Brian K. Kobilka - US grants

The beta 2 adrenergic receptor has been one of the most extensively studied members of the large family of G protein coupled receptors. Structural domains involved in ligand binding, G protein coupling, regulatory phosphorylation, and agonist mediated receptor internalization have been characterized by chimeric receptor and metagenesis studies. Much of what has been learned about the structural domains of the beta 2 receptor has been shown to be generalized to other members of the family of G protein coupled receptors. While much progress has been made towards understanding the pharmacology and cellular physiology of G protein coupled receptors, the three dimensional structure of the receptor has not been well characterized and the molecular mechanism by which these receptors transmit signals across a lipid bilayer is not known. The overall goal of this research proposal is to further characterize the three dimensional structure of the beta 2 adrenergic receptor, and to identify the structural changes that occur during agonist activation. The Specific Aims include: 1. Develop modified forms of beta 2 adrenergic receptors that will facilitate studies of receptor structure. 2. Determine if receptor oligomerization occurs and is required for signal transduction. 3. Examine the non-covalent interactions between hydrophobic segments 1-5 (domain A) and hydrophobic segments 6 and 7 (domain B), and the role of the structural integrity of the third intracellular loop joining these two domains in receptor function and stability. 4. Characterize the intramolecular relationships of different structural domains in the beta 2 adrenergic receptor, and identify agonist and antagonist specific changes in these structural relationships.

DESCRIPTION (Adapted from Investigator's Abstract): G protein coupled receptors (GPCR) represent one of the largest families of integral membrane proteins and are responsible for the majority transmembrane signal transduction. GPCRs share a common structural motif consisting of 7 membrane spanning domains with an extracellular amino terminus and intracellular carboxyl terminus. Much of what we know about the structure of G protein coupled receptors comes from studies on rhodopsin, a special member of the GPCR family capable of detecting a single photon. The beta 2 adrenergic receptor (beta 2AR) is the first ligand-activated G protein coupled receptor (GPCR) to be cloned and one of the best characterized members of this large family of integral membrane proteins. It serves as a paradigm for GPCR activation and regulation. My lab has a long-standing interest in understanding the structure and mechanism of action of the beta 2 AR. The proposed studies are designed to elucidate the mechanism by which agonist binding leads to G protein activation. We will use biophysical techniques to directly monitor ligand- and G protein-induced conformational changes in purified beta 2 AR in real- time. The Specific Aims include: 1. To characterize ligand-induced conformational changes in the beta 2 AR. 2. To determine the effect of beta 2 AR-Gs coupling on receptor structure and response to ligands. 3. To identify distinct conformational studies in the beta 2 AR.

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) constitute the largest family of hormone and neurotransmitter receptors. The specificity of signal transduction by these receptors is determined in part by the specificity of receptor-ligand binding, as well as the specificity of receptor-G protein interactions. However, many GPCRs can couple to more than one G protein, and individual G proteins can modulate the activity of multiple effector systems. The specificity of GPCR signaling in vivo therefore depends on additional factors such as the availability of specific G proteins and effectors, and the location of these signaling molecules relative to each other and the receptor. There is a growing body of evidence indicating that GPCRs and their associated down-stream signaling molecules exist as protein complexes held together by direct interactions or through scaffolding proteins or interactions with the cytoskeleton. These signaling complexes are likely to be cell-type specific in vivo, so that a given GPCR will activate signaling pathways in a cell-type specific manner.We have chosen betaAR signal transduction in cardiac myocytes as a model system to study the role of signaling complexes in differentiated cells. Beta1 and beta2 Adrenergic receptors (beta1AR and beta2AR) are highly homologous GPCRs activated by adrenaline and noradrenaline. These receptors have similar pharmacologic properties, and both couple preferentially to the Gs. Despite their structural and functional similarities, they have distinct signaling behavior in cardiac myocytes. Our preliminary studies provide evidence that the functional differences between beta1and beta2 can be attributed to the existence of subtype-specific signaling complexes in neonatal cardiac myocytes. The goals of this proposal are: to further characterize the subcellular location and functional properties of these signaling complexes; to investigate how these complexes either facilitate or restrict receptor signaling; to identify the structural domains of the receptor that are needed for interaction with the other components of the signaling complex; and to identify cellular proteins that define the subtype specific signaling complex. A better understanding of the organization of signaling complexes for receptors such as the beta1AR and beta2AR may provide new avenues to pharmacologically intervene in GPCR signaling.

DESCRIPTION (provided by applicant): The opioid receptors belong to the family G protein coupled receptors (GPCRs), the largest family of membrane proteins in the human genome. Opioid receptor agonists are widely used for the management of pain. However, these drugs are also highly addictive and their clinical efficacy is often limited by the development of tolerance. There is a growing body of evidence that opioid receptors and other GPCRs are conformationally complex molecules. Structurally different agonists may induce functionally distinct receptor conformational states that differ in their interactions with G proteins, kinases, arrestins and possibly other signaling molecules. Therefore, the agonist-specific conformational state will determine the clinical efficacy of a drug. In addition to their clinical importance, opioid receptors are particularly valuable as a model system for understanding the mechanism of GPCR activation because of the broad spectrum of opioid ligands including peptide and small molecular weight agonists and antagonists. A better understanding of the opioid receptor structure and drug-induced conformational changes will impact efforts to develop more effective drugs. The goal of this Stage I CEBRA proposal is to demonstrate the feasibility of using fluorescence spectroscopy to study ligand-induced conformational changes in the mu-opioid receptor (MOR) and the delta-opioid receptor (DOR). Experimental Aims include: 1. Develop methods for the production and purification of functional MORs and DORs. 2. Develop methods for the functional reconstitution of MOR and DOR in to phospholipid vesicles and characterize the effect of lipid composition on receptor function. 3. Site-specific labeling of purified opioid receptors with thiol-reactive probes. 4. Study ligand-induced conformational changes in MOR and DOR labeled with conformationally sensitive fluorescent probes. Successful completion of these aims will provide the preliminary data for a more comprehensive study of opioid receptor structure and conformational dynamics in a Stage II CEBRA project.

The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), and GPCRs represent the largest group of targets for drug development. My laboratory has a long-standing interest in elucidating the structure and mechanism of action of GPCRs using the 132 adrenoceptor ([52AR) as a model system. The 15_AR responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine. It has been one of the most extensively characterized members of the GPCR family, and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. My lab has developed approaches to monitor directly ligand-induced conformational changes in purified [52AR protein. Our experiments reveal that the [52AR is a dynamic molecule with complex behavior. We found that agonists and partial agonists induce ligand-specific conformational states, and that agonist activation proceeds through intermediate conformational states. The studies outlined in this proposal will extend these observations by obtaining a high-resolution three-dimensional structure of the beta2AR; by characterizing the dynamic properties of the 7TM segments of the beta2AR; and by mapping the structural changes induced by different classes of ligands (agonists, partial agonists, neutral antagonists, and inverse agonists). Many of the findings will apply to the large number of closely related monoamine receptors and to GPCRs in general. Moreover, the methodologies developed for characterizing [52AR structure will likely be applicable to other GPCRs. A better understanding of the three-dimensional structure and mechanism of activation of GPCRs will further the potential of structure-based drug design and in silico screening for GPCR targets, leading to more rapid development of highly selective and effective drugs.

DESCRIPTION (provided by applicant): Chimeric Proteins To Enhance Crystal Formation Of G Protein Coupled Receptors. This R21 proposal is in response to the Program Announcement on Membrane Protein Production and Structure Determination (RFA Number: RFA-RM-04-026). The overall goal of this proposal is to develop a general approach to obtain high-resolution structures of G protein coupled receptors (GPCRs) that can be used for structure-based drug development. The majority of hormones and neuretransmitters communicate information to cells via GPCRs. GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug development. We propose to use the human beta2AR as a model system to develop a method for generating diffraction quality crystals of GPCRs by increasing their stability and their hydrophilic (polar) surface area. This will be accomplished by generating structurally integrated chimeric proteins in which a soluble protein is integrated into a GPCR by replacing the third intracellular loop (ICL3) and the carboxyl terminus. This approach will address two important obstacles in the generation of GPCR crystals: (1) The ICL3 and carboxyl termini of GPCRs are the most dynamic (flexible) and unstructured domains in most GPCRs. These domains will be replaced by a highly structured homogeneous protein. The three splice sites will ensure a rigid structural coupling between the soluble protein and the GPCR. (2) The soluble protein component of the chimera will significantly increase the polar surface area available for crystal lattice contacts. If successful with the beta2AR, this approach will likely be generalizable to other GPCRs. Specific aims include: Aim 1. Use molecular modeling tools to identify soluble proteins that can be fused to the beta2AR to generate stable, functional chimeras. Aim 2. Generate, express and optimize fusion proteins. Lay summary. The goal of this proposal is to develop a general method to obtain high-resolution structures of G protein coupled receptors (GPCRs). These structures should facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

DESCRIPTION (provided by applicant): Chimeric Proteins To Enhance Crystal Formation Of G Protein Coupled Receptors. This R21 proposal is in response to the Program Announcement on Membrane Protein Production and Structure Determination (RFA Number: RFA-RM-04-026). The overall goal of this proposal is to develop a general approach to obtain high-resolution structures of G protein coupled receptors (GPCRs) that can be used for structure-based drug development. The majority of hormones and neuretransmitters communicate information to cells via GPCRs. GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug development. We propose to use the human beta2AR as a model system to develop a method for generating diffraction quality crystals of GPCRs by increasing their stability and their hydrophilic (polar) surface area. This will be accomplished by generating structurally integrated chimeric proteins in which a soluble protein is integrated into a GPCR by replacing the third intracellular loop (ICL3) and the carboxyl terminus. This approach will address two important obstacles in the generation of GPCR crystals: (1) The ICL3 and carboxyl termini of GPCRs are the most dynamic (flexible) and unstructured domains in most GPCRs. These domains will be replaced by a highly structured homogeneous protein. The three splice sites will ensure a rigid structural coupling between the soluble protein and the GPCR. (2) The soluble protein component of the chimera will significantly increase the polar surface area available for crystal lattice contacts. If successful with the beta2AR, this approach will likely be generalizable to other GPCRs. Specific aims include: Aim 1. Use molecular modeling tools to identify soluble proteins that can be fused to the beta2AR to generate stable, functional chimeras. Aim 2. Generate, express and optimize fusion proteins. Lay summary. The goal of this proposal is to develop a general method to obtain high-resolution structures of G protein coupled receptors (GPCRs). These structures should facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

DESCRIPTION (provided by applicant): Chimeric Proteins To Enhance Crystal Formation Of G Protein Coupled Receptors. This R21 proposal is in response to the Program Announcement on Membrane Protein Production and Structure Determination (RFA Number: RFA-RM-04-026). The overall goal of this proposal is to develop a general approach to obtain high-resolution structures of G protein coupled receptors (GPCRs) that can be used for structure-based drug development. The majority of hormones and neuretransmitters communicate information to cells via GPCRs. GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug development. We propose to use the human beta2AR as a model system to develop a method for generating diffraction quality crystals of GPCRs by increasing their stability and their hydrophilic (polar) surface area. This will be accomplished by generating structurally integrated chimeric proteins in which a soluble protein is integrated into a GPCR by replacing the third intracellular loop (ICL3) and the carboxyl terminus. This approach will address two important obstacles in the generation of GPCR crystals: (1) The ICL3 and carboxyl termini of GPCRs are the most dynamic (flexible) and unstructured domains in most GPCRs. These domains will be replaced by a highly structured homogeneous protein. The three splice sites will ensure a rigid structural coupling between the soluble protein and the GPCR. (2) The soluble protein component of the chimera will significantly increase the polar surface area available for crystal lattice contacts. If successful with the beta2AR, this approach will likely be generalizable to other GPCRs. Specific aims include: Aim 1. Use molecular modeling tools to identify soluble proteins that can be fused to the beta2AR to generate stable, functional chimeras. Aim 2. Generate, express and optimize fusion proteins. Lay summary. The goal of this proposal is to develop a general method to obtain high-resolution structures of G protein coupled receptors (GPCRs). These structures should facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to study the effect of sympathetic innervation on the formation of 21 and 22 adrenoceptor (AR) signaling complexes in the heart. We will investigate how these complexes either facilitate or restrict receptor signaling; we will identify the structural domains of the receptors that are needed for interaction with the other components of the signaling complex; and we will identify cellular proteins that define these subtype-specific signaling complexes. 21ARs and 22ARs are prototypical G protein coupled receptors (GPCRs), the largest family of hormone and neurotransmitter receptors in the human genome. These receptors are essential for the physiologic regulation of cardiac function in response to catecholamines (adrenaline and noradrenaline) released from sympathetic nerves. Recent studies suggest that 21ARs and 22ARs play distinct roles in the pathogenesis of heart failure, a growing health problem in the United States. We have developed an experimental system to study the important interface between sympathetic nerves and the heart using co-cultures of neonatal cardiac myocytes and sympathetic neurons. Our preliminary studies show that 21ARs and 22ARs have differential subcellular targeting relative to these synapses, and that signaling complexes form at the sites of synapse formation. The following aims are designed to characterize these signaling complexes. [unreadable] Aim 1. Determine the structural basis for subtype specific targeting and trafficking of 21ARs and 22ARs in cardiac myocytes. Aim 2. Determine the functional significance of subtype-specific localization of 21ARs and 22ARs relative to sympathetic synapses. Aim 3. Characterize protein components of the 21AR and 22AR signaling complexes in cardiac myocytes innervated by sympathetic neurons. Aim 4. Determine the functional significance of interacting proteins identified in Aim 3 on 21AR and 22AR signaling and trafficking in cardiac myocytes, and verify their existence in signaling complexes in the adult heart. [unreadable] The proposed studies will provide new information about how the brain regulates heart function. We will characterize the mechanism by which noradrenaline and adrenaline released from sympathetic nerves alters heart function by activation of two specific adrenergic receptors. This research will further our understanding of the development of diseases such as heart failure and sudden death. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), and GPCRs represent the largest group of targets for drug development. The 22 adrenoceptor (22AR) has been one of the most extensively characterized members of the GPCR family. It responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine; and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. The coupling of the 22AR to Gs, the stimulatory protein for adenylyl cyclase, was one of the first hormone activated signaling pathways to be discovered and serves as a paradigm of GPCR signaling. During the past several years the Kobilka lab has made significant progress towards characterizing the structural changes associated with agonist activation, and has recently obtained a crystal structure of the wild type 22AR in complex with a Fab fragment, as well as a crystal structure of a 22AR that has been modified to improve its structural stability. The Sunahara lab recently developed recombinant HDL phospholipid particles as an ideal biochemical system for studying structural interactions between the 22AR and Gs. This proposal represents a close collaboration that combines the expertise of these two labs to characterize the structural interactions between the 22AR and Gs. The findings will likely apply to the large number of closely related monoamine receptors and to GPCRs in general. Moreover, the methodologies developed for characterizing 22AR coupling to Gs will be applicable to other GPCRs. A better understanding of the structure and mechanism of activation of the 22AR-Gs complex will further the potential for structure-based drug design and in silico screening for GPCR targets, leading to more rapid development of highly selective and effective drugs. the specific aims are: aim 1. characterize the structural dynamics of 22ar coupling to Gs. We will use biophysical approaches to examine the structural basis of functional cooperativity observed in the 22AR-Gs complex. We will examine the effect of Gs on the structure of the 22AR, and the effect of the 22AR on the structure of Gs. We will examine coupling of Gs to 22AR monomers and oligomers. In addition, we will study the effects of ligands having different efficacies on interactions between the 22AR and Gs. Aim 2. Determine the structure of the 22ar-gs complex. We will take several complementary approaches to obtain a high-resolution crystal structure of the 22AR-Gs complex. We will also use single particle imaging by cryoelectron microscopy to study the structure of the 22AR-Gs complex in a native lipid environment. [unreadable] [unreadable] Public Health Relevance: The goal of this proposal is to determine the mechanism by which G protein coupled receptors (GPCRs) respond to hormones and neurotransmitters, and modify the function of cells. This information will facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to enable the application of Nuclear Magnetic Resonance (NMR) spectroscopy to investigate G protein coupled receptor (GPCR) structure and facilitate drug discovery. G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are the largest group of targets for novel therapeutics. The focus of research in my lab has been to characterize the structure and mechanism of activation of GPCRs using the [unreadable]2 adrenergic receptor ([unreadable]2AR) as a model system. The [unreadable]22AR is one of the most extensively characterized members of the GPCR family. It responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine, and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. During the past several years my lab has made significant progress towards obtaining a high-resolution structure of the [unreadable]2AR by X-ray crystallography, as well as characterizing the structural changes associated with agonist activation. Biophysical studies on purified [unreadable]2AR protein provide evidence that GPCRs are conformationally complex molecules. This conformational complexity contributes to the challenges of drug discovery: in identifying lead compounds, and in developing leads into selective, effective and safe drugs. Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool that has the potential to provide high-resolution structural information about receptor-drug interactions and about the dynamic aspects of GPCR structure. The goal of this R21 proposal is to develop expression technology to make NMR analysis of GPCRs and other membrane proteins economically tractable, and to enable the routine use of NMR for GPCR structural biology and drug discovery programs. We propose to achieve this goal by developing Trichoplusia ni (T. ni) insect larvae as an expression and isotope labeling system for GPCRs. T. ni are a natural host for baculovirus and have been used to express recombinant proteins in milligram quantities. Moreover, T. ni eat a variety of plant material and can be fed inexpensive sources of 13C and 15N: the isotopes needed for high-resolution NMR. Specific Aims include: AIM 1 - Develop economical Trichoplusia ni diet for 13C and 15N isotope labeling of recombinant proteins expressed in baculovirus infected larvae. AIM 2 - Develop efficient protocols for large-scale baculovirus-mediated expression of the [unreadable]2AR in Trichoplusia ni larvae. AIM 3 - Optimize purification of functional [unreadable]2AR from Trichoplusia ni larvae. PUBLIC HEALTH RELEVANCE GPCRs represent the largest family of membrane proteins in the human genome and the largest group of targets for drug discovery. The proposed studies will develop the technology to make labeling of GPCR protein with 13C and 15N economically tractable and broadly applicable, and will enable the routine use of Nuclear Magnetic Resonance spectroscopy for GPCR structural biology and drug discovery programs. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this R21 proposal is to obtain a high-resolution crystal structure of the angiotensin II type 1 receptor (AT1R). The AT1R is a member of the Class A G protein coupled receptor (GPCR) superfamily that plays important roles in the regulation of cardiovascular function. Drugs acting on the AT1R are currently used in the treatment of hypertension and heart failure. There are three distinct classes of drugs for the AT1aR: (1) classical antagonists, angiotensin receptor blockers (ARBs) that stabilize the receptor in an inactive conformation, (2) the endogenous agonist angiotensin II that stabilizes an active conformation of the receptor capable of signaling through both the G proteins and the ? -arrestins, and (3) a highly specific ? -arrestin biased agonist that stabilizes a conformation of the receptor that is capable of signaling exclusively through ? -arrestins without any detectable activation of G proteins. Such ??arrestin biased agonists have unique pharmacological and therapeutic properties, distinct from classical agonists or antagonists, e.g. they lower blood pressure (like antagonists) but increase cardiac performance (like agonists). Little is known about the structural basis by which these different types of ligands regulate receptor function. We propose to begin a detailed investigation of the structural biology of the AT1aR through an R21 mechanism. Our goal for this proposal is to demonstrate that we can obtain diffraction quality crystals and a high-resolution structure of the AT1R bound to a high-affinity antagonist. The proposed work is high-risk and high-impact. If successful, the outcome of this R21 proposal will enable us to obtain funding for a more thorough structural characterization of the AT1aR in different conformational states. The long-term objective of this proposal is to determine the high-resolution crystal structures of the AT1aR in three different conformations; the inactive conformation stabilized by an ARB; the classical active conformation stabilized by angiotensin II; and an active conformation capable of signaling through only 2- arrestins stabilized by a ? -arrestin biased agonist. These structures will facilitate the development of safer and more effective therapeutics for heart failure and hypertension. Specific Aims include: 1) Generate an AT1aR-T4lysozyme fusion protein and adjust the linkers between these two proteins to optimize AT1aR functional expression and stability. 2) Establish conditions for expression and purification of AT1aR for crystallography trials. 3) Crystallize and determine the X-ray crystal structure of the AT1aR.

This International Collaboration in Chemistry (ICC) award in the Chemistry of Life Processes (CLP) program in the Division of Chemistry, supports collaborative work by Professors Brian Kobilka of Stanford University and Brian Shoichet of University of California, San Francisco in the United States, and Professor Takuya Kobayashi of Kyoto University in Japan, who is supported by the Japan Society for the Promotion of Science (JSPS). The goal of this proposal is to elucidate the chemical basis for the effect of allosteric ligands on muscarinic receptor structure and to develop new allosteric probes. Muscarinic receptors are members of the family of G protein coupled receptors (GPCRs). GPCRs are nature's most versatile chemical sensors. There are over 800 GPCRs in the human genome and they respond to a broad spectrum of chemical entities. Among the large family of GPCRs, the muscarinic receptors stand out for their ability to detect and respond to two distinct chemical entities that interact with two different binding sites: the orthosteric and allosteric binding pockets. The orthosteric binding pocket is the binding site for the neurotransmitter acetylcholine. The amino acids that form the orthosteric binding site are highly conserved among all five muscarinic receptor subtypes. As such, developing subtype selective chemical probes that regulate muscarinic receptor function has not been successful. There is a higher degree of chemical diversity outside of the orthosteric site including potential allosteric binding sites. Thus, allosteric ligands have the potential for highly subtype-selective chemical interactions with receptors. However, much less is known about the structural basis of allosteric ligand binding or how chemical interactions between amino acids that form this allosteric site and allosteric ligands lead to changes in receptor function.

The collaborative US-Japanese team will use the M2 muscarinic receptor as a model system for the proposed project, the methods developed during this collaboration will be generally applicable to other GPCRs, and allosteric tools developed during this project may ultimately be developed into more effective therapeutics. The proposed research will take advantage of complementary expertise in the three collaborating labs using pharmacological, biophysical and computational approaches to develop new highly selective chemical tools that can be used to modulate the activity of muscarinic receptors. Postdoctoral fellows from the three collaborating institutions will gain experience in all aspects of the proposed research.

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the structural basis by which G protein-coupled receptors (GPCRs) activate specific G proteins. The majority of hormones and neurotransmitters communicate information to cells via GPCRs, and GPCRs represent the largest group of targets for drug development. Our laboratories have a long-standing interest in elucidating the structure and mechanism of G protein activation by GPCRs. During the previous funding period we succeeded in obtaining the first crystal structure of a GPCR-G protein complex: the beta2 adrenoceptor (?2AR) in complex with Gs, the stimulatory protein for adenylyl cyclase. This structure provides important mechanistic insight into G protein activation, but at the same time raises new questions that will be addressed in this competitive renewal. Specific Aims include: Aim 1. Determine the structural basis of GPCR-G protein coupling specificity. The structure of the ?2AR-Gs complex provided the first high-resolution insights into transmembrane signaling by a GPCR. However, additional GPCR-G protein complexes will be required to understand the structural basis for G protein coupling specificity, and to determine if the mechanistic insights obtained from the ?2AR -Gs structure are generalizable to other GPCR-G protein pairs. We therefore propose to obtain three additional GPCR-G protein complex structures: (1) the vasopressin receptor-Gs complex; (2) the structure of the ?2AR-Gi complex; and (3) the structure of the M2R-Gi complex. Aim 2. Characterize the formation of the ?2AR-Gs complex from the GDP bound Gs heterotrimer. The ?2AR- Gs crystal structure represents a single state in a complex cycle of events. The process of complex formation and dissociation remains poorly understood. These are dynamic process that may not be addressable by crystallography; however, the ?2AR-Gs structure will provide the basis for designing and interpreting biochemical and biophysical studies to characterize the mechanism of complex formation and dissociation. In Aim 2 we will characterize the low affinity interactions between the ?2AR and GDP bound Gs. These interactions may play a role in G protein coupling specificity. Aim 3. Characterize the process of ?2AR -Gs dissociation following GTP binding. The goal of this Aim is to understand how the ?2AR -Gs complex dissociates into active signaling proteins upon binding GTP and to identify persisting interactions between any of the three components: ?2AR, G?s and G??. Aim 4. Characterize the dynamic behavior of the G?s alpha helical domain. The most surprising and unexpected feature of ?2AR-Gs structures is the flexible link between the two domains that make up G?s: the Ras-like GTPase domain and the alpha helical domain (AHD). This subaim will further characterize the interactions between these two domains in the ?2AR-Gs complex as well as in GTP and GDP bound states. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to determine the mechanism by which G protein coupled receptors (GPCRs) activate specific cellular G proteins in response to hormones and neurotransmitters, and modify the function of cells. This information will facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease.

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the structural basis for opioid receptor pharmacology and function. Opioid receptors constitute the major and the most effective target for the treatment of pain. The use of opioid drugs acting at these receptors is however a leading cause of death by overdose in Europe and North America. Both beneficial and adverse effects of illicit opioid drugs (opium, heroin) as well as approved therapeutics (morphine and codeine) are mediated by the activation of mu opioid receptor (¿-OR), a member of the G protein-coupled receptors (GPCRs) superfamily. To understand the structural basis for ¿-OR function, we obtained the first high-resolution crystal structure of an inactive state of this receptor along with a closely relate family member the delta-opioid receptor (¿-OR). These structures provide new insights into OR preference for specific antagonist drugs but do not address important questions regarding OR activation mechanisms and, in particular, opioid drug efficacy. We therefore propose to characterize the structural basis of opioid receptor activation using a combination of biochemical and biophysical approaches. Specific Aims include: Aim 1. Determine active state structures of ORs The inactive state structures of the ¿-OR and ¿-OR provided the first structural insights into the binding mode of morphinan antagonists. The goal of this aim is to obtain structural insights into the process of opioid receptor activation of the G protein Gi. We will initially focus on the use of crystallography and single particle electron microscopy to obtain three-dimensional structures of the ¿-OR-Gi and ¿-OR-Gi complexes. We will also develop a panel of camelid antibody fragments (nanobodies) that stabilized ligand-specific conformational states for crystallography. These will be used to determine the structural basis for the different functional properties of opioid receptor agonists. Aim 2. Conformational dynamics of OR structure and activation. OR activation and more generally GPCR activation involves a complex allosteric coupling between ligand binding and G protein coupling domains that is poorly understood. Crystal structures offer a limited number of static snapshots of this dynamic process. We therefore propose to develop and apply biophysical approaches to characterize the structural plasticity and dynamic properties of ORs and to determine how this is translated into signaling complexity and ligand efficacy. Inactive state crystal structures will constitute an important starting point for designing and interpreting biochemical and biophysical studies, some of which include fluorescence, EPR and NMR spectroscopy. These studies will provide new insights into opioid ligand efficacy and the differences between small molecule and peptide ligands.

PROJECT SUMMARY (See instructions); The goal of Project 3 is to provide high-resolution structural information that will: (1) further our understanding ofthe mechanism by which allosteric modulators regulate GPCR function, (2) form the basis for docking experiments to identify new subtype selective allosteric modulators, and (3) provide structural information for optimizing docking hits. The X-ray structures of GPCRs such as the P2AR (in both inactive and active states), the muscarinic M2, and M3, and the p- and 5-opioid receptors have enabled this project. These targets were initially selected for structural studies in the Kobilka lab based on their interesting pharmacological properties: the P2AR, an established model system for GPCR signaling; the muscarinic receptors, model systems for allosteric regulation; and the opioid receptors, which have been shown to function as homo- and hetero-oligomers, and for which there is a wealth of both peptide and small molecule ligands. The existing structures will form the basis for initial docking studies proposed in Project 1. We will work with Project 1, Project 2, and the Medicinal Chemistry core to identify and optimize novel ligands through iterative rounds of crystallography and chemical optimization. Finally, the Kobilka lab will work with the Sunahara (Project 2) and Shoichet (Project 1) labs to correlate chemical structure and functional properties with structural changes and protein dynamics determined by biochemical and biophysical approaches. Specific Aims: Aim 1. Determine active-state structures ofthe M2 and M3 muscarinic receptors. Aim 2. Obtain structures of receptors bound to positive and negative allosteric modulators. Aim 3. Determine structures of intermediate conformational states ofthe B2AR. Aim 4. Determine the structure of ligands bound to the B-opioid receptor oligomerization interface. Aim 5. Characterize the effect of allosteric modulators on receptor structure and dynamics.

PROJECT SUMMARY (See instructions): Our long term goal is to leverage GPCR structures to discover new chemotypes, and use these to as leads and probes to disentangle signaling pathways. Libraries of over 4.4 Million commercially available molecules are docked against GPCRs, typically targeting allosteric sites, and those that rank well by a physics-based complementarity score are acquired for testing. Those that are confirmed are optimized for affinity, specificity, and permeability. We begin relatively conservatively, seeking ligands with new chemotypes and physical properties for the orthosteric site of muscarinic receptors, and build on this to target the allosteric sites newly revealed in the structures. The specific aims are: 1. Novel chemotypes for the orthosteric sites ofthe muscarinic receptors. We are particularly focused on chemotypes with new physical properties (e.g., uncharged ligands) and sub-type specificities. 2. Ligands to the Gs-binding site ofthe B2-AR. Combined with an orthosteric ligand, and potentially on their own, molecules that bind to this site will bias signaling down non-G-protein pathways, such as that of arrestin. 3. Allosteric ligands ofthe muscarinic receptor. The high sequence identity in the orthosteric site ofthe five muscarinic subtypes has interfered with the discovery of specific ligands. Sequence is much less conserved in the allosteric sites revealed in the new structures, and we are targeting these for sub-type specific ligands. 4. Dimer-site ligands for the u-opioid receptor. Molecules that bind to this site will stabilize dimer vs. momomer signaling, suggesting a new route to specificity among opioid receptors and new tools to investigate the role of oligomers in GPCR signaling. Whereas these goals are ambitious, extensive preliminary results, in collaboration with the Kobilka and the Sunahara labs, support their feasibility.

PROJECT SUMMARY (See instructions): The overall goal of this proposal it to develop structure-based approaches to discover new G protein coupled receptor (GPCR) ligands having new signaling properties and specificities. GPCRs are involved in regulating virtually every aspect of physiology and are pivotal targets for drug discovery. Until now, ligand discovery efforts for GPCR has been empirically driven, and though this has had successes, it has restricted the field to sites precedented by canonical, often natural ligands. Considering the remarkable progress in identifying new GPCRs over the past two decades, drug discovery for this family of receptors using classical approaches has been disappointing. Most available ligands act at orthosteric sites, competing directly with the natural hormones and neurotransmitters. In the rare circumstances that they bind allosterically, their discovery has been fortuitous, their optimization difficult, as has been the dissection of their signaling. The recent efflorescence of GPCR X-ray structures was followed by the application ligand docking methods demonstrating the feasibility of this approach for the discovery of novel orthosteric ligand chemotypes for several GPCRs. We propose an integrated program of structure-based exploitation of GPCRs for new ligand chemotypes with an emphasis on allosteric ligands, their testing for new signaling properties, the determination of their structures bound to their GPCRs, and their optimization for affinity and signaling. This proposal builds on a network of existing collaborations among the labs of Kobilka, Shoichet, Sunahara and Gmeiner over the past four years. These four investigators bring together a unique combination of expertise in GPCR structural biology, ligand docking, GPCR pharmacology and function, and medicinal chemistry. Preliminary studies from this group demonstrate the feasibility and potential value of this approach.

DESCRIPTION (provided by applicant): Protease-activated receptors (PARs) are G protein-coupled receptors (GPCRs) that permit thrombin and other extracellular proteases to regulate cellular behaviors. Together with the coagulation cascade, PARs link tissue injury to cellular responses that help orchestrate hemostasis and thrombosis, inflammation, cytoprotection, repair, and pain perception. Given these and other roles, PARs are potential drug targets. Available evidence supports a model in which thrombin activates the prototypical PAR, PAR1, by cleaving the N-terminal exodomain of the receptor at a specific site to generate a new N-terminus that then functions as a tethered peptide agonist, binding intramolecularly to the receptor's heptahelical bundle to effect receptor activation. Structures that test this model and reveal how the tethered ligand binds and how such binding drives transmembrane domain (TM) movement and G protein activation are lacking, as are structures to support development of pharmaceuticals targeting PARs. Building on our recent crystal structure of inactive-state PAR1 complexed with the antagonist vorapaxar, we propose studies to illuminate the mechanism of PAR activation, signaling and antagonism at a structural level. We will 1) Solve crystal structures of thrombin- activated PAR1 in complex with Gi and either Gq or G12/13. 2) Determine the basis for vorapaxar's specificity for PAR1 over closely related receptors and the route of vorapaxar entry (from the plasma membrane or the extracellular space), and 3) Solve the crystal structure of a PAR2-antagonist complex. Cutting edge crystallographic approaches, including use of stabilizing nanobodies and single particle EM to assess complexes, will be employed. Molecular dynamics simulations will aid design and interpretation of mutational studies. Our studies of PAR1 will reveal the mechanism by which the PAR1 tethered agonist binds and triggers TM movement and G protein activation, the structural basis for PAR1's promiscuous coupling to multiple G protein subtypes (Gi, Gq, and G12/13), a novel route of antagonist entry and the importance of entry route for specificity. Detailed studies of the PAR1-vorapaxar structure and the PAR2 crystal structure will provide an entry to structure-based discovery and optimization of better PAR antagonists needed to explore the role of these receptors in human disease. Our studies will provide the first structure of a peptide agonist- GPCR-G protein complex and the first structural insight into whether distinct conformers of individual GPCRs recognize different G proteins.

Project Summary G protein coupled receptors (GPCRs) are the largest family of receptors for hormones and neurotransmitters and therefore the largest group of targets for new therapeutics for a very broad spectrum of diseases including neuropsychiatric, cardiovascular, pulmonary and metabolic disorders, cancer and AIDS. While initially thought to signal exclusively though G proteins and function as two-state switches activated by hormones and neurotransmitters, research over the past 30 years has revealed that most GPCRs have complex and diverse signaling behaviors. A single GPCR can activate more than one G protein subtype as well as G protein independent signaling pathways such as arrestins. Many GPCRs exhibit basal, agonist independent activity. When considering one of the several possible downstream signaling pathways, a drug acting at the orthosteric binding pocket may exhibit one of four efficacy profiles. It may behave as an inverse agonist, suppressing basal activity, a full agonist, maximally activating the pathway, a partial agonist, promoting submaximal activity even at saturating concentrations, or a neutral antagonist, having no effect on basal signaling, but blocking the binding of other orthosteric ligands. The efficacy profile of a given ligand may differ for different signaling pathways such that a drug may behave as an agonist for a specific G protein subtype or arrestin while have no effect or inhibiting other signaling pathways. This pathway selective (or biased) signaling has become an important consideration for drug discovery, since one signaling pathway may produce therapeutic effects while another may lead to adverse effects. During the previous funding period we have applied crystallography and several biophysical methods to characterize the structure and dynamic character of the ?2 adrenergic receptor (?2AR). These studies provide evidence that the ?2AR is highly dynamic and conformationally complex. We hypothesize that this complexity is essential for their functional versatility, and believe that a more detailed understanding of this complex conformational landscape will provide mechanistic insights into targeted activation of a specific pathway with biased ligands. The goal of this proposal will be to understand the structural basis for GPCR signaling through multiple pathways using methods that will provide high-resolution structural constraints and characterize protein dynamics under more physiologic conditions.