Kendall J. Blumer, Ph.D. - US grants

Many hormones, autacoids, neuroregulatory agents, and sensory stimuli act through cell-surface receptors that are coupled to intracellular guanine nucleotide-binding proteins (G proteins). Receptors of this family mediate diverse physiological processes in man, including regulation of blood pressure, inflammation, cardiac rhythm, synaptic response and neuronal plasticity, sensation of light and olfactants, and regulation of cellular proliferation. The mechanisms and regulation of signal transduction between receptors, G proteins and their intracellular effectors are unresolved issues of key importance from the standpoint of developing pharmacological agents targeted to specific receptor subtypes, and understanding (and perhaps controlling) the transforming activity of certain cellular oncogenes. Although the structural features of receptors and G proteins that mediate transmembrane signaling are beginning to emerge, the mechanisms by which receptors, G proteins and effectors are activated and regulated are poorly understood. The long-term goal of this research program is to establish the fundamental molecular principles by which G protein-coupled signaling systems are regulated. for these purposes a system offering unique experimental advantages will be used: the alpha-factor mating pheromone signal transduction pathway of the yeast Saccharomyces cerevisiae. Genes encoding the alpha-factor receptor and the alpha, beta and gamma subunits of its cognate G protein have been identified. Pharmacological and biochemical assays for receptor/G protein function are now well-developed. These biochemical techniques will be combined with those of classical yeast genetics, molecular genetics and yeast mating physiology to select and characterize mutant receptors and G protein subunits that alter the signal transduction process in novel and illuminating ways. Also, the potential nature of intracellular effectors will be explored. Specific objectives are to: 1. Identify structural elements of the alpha- factor receptor involved in transmembrane signaling by selecting for receptor variants that: i) no longer activate G protein; ii) constitutively activate G protein without binding agonist; and iii) have gained the ability to respond to alpha-factor antagonists. 2. Establish whether receptor mutations affect G protein interaction or activation by using: i) techniques of allele-specific suppression to obtain G protein variants that define specific protein-protein interactions; ii) kinetic, equilibrium, and competition binding experiments to monitor receptor-G protein coupling in vitro; iii) of agonist-stimulated GTPase or GDP/GTP exchange to monitor G protein activation. 3. Explore the potential intracellular second messenger system(s) that mediate response to alpha- factor by determining whether: i) the alpha-factor receptor and its cognate yeast G protein subunits expressed in Xenopus oocytes modulate effectors that regulate ion channel activity; and ii) yeast cell stimulated with alpha-factor produce putative second messengers through the hydrolysis of membrane phospholipids.

This award provides funds to the Division of Biology and Biomedical Sciences at Washington University to continue a successful REU Site. Each summer, about 36 undergraduates from diverse collegiate and ethnic backgrounds will spend 10 weeks engaged in developmental biology research at the University. Mentors will be selected by the students from about 40 faculty members who have active, well-funded research groups and who study developmental mechanisms in a wide range of experimental systems. The emphasis will be on getting each student actively engaged in increasingly independent research in one of these productive research groups. However, to permit students to view their own research in a broader context, the program will provide five types of supplemental activities: (i) workshops dealing with important model organisms, methods and paradigms in contemporary developmental biology research, (ii) faculty seminars designed specifically for the students, (iii) participation in the existing Developmental Biology Journal Club, (iv) a parallel journal club run by and for the students themselves and (v) an end-of-summer research retreat at which all students will present papers summarizing their research accomplishments. Opportunities for frequent peer-group social interactions will also be provided.

DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) and their signaling pathways are the targets of many current therapeutics as well as drugs of abuse. Most currently used therapeutics were developed decades ago when few components of GPCR signaling systems were known. However, new therapeutics, such as agonists biased to evoke GPCR-arrestin signaling, are being developed as a consequence of identifying novel components and regulators of GPCR signaling networks. Such novel targets potentially enable long-standing problems associated with current therapeutics, such as drug tolerance, side effects or addiction, to be reduced or eliminated. This project addresses these long-term goals by identifying novel mechanisms that control GPCR signaling in the nervous system. It focuses on the R7 RGS family of G protein regulators, which have been shown genetically to be critical intracellular regulators of GPCR signaling throughout the nervous system, and which regulate the biological effects of opioids and amphetamines, and side effects of L-DOPA. The Aims will determine how R7 RGS proteins under the control of a palmitoylated allosteric regulator called R7BP (R7 RGS-binding protein) regulate the activity of adenylyl cyclases and cAMP signaling in neuronal cells. They will identify novel enzymes that regulate palmitate turnover on R7BP and establish the functions of these enzymes as regulators of GPCR signaling in neuronal cells. Lastly, they will determine whether global or local cycles of palmitate turnover regulate the intracellular trafficking and function of R7BP-bound R7 RGS complexes. New knowledge gained by this project will provide deeper understanding of neurobiological signaling processes controlled by R7 RGS proteins, new models for elucidating the functions of palmitate turnover in diverse aspects of cell signaling and disease, and new drug targets that potentially enhance the action or reduce the side effects of GPCR-targeted neurotherapeutics.

Signaling pathways employing heterotrimeric guanine nucleotide-binding proteins (G proteins) and mitogen-activated protein kinases (MAP kinase or MAPK) allow mammalian cells to sense light and olfactants, regulate synaptic transmission, control cardiac rate and force, modulate blood clotting, regulate synaptic transmission, control cardiac rate and force, modulate blood clotting, regulate smooth muscle contraction , and control cell proliferation and differentiation. Mutant forms of G proteins, their receptors, and downstream effectors cause various diseases in human, including endocrine disorders, retinal degeneration, and pituitary, thyroid, adrenal cortical and ovarian tumors. These signaling pathways therefore provide important pharmacological targets for controlling human diseases. This project focuses on the mechanisms whereby; 1) agonists activate their cognate G protein-coupled receptor; and 2) G protein betagammma subunits activate a specific MAPK pathway. The mating pheromone response pathway of the yeast Saccharomyces cerevisiae is used as a model that offers genetic and biochemical tools unavailable in mammalian systems. In the short-term, six specific aims will be addressed; 1) define receptor domains that govern the activation process by generating and characterizing an extensive collection of constitutively active receptor mutants; 2) determine whether mutant receptors that cause constitutive signaling in vivo are constitutively active in vitro in a purified reconstituted system; 3) determine whether Gbetagamma subunits or members of the rho family of small GTP-binding proteins bind and/or activate Ste20p protein kinase in vitro; 4) determine whether Gbetagamma subunits target Ste20p to the plasma membrane, and whether constitutive targeting of Ste20p to the membrane bypasses the requirement for Gbetagamma subunits; 5) define domains of Ste20p that are important for regulation by Gbetagamma subunits and/or rho family members; 6) if Ste20p is not the direct target of Gbetagamma subunits, then direct targets will be identified by using Gbetagamma affinity columns, isolating Gbetagamma-effector complexes, or using two- hybrid screens. Given the extensive similarities between G protein- and MAP kinase-lined signaling pathways in yeast and mammalian cells, these studies will reveal fundamental mechanisms governing signaling by receptors and Gbetagamma subunits in mammalian cells.

G protein dependent signaling pathways regulate biological processes triggered by many hormones, inflammatory mediators, neurotransmitters and sensory stimuli, thereby coordinating a diverse array of cell, tissue and organ functions in the adult. These pathways also regulate embryonic development. Perturbations of G protein signaling pathways are associated with many human diseases, including cancer, heart failure, asthma and endocrine disorders. G protein signaling pathways are the targets of more than half of the drugs used today in clinical medicine. Therefore, basic research of G protein signaling pathways will continue to reveal novel disease mechanisms and fuel the discovery process for novel therapeutic agents used to treat a variety of human diseases. The long term goal of this project is to delineate the mechanisms whereby receptors and G proteins function in cell-cell signaling pathways that control the proliferation, differentiation and morphogenesis of eukaryotic cells. Two major challenges are to determine how receptors activate G proteins, and how downstream components of the signaling pathway regulate cell motility and morphogenesis. To answer these questions, this project uses the mating pheromone response pathway of the yeast S. cerevisiae as a model that is directly applicable to cells of many other eukaryotic organisms including humans. Studies of G protein signaling in yeast have revealed novel mechanisms that later have been shown to be used in human cells. The following specific aims are proposed: 1. How do receptors activate G proteins? 2. Does oligomerization regulate receptor assembly, trafficking to the plasma membrane, signaling, or downregulation? 3. How do PAK family kinases regulate actin cytoskeletal organization?

As transducers of sensory stimuli and extracellular signals, G protein coupled receptors and the signaling pathways they activate are important regulators of cell function throughout development and adult life. They regulate cell differentiation, proliferation, motility and membrane excitability, which have key roles in living systems. A fundamental challenge is to determine how cells elaborate specific and temporally regulated responses when challenged with stimuli that trigger G protein signaling pathways. New insights into the mechanisms governing the specificity and temporal regulation of G protein signaling pathways have been provided by the recent discovery of RGS proteins (regulators of G protein signaling), a novel family of more than 20 regulatory proteins. RGS proteins can limit the strength or duration of cellular responses by acting as GAPs (GTPase activating proteins) that stimulate the ability of G protein alpha subunits to hydrolyze GTP. Certain RGS family members can act as effectors, mediating specific responses triggered by agonist stimulation. Because RGS proteins probably have additional functions, further studies of these proteins should yield novel insights into the mechanisms whereby G proteins control cell function.

The long term objective of this project is to delineate the mechanisms whereby cell function is regulated by RGS proteins. Currently the focus of this project is on RGS2, which is expressed in various organs and tissues where it potentially regulates a number of G protein dependent signaling pathways. This project will determine if the selectivity of RGS2 toward Gq is an important determinant of biological function. These studies will aid in our understanding of how signals are transmitted and regulated in cells.

DESCRIPTION (provided by applicant): G protein dependent signaling pathways regulate biological processes triggered by many hormones, inflammatory mediators, neurotransmitters and sensory stimuli, thereby coordinating a diverse array of cell, tissue and organ functions in the adult. These pathways also regulate embryonic development. Perturbations of G protein signaling pathways are associated with many human diseases, including cancer, heart failure, asthma and endocrine disorders. G protein signaling pathways are the targets of more than half of the drugs used today in clinical medicine. Therefore, basic research of G protein signaling pathways will continue to reveal novel disease mechanisms and fuel the discovery process for novel therapeutic agents used to treat a variety of human diseases. To reveal novel and fundamentally important mechanisms of G protein-coupled receptor (GPCR) signaling and regulation, this project uses yeast GPCRs as simple and experimentally tractable models that are broadly applicable throughout biology. The proposal builds on preliminary data showing that a yeast GPCR is dimeric or oligomeric in vivo, and that endocytic transport of this GPCR requires actin polymerization stimulated by a WASp homolog that activates the Arp2/3 complex. Specific aims are to: 1) Determine whether GPCR biogenesis, signaling and/or endocytosis requires formation of receptor dimers and/or oligomers. 2) Determine whether heterooligomerization between two different GPCRs regulates receptor biogenesis, signaling or endocytosis. 3) Determine the mechanism by which a WASp homolog promotes actin polymerization-dependent transport of GPCR-bearing endosomes. Completion of this project will yield new concepts and mechanisms important for designing new therapeutics targeted to GPCRs and for understanding disease mechanisms caused by dysfunctional GPCR signaling.

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify functions of RGS proteins and the mechanisms that regulate them. The focus of the present application is RGS2, which has been linked genetically in mice and humans to hypertension. The central hypothesis is that RGS2 regulates blood pressure by carrying out discrete functions in vascular smooth muscle, vascular endothelium, and kidney. This project focuses on the ability of RGS2 to regulate vascular contraction and relaxation by functioning in vascular smooth muscle, endothelium and to regulate fluid transport in renal nephron. It uses cell type- specific RGS2 knockout mice in conjunction with biochemical, cell biological, physiological and imaging methods to address the following Specific Aims: 1) determine how RGS2 degradation is regulated in vascular smooth muscle; 2) determine the relative contributions of RGS2 in vascular smooth muscle, endothelium and nephron in blood pressure control; 3) determine how RGS2 promotes endothelium-dependent vascular relaxation; and 4) determine whether RGS2 regulates the ability of vasoconstrictors to regulate RhoA signaling ex vivo and in vivo. Accordingly, this project advances understanding of blood pressure control mechanisms and their dysregulation in hypertension, which may contribute to the identification of new hypertension therapies. PUBLIC HEALTH RELEVANCE: Hypertension affects 50 million Americans, making this condition a leading mortality risk factor due to its association with greatly increased risk of cardiovascular disease, renal failure, diabetes and stroke. Hypertension causes enormous clinical and societal burden because ~70% of the patient population is poorly treated by currently available therapeutics. Improved treatment is likely to occur when therapeutics can be tailored to a patient's genetics. This project advances this goal by determining how a newly identified hypertension-linked gene---RGS2-regulates blood pressure.

Project Summary/Abstract Therapeutic development in a broad spectrum of diseases often involves drugs that target G protein- coupled receptors (GPCRs). However, despite the importance of GPCRs in disease pathogenesis or progression, receptor-targeted drugs often have surprisingly limited therapeutic effect. One reason is that multiple GPCRs with redundant functions drive disease pathogenesis or progression, as occurs in Alzheimer's, inflammatory disorders and many cancers. Thus, effective therapy would require concurrent targeting of multiple GPCRs, which often cannot be achieved because drugs targeting certain GPCRs do not yet exist, or all disease-driving GPCRs have yet to be identified. In other diseases, including uveal melanoma, hormone- secreting pituitary tumors and ~10-15% of all cancers, pathogenesis is driven independently of GPCRs by constitutively active mutant G protein ?-subunits. Here, GPCR-targeted drugs are inappropriate because they cannot prevent activation of mutant G proteins. However, both types of therapeutic roadblocks could be overcome by pharmacologically targeting G proteins instead of GPCRs. In addition to their clinical/translational potential, pharmacological agents that directly target specific G proteins would be extremely valuable as probes in basic science. They would provide simple, fast, cheap and reliable tools to identify novel functions of G proteins in normal physiology and in animal models of many diseases, in contrast to conventional knockout or knockdown strategies, which are slow, expensive, or suffer from compensatory or off-target effects. Furthermore, understanding how pharmacological agents inhibit specific G proteins will reveal fundamentally new mechanistic principles that control G protein activity. Accordingly, this project aims over the long term to develop a panel of pharmacological agents, each of which directly and selectively inhibits specific G protein ?-subunit subtypes, and describe their mechanisms of action in detail. Its foundation is a pair of nearly identical cyclic depsipeptide natural products that are bioavailable, potent and highly selective inhibitors of the Gq/11 subfamily of G protein alpha-subunits. Using a combination of synthetic organic chemistry, computational biology and G protein functional assays, the project team will pursue Specific Aims that will: 1) determine how these molecules inhibit Galpha activation; 2) identify features of these inhibitors that determine potency, pseudo-irreversibility and Galpha selectivity; and 3) identify synthetic analogs of these inhibitors that target constitutively active Gq/11. These Aims are founded on preliminary data showing that: 1) the project team has established the first robust, scalable route for synthesizing analogs of these inhibitors, as required for preclinical and, eventually, clinical studies; 2) the naturally produced inhibitor potently targets cells driven by constitutively active mutant Gq/11; and 3) the naturally produced inhibitor works by a mechanism that can be adapted to every Galpha subtype.

Project Summary Uveal melanoma (UM) is the most common intraocular tumor in adults. Nearly half of UM patients develop metastatic disease and survive one year or less. No effective therapy exists. Current interventions impair or destroy vision, yet do not improve morbidity or mortality caused by metastatic disease. Clinical trials of cytotoxic chemotherapeutics and immune checkpoint inhibitors have shown little efficacy. Mutant constitutively active forms of G?q/11 that drive oncogenesis in ~90% of UM patients thus far have been undruggable. Inhibitors of signaling molecules downstream of these oncoproteins have failed to demonstrate significant clinical benefit. Effective therapy may require breakthroughs that enable direct targeting of constitutively active G?q/11 or necessary but as yet unidentified downstream signaling cascades. This project fills these gaps by showing for the first time that constitutively active G?q/11 can be trapped pharmacologically in the inactive GDP-bound state, thereby attenuating downstream signaling. The inhibitor causes G?q/11-driven UM cells to arrest growth, die, or re-differentiate into melanocytic cells, whereas it has no effect on BRAF-driven UM cells. Inhibitor-treated UM cells has revealed a novel oncogenesis mechanism in which signaling by constitutively active G?q/11 antagonizes epigenetic silencing mediated by polycomb repressive complex 2 (PRC2) to drive de-differentiation. Based on these breakthroughs, the following Aims will be pursued: 1) Identify novel druggable targets that mediate signaling between constitutively active G?q/11 and PRC2 in UM cells; 2) Determine whether the G?q/11 inhibitor provides vision-sparing therapeutic benefit in mouse models of primary and metastatic UM; and 3) Set the stage for clinical trials by determining which clinical subclasses of human primary UM tumors respond ex vivo to the G?q/11 inhibitor. In summary, this project provides unprecedented opportunity to determine whether direct pharmacological inhibition of mutant constitutively active G?q/11 could provide the first effective and potentially vision-sparing therapeutic option for treating UM.