Michael R. Koelle - US grants

The long-term goal of this proposal is to understand how neurotransmitters act through G protein-coupled receptors to modulate the activities of neurons. Our studies focus on a neurotransmitter signaling pathway that regulates activities of the neurons and muscles of the egg-laying system of C. elegans. We have identified mutations that disrupt the regulation of egg-laying behavior and used them to identify and study genes encoding components of this G protein signaling pathway. The genes analyzed so far have close homologs expressed in the mammalian brain, suggesting that C. elegans will prove a useful model for understanding neurotransmission through G proteins in humans. The first major aim of this proposal is to systematically exploit the molecular genetic system we have developed to analyze this neurotransmitter signaling pathway. We will carry out saturating genetic screens to identify as many of the signaling genes as possible. We will clone and molecularly analyze these genes. Our goal is to eventually reduce our understanding of this G protein signaling pathway to a biochemical level. The potential of this approach is illustrated by the fact that we have already used it to identify a protein, EGL-10, that inhibits signaling by a heterotrimeric G protein in the C. elegans egg-laying system. EGL-10 is a prototype for a large family of proteins we identified in worms and humans that we have named the "regulator of G protein signaling" (RGS) proteins. These RGS proteins may regulate may or all G protein signaling pathways. The second major aim of this proposal is to determine why there are so many RGS proteins. Are they simply redundant, or does each have a specific biological function, perhaps because each is restricted to interacting with a particular G protein or set of G proteins? We will examine this issue by determining the genetic functions and expression patterns of the 11 worm RGS proteins and by studying the biochemical specificity of the interactions between C. elegans RGS and G proteins in vitro. this analysis should reveal the logic by which the family of RGS proteins regulates multiple signaling pathways in the worm. Because many C. elegans G proteins and RGS proteins are closely related to corresponding human homologs, our analysis should shed light on analogous human signaling pathways and direct their further study.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand how neurotransmitters signal through G protein coupled receptors to modulate the activities of neurons. Egg-laying behavior in C. elegans is regulated by neurotransmitter signaling through the G proteins Ga0 and Ga. To identify novel G protein signaling components and understand their mechanisms of action, we: isolate and analyze mutations that disrupt C. elegans egg-laying behavior; 2) clone the genes identified by these mutations; 3) purify the signaling proteins encoded by the cloned genes; and 4) study the properties and interactions of the purified proteins. The signaling proteins analyzed so far have close homologs expressed in the mammalian brain, suggesting that C. elegans is a useful model for understanding neurotransmission through G proteins in humans. The potential of our approach is illustrated by the fact that we have already used it to discover a novel class of regulators of G protein signaling (RGS proteins) that inhibit signaling by acting as G protein GTPase activators. The first major aim of this proposal is to exploit C. elegans genetics to identify and analyze more G protein signaling pathway components. We recently isolated mutations in at least three novel signaling genes. We will clone and molecularly analyze these genes. We also recently cloned an additional genetically identified signaling gene, egl-47, that encodes two putative G protein-coupled receptors, and propose further analysis of their functions. As an additional genetic project, we will knock out the genes encoding most or all of the 13 RGS proteins of C. elegans and use these mutants to define the in vivo functions of RGS proteins. The second major aim of this proposal is to carry out biochemical studies of the signaling proteins already identified. First, we will carry out enzymological studies of diacylglycerol kinase-1, which genetic and biochemical experiments show is a direct effector of Ga0. We will characterize the activities of the purified enzyme and its activation by purified Galphao. Second, we will study the in vitro activities of the RGS proteins EGL-lO and EAT-16. They contain a G gamma-like domain via which they complex with the GB5-like subunit GPB-2 to regulate Galphao and Galphaq. These proteins may form a novel type of G protein heterotrimer containing a Ga subunit, the GB protein GPB-2, and an RGS protein rather than a conventional gamma subunit. We will examine formation of complexes between the RGS, GB and Ga subunits in vitro and determine which regions of the RGS proteins direct them to act specifically on their genetically identified Ga targets.

DESCRIPTION (provided by applicant): We seek to understand how neurotransmitters signal through heterotrimeric G proteins to modulate the activities of neurons. We will focus on the mechanism of signaling by serotonin, defects in which are thought to underlie major depression in humans. Our goal is to understand how the many G protein coupled serotonin receptors function together to mediate appropriate responses to this neurotransmitter, and to find out what happens downstream of receptor activation. C. elegans uses serotonin as a neurotransmitter, and we will use this model system to investigate the mechanism of three different serotonin signaling events. In our first aim, we will characterize a signaling complex that mediates serotonin signaling onto the C. elegans egg-laying muscles. We will test a model in which multiple serotonin receptors and an ERG potassium channel are co-localized to postsynaptic structures so that localized signals produced by serotonin receptor signaling can inhibit the ERG channel to promote muscle contraction. In the second aim, we will study the mechanism by which serotonin released onto the egg-laying muscles also signals back onto the neurons that release it via autoreceptors to feedback inhibit further serotonin release. We will use a combination of genetic and biochemical studies to analyze two new types of signaling regulators that our preliminary results show enhance such feedback signaling: a GPR/GoLoco protein which binds G1 subunits, and the RIC-8 protein which acts as a G protein nucleotide exchange factor. In the third aim, we will study the mechanism by which serotonin signals to slow down C. elegans locomotion behavior. We have used a genetic screen to identify genes required for this serotonin signaling event. We found that two different serotonin receptors are each required for the effect of serotonin on locomotion: knocking out either receptor alone or both together leads to the same strong defect in serotonin response. We will study how these two different serotonin receptors work together. We will also comprehensively analyze a new protein identified by our screen that is required specifically for serotonin signaling, but that is not similar to any protein previously known to be involved in serotonin signaling. The mechanisms of serotonin signaling are highly conserved between C. elegans and humans and the insights made possible by the power of the C. elegans model system should shed light on new details of human serotonin signaling. This will ultimately help to understand the causes of depression and the interventions that can be used to treat it.

The long-term objective of this proposal is to understand the molecular basis of signaling specificity by fibroblast growth factor receptors (FGFRs). Fibroblast growth factors (FGFs) initiate many different types of biological events, including cell proliferation, angiogenesis, differentiation, cell migration, and cell survival. While much work has been done to elucidate one key FGFR signaling pathway, we have only a rudimentary understanding of the mechanisms that confer specific outcomes to FGF triggered events. FGFs play important roles in human health and disease. FGFs are known to be involved in many important developmental and homeostatic events, and aberrant FGF signaling is responsible for a number of skeletal and craniofacial syndromes. Studies in model organisms have helped elucidate the function of FGFRs and the signaling pathways they utilize. This proposal seeks to extend this understanding by analyzing FGFR signaling specificity in the nematode Caenorhabditis elegans, a model system with tremendously reduced cellular and molecular complexity. C. elegans possesses a single FGFR (EGL-15) and two known FGFs (LET-756 and EGL-17) that mediate several distinct functions: (1) an essential function; (2) guidance of the migrating sex myoblasts (SMs); and (3) the inhibition of sex muscle differentiation. This proposal will focus on how the ligands, specificity determinants on the receptor, and different signal transduction components combine to generate specific biological outcomes of FGFR signaling. We will analyze the specificity determinants on EGL-15 by structure/function studies. We will identify other components required for specific EGL-15 functions by using genetic screens for modifiers of EGL-15-induced phenotypes and by two-hybrid screens for proteins that interact with the specificity determinants on EGL-15. In this way, we will gain a clearer understanding of how specific responses are elicited by the activation of FGF receptors.

DESCRIPTION (provided by applicant): Regulator of G protein Signaling (RGS) proteins downregulate signaling through heterotrimeric G proteins by acting as G protein GTPase activators. They thus regulate signaling by serotonin, dopamine, and other neurotransmitters critical to human mental health. Some of the most important questions about RGS proteins remain unanswered. How are RGS proteins controlled so they can downregulate neural signaling only under appropriate circumstances? What do the various domains and subunits of RGS proteins do? This proposal seeks to answer these questions by carrying out a detailed analysis of two C. elegans RGS proteins, EGL-10 and EAT-16. These are so-called R7 RGS proteins, which have a domain similar to G protein 3 subunits and are constitutively bound to a divergent G2-like protein called G25. Genetic experiments in C. elegans strongly support the hypothesis that G25/RGS dimers function like the 23 component of conventional G123 heterotrimers, and thus assemble under certain circumstances with G1 proteins to form G1/25/RGS "unconventional heterotrimers". The unconventional heterotrimer model can explain 10 years of genetic results with EGL-10 and EAT-16 and provides a framework for understanding the whole purpose for which R7 RGS proteins exist. The proposed experiments will test the unconventional heterotrimer model using three independent lines experiments to analyze properties of the protein complexes formed by EGL-10 and EAT-16. The first aim will use co-immunoprecipitation experiments to test whether unconventional heterotrimers exist in C. elegans protein lysates, and to examine the properties of these protein complexes. A novel approach for rapidly generating biochemical quantities of lysates from transgenic C. elegans will be used to facilitate these experiments. The second aim will use both biochemical and genetic experiments to analyze the effects of an unusual mutant of G25 predicted to specifically disrupt unconventional heterotrimers. The third aim seeks to generate purified recombinant unconventional heterotrimer complexes and to analyze their biochemical properties. PUBLIC HEALTH RELEVANCE: Misregulation of signaling in the brain by neurotransmitters such as serotonin and dopamine leads to disorders such as depression and schizophrenia. This proposal seeks to understand the mechanisms that normally control levels of signaling by these neurotransmitters. This may lead to understanding the causes of human mental disorders and to therapies for their treatment.

[unreadable] DESCRIPTION (provided by applicant): The most commonly used signal transduction mechanism in eukaryotes involves seven transmembrane receptors that activate heterotrimeric G proteins. G proteins mediate the effects of a vast array of signaling molecules including neurotransmitters, hormones, cytokines, the majority of pharmaceutical and addictive drugs, as well as the senses of vision and olfaction. In 1996, Regulators of G Protein Signaling (RGS) proteins were discovered as a fundamental component of the G protein signaling mechanism. Dozens of RGS proteins exist in humans. They are defined by a G protein GTPase activation domain that acts to terminate signaling, but often have multiple other domains whose functions are the subject of active investigation. This application seeks support for the Third RGS Protein Colloquium, to be held April 4-5, 2008 in San Diego, CA. The Colloquium will be satellite of the Experimental Biology 2008 conference. Its association with this massive conference ensures a high level of publicity for the Colloquium and will increase participation by making attendance convenient. Financial support will be used to fund travel to the meeting by speakers, and to encourage the participation of young scientists by reducing their costs. The RGS Protein Colloquia have emerged as the sole meeting devoted to RGS proteins, and provide an essential forum to allow investigators in this field to interact with each other, most of them young and/or new to this fast-growing field. A group of outstanding speakers have already committed to the Colloquium, including established leaders in the RGS field as well as new investigators. A set of additional speakers will be chosen from among meeting registrants to provide more opportunities for investigators who are young or new to the field. Two poster sessions will provide opportunities for yet more young scientists to present their work and also will facilitate interaction among all attendees. Sessions at the meeting will be devoted to the following topics: 1) RGS structure/function; 2) RGS targeting/cellular localization; 3) Novel interactions/functions; 4) RGS action in vivo. Meeting materials will be made available to the public via the conference web site to increase the impact of the Colloquium. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Altered signaling by neuromodulators, such as biogenic amines or neuropeptides, underlies human brain diseases including mood disorders and Parkinson's disease, yet our understanding of exactly how neuromodulators produce proper functioning of neural circuit's remains vague. How do neuromodulators collaborate with fast-acting neurotransmitters to create patterns of activity in neural circuits that underlie complex behaviors? Our goal is to understand for the first time precisely how neuromodulators function in a model neural circuit. The egg-laying circuit of C. elegans contains just three types of neurons and one set of postsynaptic muscles. This circuit involves just one fast-acting neurotransmitter, acetylcholine, plus a set of neuromodulators: the biogenic amines serotonin and tyramine, and several neuropeptides. The circuit generates a two-state behavior that alternates between a ~2.5 minute active phase in which rhythmic egg-laying contractions occur, and a ~20 minute inactive phase. Over the past 16 years, we have developed a battery of C. elegans mutants that show increased or decreased egg laying due to defects in specific signaling molecules in the circuit. We have also developed cell-specific promoters that allow us to express any protein we want in any individual cell type of the egg-laying system. We have now developed new tools to optogenetically manipulate activity of the individual cell types and to use the genetically-encoded Ca2+ sensor GCaMP to quantitatively analyze activity of each cell type within freely-behaving animals. These new tools have brought us to a tipping point at which we are ready to delineate how each cell and each neurotransmitter in the circuit act to together produce the activity pattern of the circuit. We expect a newly meaningful understanding of neuromodulators will be revealed now that we can finally analyze their function in the context of detailed investigation of a circuit they modulate. We will generate this understanding through three aims, each focused on dissecting the function one of the three neuron types in the circuit: Aim 1. We will determine how serotonin and the neuropeptide NLP-3, co-released from the HSN motor neuron, signal onto other cells in the circuit to initiate the active phase of egg laying. Aim 2. W will determine how acetylcholine, released from the VC motor neurons within the active phase, acutely triggers egg-laying contractions only when serotonin and NLP-3 have activated the circuit. Aim 3. We will determine how the uv1 neuroendocrine cells release tyramine to lengthen the inactive phase of egg laying and to space out egg-laying events during the active phase.

The long-term goal of this project is to understand the molecular mechanisms of neurotransmitter signaling through heterotrimeric G proteins. A set of >100 G protein coupled neurotransmitter and neuropeptide receptors signal in the brain by activating a small set of heterotrimeric G proteins. By far the most abundant neural G protein is G?o, which inhibits neural function by a mechanism that remains poorly understood. Our genetic work in C. elegans suggests that signaling by G?o in given neuron is not predominantly activated by any one receptor, but rather may result from the additive effects of many different receptors. Our first aim is to understand how many different receptors present in a single neuron can all activate one G protein to do something that makes biological sense. We will create a GPCR expression atlas and use it to identify all the receptors expressed in a model C. elegans neuron, knock them out in combinations, and analyze the resulting effects on G?o signaling. Our second aim is to characterize how acetylation of G?o is used to regulate neural signaling. We recently used mass spectrometry to show that G?o in both worms and mouse brain is post- translationally modified by acetylation on multiple lysine residues. Knocking out a lysine acetyltransferase enzyme that creates these modifications results in specific defects in serotonin signaling. We will determine how and why acetylation is used to regulate G?o signaling. Our third aim is to identify the long-sought effectors through which G?o signals. A mystery in G?o signaling is that no downstream ?effector? protein activated by directly binding to G?o has yet been identified. Genetic studies in C. elegans suggest that such an effector should exist, yet a variety of approaches have failed to identify it. In the course of our mass spectrometry analysis of G?o, we found that specific neural signaling proteins can co-purify with G?o at sub-stoichiometric levels. We will test the hypothesis that these proteins are G?o effectors. Aim 1. We will identify every cell in C. elegans expressing its 26 G protein coupled small molecule neurotransmitter receptors, and document and partially identify the cells expressing >100 neuropeptide receptors. We will demonstrate the utility of this GPCR atlas by identifying the receptors expressed in a specific pair of neurons that control egg laying, and then characterize knockouts for these receptors to determine how the receptors function together. Aim 2. We will determine how acetylation of G?o by the lysine acetyltransferase ?elongator' affects serotonin signaling, and how this acetylation is regulated. Aim 3. We will determine if signaling proteins that co-purify with G?o function as its long-sought effectors.

Abstract of the funded grant: The long-term goal of this project is to delineate at an unprecedented level of precision how all the neurotransmitter signaling events within a model neural circuit together produce its dynamic pattern of activity. Neural circuits are a basic unit of brain function and altered signaling within neural circuits can disrupt circuit function to produce mood disorders and other human brain diseases. To date, our understanding of such disorders remains vague because we understand only a few individual features within each of the many different neural circuits that have been studied. Therefore, our approach is to analyze all the signaling events within one simple neural circuit, anticipating that this will yield new insights into how neural circuits in general work. This approach is inspired by past successes in other areas of biology in which deep analysis of a simple model in a genetically tractable organism (e.g. the lac operon in E. coli or pattern formation in the Drosophila embryo) led to conceptual breakthroughs that generalized to human biology. Thus, we are intensely studying the simple egg-laying circuit of C. elegans, an experimental system in which we have developed a powerful combination of genetic, optogenetic, chemogenetic, and Ca2+ imaging approaches that together can provide unprecedented mechanistic insights into circuit function. Our analysis has already discovered two instances in which a small-molecule neurotransmitter signals alongside co-released neuropeptides, and also that ongoing circuit activity responds to homeostatic feedback from the postsynaptic cells. These features are likely conserved in other neural circuits. In the next period of this project, we aim to understand how the egg-laying circuit turns itself on. This circuit alternates between ~20 minute inactive phases and ~2.5 minute periods of rhythmic activity. Our recent work shows that the two HSN command neurons release a combination of serotonin and neuropeptides encoded by the nlp-3 gene to activate the circuit. These neuromodulators signal through a set of at least five receptors to alter the postsynaptic muscle response to acetylcholine released from presynaptic motor neurons. Aim 1. We will determine how the HSN neurons ?decide? when to release serotonin and NLP-3 neuropeptides to activate the circuit. Aim 2. We will determine how a diverse set of receptors for serotonin and NLP-3 neuropeptides alter activity of various specific cells of the circuit to make the circuit active. Aim 3. We will identify the cells and signals that act with HSN-released serotonin and NLP-3 neuropeptides to