium called the Hitchhiker's Guide to the Biomolecular Galaxy. Examples of key activities that support the professional development of these trainees are exercises in teamwork built into program coursework and symposium planning, active development and implementation of detailed individual development plans reviewed and revised annually in collaboration with the mentor, personalized teaching and/or internship opportunities, a grant-writing class tailored to biophysical topics, training in the responsible conduct of research, and participation in the recruitment and retention of underrepresented and/or disabled students. By leveraging Purdue's expertise in Biology Education and self- assessment, the training program and its individual activities will be evaluated annually and refined to ensure that the program is meeting its objectives and the needs of the scientific community.