1992 — 1996 |
Javitch, Jonathan A |
K21Activity Code Description: To foster the development of outstanding scientists with potential for making important contributions to the fields of alcoholism, drug abuse or mental health (ADM) research. Primarily intended to meet the need for supervised research experience for highly promising biological or behavioral scientists who need further supervised research experience. |
Molecular Recognition of Antipsychotic Drugs @ Columbia Univ New York Morningside
Research methodology in protein chemistry, molecular biology, immunology and physiology will be applied to a receptor system relevant to psychosis. This will complement Dr. Javitch's previous training in pharmacology, neuroscience, and clinical psychiatry, and thereby facilitate his transition to an independent investigator in the basic science of psychiatric disorders. In addition to his research training, Dr. Javitch will continue a small commitment to clinical practice and teaching. The training supported by the SDA will prepare him to apply a molecular approach to studying the role of receptors in psychiatric illness. Three types of D2-like receptor (D2, D3 and D4) have been identified by cloning and sequencing. The pharmacology of these receptor types is similar, except for several differences which may have profound clinical ramifications. It is unclear which type, or combinations of types, is relevant to the etiology and treatment of psychosis, and the clinically relevant pharmacology cannot be inferred from the sequences alone. The specific intent of this proposal is to develop an understanding of how the structures of the D2, D3 and D4 receptors that form the binding sites for agonists, typical neuroleptics and atypical neuroleptics will be identified. D2 receptor will be prepared from bovine striatum, and D2, D3 and D4 receptor will be expressed using the baculovirus expression system. Specific residues of the receptor types will be radiolabeled by covalent modification, and the protein will be cleaved. The fragments will be separated and identified with polyclonal antibodies produced to synthetic peptides unique to each fragment. Specific residues will be identified by protein sequencing. These results will provide a basis for further analysis of the identified sites by site-directed mutagenesis and expression in cell lines, thereby laying a foundation for understanding the important pharmacological and physiological differences among the types of the dopamine D2-like receptors.
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1 |
1995 — 2001 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure of the Dopamine D2 &D4 Receptor Binding Sites @ Columbia University Health Sciences |
1 |
1997 — 2006 |
Javitch, Jonathan A |
K02Activity Code Description: Undocumented code - click on the grant title for more information. |
Molecular Recognition of Antipsychotic Drugs and Cocaine @ Columbia University Health Sciences
DESCRIPTION (Adapted from applicant's abstract): The catecholamine dopamine plays a major role in the regulation of cognitive, emotional and behavioral functions. Abnormalities in its regulation have been implicated in a number of psychiatric and neurological disorders. Dopamine exerts its actions at a number of G-protein-coupled receptors, including the D24-like (D2, D3, D4) and D1-like (D1, D5) receptors. Antipsychotic medications potently inhibit D2-like receptors, and it is unclear which types or combinations of types are relevant to the etiology and treatment of psychosis. Furthermore, the structural bases of the differences in pharmacological specificity among these receptors is unknown. After dopamine's release, its concentration in and around the synapse is rapidly reduced by the dopamine transporter (DAT). This protein is the major molecular target of several psychoactive drugs, including cocaine. The availability of a cocaine antagonist which was itself not an uptake blocker, and therefore not rewarding, would be a valuable addition to the therapeutic armamentarium against cocaine abuse and its attendant complications. The rational design of such agents would be greatly facilitated by an appreciation of the structural bases of substrate recognition and inhibition by cocaine and other psychostimulants. The candidate has used a new approach to obtain information about the structure of binding sites by systematically identifying the residues which line the site. This approach combines site-directed mutagenesis to substitute cysteine for putative membrane-spanning segment residues, expression of the mutant, and probing the aqueous surface accessibility of the cysteine residue by its ability to react with small, polar, charged, sulfhydryl-specific reagents. The long-term goals of this project are (a) to understand the structural bases of agonist and antagonist binding and specificity in the dopamine D2-like receptors and related biogenic amine receptors, (b) to determine how agonist binding is transduced into G-protein activation, and (c) to determine the structural bases of the transport of substrate by the dopamine transporter and its inhibition by drugs such as cocaine. Thus, I propose the following specific aims: (1) To identify the amino acid residues in membrane-spanning segments forming the surface of the binding-site crevice of the dopamine D2 receptor. (2) To identify conformational changes of the membrane-spanning segments concomitant with changes in the functional state of the receptor. (3) To identify amino acid residues forming the surface of the binding site and transport pathway of the DAT, focusing on residues which have a greater effect on cocaine binding than on transport. (4) To use computational molecular modeling to interpret our experimental results in a structural context.
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1 |
1998 — 2002 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dopamine Transporter--Substrate &Cocaine Binding Sites @ Columbia University Health Sciences
DESCRIPTION (Applicant's Abstract): Dopamine reuptake at the plasma membrane by the dopamine transporter (DAT) is a major mechanism for terminating dopaminergic synaptic transmission. DAT and the related sodium- and chloride-coupled neurotransmitter transporters combine functional aspects of both G-protein-coupled-receptors and ion channels: namely binding sites for substrate, inhibitors, and ions, and a gated channel or transport pathway through which substrate and ions move. Binding of substrate, sodium and chloride mediates a conformational change which exposes the substrate and ions to the intracellular environment where they are released. Therefore, a water-accessible transport pathway must be formed among the membrane-spanning segments. This pathway should be accessible to hydrophilic reagents applied extracellularly. Although they may not be identical, the binding sites for substrate, ions and inhibitors, such as cocaine, likely lie, at least in part, within this transport pathway. We have developed an approach, the substituted-cysteine-accessibility method, to obtain information about the structure of binding sites and channels by systematically identifying the residues which line the site or channel. Our approach combines: site-directed mutagenesis to replace putative membrane-spanning segment residues, one at a time, with cysteine; heterologous expression of the mutant; and probing the aqueous surface accessibility of the engineered cysteine residue by its ability to react with small, charged, hydrophilic, lipophobic, sulfhydryl-specific reagents. The long-term goals of this project are to determine the structural bases of the transport of substrate by DAT and its inhibition by drugs such as cocaine. The specific aims are: l) To identify the amino acid residues forming the surface of the cocaine binding site, the dopamine binding site, and the transport pathway in DAT. 2) To determine the secondary structure of the membrane-spanning segments containing these residues. 3) To identify conformational changes of the membrane-spanning segments associated with transport. The approach outlined in this proposal will enable us to create a low resolution structural model of DAT, thereby laying a foundation for understanding, at the molecular level, the binding and transport of dopamine and its inhibition by cocaine. This approach might lead to a differentiation of the binding sites for cocaine and for dopamine and thereby facilitate the development of cocaine antagonists which do not inhibit dopamine transport. Furthermore, the approach will provide insights into structure-function relationships for other members of the neurotransmitter transporter family, such as the serotonin transporter and norepinephrine transporter, which are targets for a wide variety of antidepressant drugs.
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1 |
2001 — 2007 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Archaeal &Bacterial Homologs of Dopamine Transporter @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): The dopamine transporter (DAT) is the major molecular target responsible for both the rewarding properties and abuse potential of cocaine and related psychostimulants. The homologous neurotransmitter transporters (NTs) for serotonin and norepinephrine, SERT and NET, are primary targets of antidepressant drugs. These integral membrane proteins couple the accumulation of neurotransmitter to the movement of sodium ions down their concentration gradient. Progress in the study of their molecular structure and transport mechanisms has been hampered by an inability to develop high-level expression systems for these proteins and the subsequent lack of sufficient functional, purified protein. Bacterial membrane proteins are generally more amenable to structural analysis and high-level expression than are their eukaryotic counterparts. We have recently identified an entire family of proteins in archaea and in bacteria (currently 73 proteins from 45 different organisms) that are homologous to DAT. The sequence identity to DAT for the most similar proteins is approximately 25 percent, making it very likely that they have a similar structure. Our strategy was to develop a high-level expression system with one or more of these proteins to obtain adequate amounts for direct structural studies. During the first 1.5 years of our Stage I Cutting-edge Basic Research Award (CEBRA), we have: a) cloned 17 of these genes from various bacterial and archaeal genomes, b) heterologously over-expressed 12 of these in the membrane of E. coil, c) shown that one of these gene products, TnaT, is a sodium-dependent tryptophan transporter, confirming that these genes encode proteins with functions similar to the NTs, and reaffirming their value as models for direct structural analysis, d) purified full-length TnaT from the membrane to near homogeneity in yields of approximately 0.6 mg/I culture, e) constructed a cysteine-less TnaT that is functional and expresses at near wild-type levels, and f) constructed strategically placed individual cysteine mutants that express and function normally. In this Stage II CEBRA proposal the specific aims are: 1) To identify residues within or very near the substrate binding site in TnaT, a sodium-dependent tryptophan transporter from Symbiobacterium thermophilum, using mass spectroscopic analysis of azido-tryptophan analogs photo-incorporated into TnaT. 2) To identify a drug-like inhibitor of TnaT by screening a combinatorial chemical library. 3) To identify solubilization conditions that preserve the structure and function of TnaT. 4) To establish conditions for functional reconstitution of TnaT into proteoliposomes. When these aims have been achieved, we will be in a position to choose a limited number of the bacterial transporters for use in crystallization trials as a step towards obtaining a high-resolution structure. Moreover, we will also be poised to pursue spectroscopic methods to dynamic structure. Success in either or both of these goals would revolutionize our structural understanding of the function of related human neurotransmitter transporters in away that is only a remote prospect through continued work on the eukaryotic transporters alone.
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1 |
2002 — 2006 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Structure of the Dopamine D2-Like Receptor Binding Sites @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): The long-term goals of this project are: (a) To understand the structural bases of agonist and antagonist binding and I specificity in the dopamine D2-Iike receptors and related biogenic amine receptors, and (b) To determine how agonist binding is transduced into G-protein activation. During the previous grant period we mapped the entire binding-site crevice of the dopamine D2 receptor, which is lined by the transmembrane segments (This). Our identification of all the residues that are water-accessible in this binding-site crevice led to our identification of structural determinants of pharmacological specificity in the clopamine D2 and D4 receptors. In a D2 receptor background, mutation of a cluster of residues in TM2, TM3, and TM7 to the aligned D4-receptor residues increased the affinity of the mutant D2 receptor for D4-selective ligands by three-orders of magnitude. We studied transduction in the 62 adrenergic receptor where we showed that conformational changes in TM6 are associated with receptor activation and demonstrated that the presence of an "ionic lock" between the cytoplasmic ends of TM3 and TM6 stabilizes the inactive state of the 62 adrenergic receptor. The structural implications of our work are remarkably consistent with the recent high-resolution structure of rhodopsin. Th4 is an exception in that residues facing lipid in rhodopsin are water-accessible in the D2 receptor, and our recent cross-linking data indicate that TM4 may form a D2 receptor homodimer interface. A number of class C GPCRS, such as the GABA8 receptors, are dimers, and there is increasing evidence, both in heterologous expression systems and in native tissue, for homo- and hetero-dimerization of class A receptors, including the dopamine receptors. We hypothesize that ligand binding and receptor I activation is associated with conformational changes at the TM4-dimer interface that are a link in the interaction between the two binding sites. We also hypothesize that just beyond the dimer interface the extracellular loop connecting TM4 and TM5 dives down into the binding-site crevice in the D2-like receptors, as it does in rhodopsin, and contributes to the binding site and to pharmacological specificity. We propose the following specific aims: 1) To map the interaction surface between D2 receptors in the membrane environment by cross-linking endogenous and substituted cysteines.2) To determine whether the heterodimer interface between D2 and D3 receptors as well as the homodimer interface of the homologous 62 adrenergic receptor is similar to the D2 homodimer interface. 3) To assess the effects of ligand-binding and receptor activation on cross-linking as well as the effect of cross-linking on binding and activation. 4) To determine whether interconverting the extracellular loop between Th4 and TM5 and selected amino acid residues therein alters the pharmacological specificity of D2, D3 and D4 dopamine receptors for selective ligands.
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1 |
2003 — 2007 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dopamine Transporter: Substrate &Cocaine Binding Sites @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): The dopamine transporter (DAT) mediates the inactivation of released dopamine through its reuptake. DAT is the major molecular target responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. Homologous neurotransmitter transporters for serotonin and norepinephrine are targets for antidepressant medications as well as secondary targets for psychostimulants. The long-term goals of this research are to determine the structural bases of substrate translocation and drug binding to DAT and eventually to understand whether the action of cocaine can be prevented while simultaneously preserving dopamine transport. During this grant period we showed that: substrates and non-substrate inhibitors differentially alter the conformations of DAT; within the class of inhibitors, drug-specific conformational changes are detectable and may be consistent with the drugs' distinct behavioral effects; DAT is an oligomer with a symmetrical interface in TM6 and a separate symmetrical interface in TM4, and that cocaine binding alters the TM4 interface and possibly the quaternary structure of DAT; amphetamine and cocaine redistribute DAT to/from the cell surface in opposite ways; N-terminal phosphorylation of DAT occurs in response to PKC activation, but this phosphorylation is not necessary for PKC-induced DAT internalization. We now propose to test the following hypotheses: a) Particular transmembrane segments line the transport pathway of DAT. b) The cocaine binding site is lined by residues from the third intracellular loop (IL3) as well as the transmembrane segments, c) The orientations of cocaine analogs and benztropine analogs bound within the transporter are different, and they induce/stabilize different conformations of DAT. d) Cytoplasmic loops of the transporter form part of a gate that alternately blocks and permits access from the transport pathway to the intracellular milieu. To test these hypotheses, we propose studies with the following specific aims: 1) To determine the residues in TM1, TM3, and TM5 of DAT that are water-accessible and likely to line the transport pathway. 2) To identify residues in and very near to the binding site of a cocaine analog using sulfhydryl-targeted affinity labeling. 3) To compare sulfhydryl-targeted affinity labeling using cocaine and benztropine analogs in which the sulfhydryl reactive groups have been placed in different positions on the tropane rings. 4) To map the intracellular and extracellular accessibility during different functional states of substituted-cysteines through IL1 and IL3 of DAT.
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1 |
2005 — 2009 |
Javitch, Jonathan A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Structure and Function of the Dopamine Transporter @ Weill Medical Coll of Cornell Univ
The dopamine transporter (DAT) is the plasmalemmal membrane protein that mediates the inactivation of released dopamine through its reuptake. DAT is the major molecular target responsible for the rewarding properties and abuse potential of cocaine, amphetamine, and related psychostimulants. Homologous neurotransmitter transporters for serotonin and norepinephrine are targets for antidepressant medications as well as secondary targets for psychostimulants. The long-term goals of this research are to determine the structural bases of substrate translocation and of efflux induced by psychostimulant substrates such as amphetamine. More specifically, we would like to understand whether DAT-mediated amphetamine-induced dopamine efflux can be blocked while simultaneously preserving dopamine transport and how this would alter the behavioral and neurotoxic effects of amphetamine. For the next grant period, we propose to test the following hypotheses: a) Phosphorylation of specific serines in the N-terminus of DAT switches the transporter from a "resistant" to a "willing" state for amphetamine-induced efflux, b) Phosphorylation of the N-terminus regulates its interaction with the N-terminus of another DAT at an oligomeric interface, c) Distinct kinases are responsible for regulated phosphorylation of the N-terminal serines, d) Amphetamine-induced dopamine-efflux, and not simply blockade of uptake, is responsible for some of the behavioral effects of acute and/or chronic amphetamine in vivo. To test these hypotheses, we propose studies with the following specific aims: 1) To identify the sites of DAT N-terminal phosphorylation and the functional role of phosphorylation. 2) To identify elements in the N-terminus and their interactions at an oligomeric interface with the N-terminus of another DAT and to assess the impact of phosphorylation on these interactions. 3) To identify the kinase or kinases involved in phosphorylating these N-terminal serines. There are numerous advantages to pursuing these studies as part of the proposed Program Project grant. Much of the work carried out during this grant period was conducted as part of collaborations with Aurelio Galli, Ulrik Gether, or Harel Weinstein, and substantial synergies have been achieved through our interactions. Continued building upon these synergies is reflected in the proposed aims of this project, as well as in the interplay of our related aims and, in some cases, related methodologies.
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0.924 |
2007 — 2016 |
Javitch, Jonathan A |
K05Activity Code Description: For the support of a research scientist qualified to pursue independent research which would extend the research program of the sponsoring institution, or to direct an essential part of this research program. |
Molecular Determinants For the Action of Psychostimulants @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): The catecholamine dopamine (DA) plays a key role in the regulation of cognitive, emotional, and behavioral functions. Abnormalities in its regulation have been implicated in drug abuse as well as several psychiatric and neurological disorders. DA exerts it actions at D2-like and D1-like receptors, members of the G protein-coupled receptor (GPCR) family. DA reuptake by the DA transporter (DAT) is a principal mechanism for terminating dopaminergic transmission, and this protein is the primary molecular target of amphetamine, cocaine, and other psychostimulants. The Javitch laboratory studies structure-function relationships and mechanisms of regulation of neurotransmitter transporters and related bacterial transporters, as well as mechanisms of dopamine receptor oligomerization and function. His long-term research goals are to: 1) Understand the structural bases of agonist and antagonist binding and specificity in G protein-coupled receptors, with a current focus on DA and opioid receptors. 2) Determine how agonist binding is transduced into G-protein activation and arrestin recruitment and signaling. 3) Determine the structural bases of substrate translocation and inhibitor binding to neurotransmitter transporters and the dynamics associated with transport using biophysical and structural approaches in parallel with computational analysis. 4) Determine the mechanistic bases of AMPH-induced DA efflux and the role of regulation of these processes in sensitization and substance abuse. The K Award enables the candidate to devote focused effort to the exploration of new approaches, novel systems and various multi-disciplinary methods and collaborations aimed at one of the central goals of the research program in the laboratory - the mechanisms of drugs of abuse. The candidate's laboratory is pursuing membrane protein crystallography and electron paramagnetic resonance & single molecule fluorescence spectroscopy of bacterial homologs of neurotransmitter transporters. This work is now being extended to single molecule imaging of receptors in living cells. The lab is also pursuing work in genetically modified flies and mice as model systems to probe molecular and mechanistic insights in a physiological background. These new approaches are being developed and used to maintain the candidate's research at the leading edge of the field of molecular mechanisms of drug abuse and actions of antipsychotic drugs. The support of the K05 Award would play an essential role in the candidate's continued growth by giving him the flexibility to focus on expanding his research methodologies and to fuse his own professional growth with that of his research program as well as extensive mentorship of his trainees and junior faculty.
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1 |
2007 — 2009 |
Javitch, Jonathan A |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Schizophrenia Research Fellowship @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): The purpose of this training program is to increase the number of researchers in the area of schizophrenia who have the ability to apply modern techniques of basic and clinical research into their investigations. Training in the disciplines of psychopharmacology, brain imaging, genetics, neuropathology, neuropsychology, molecular neurobiology and epidemiology are offered by participating faculty. All mentors have federally-funded research projects. The training develops research skills by supervised participation in ongoing research, but the trainee is expected to construct and execute independent projects as well. Didactic work focuses on research design and statistics, and a weekly schizophrenia research seminar reviews current schizophrenia research in all areas. Trainees for the project are primarily psychiatrists who have completed both their medical school training and 4 years of residency. These trainees have had considerable experience with the diagnosis and treatment of major psychiatric disorders, including schizophrenia, but are likely to lack specific research skills such as developing testable hypotheses, designing a feasible research study acceptable to institutional review boards, and the collection and analysis of standardized research data. In addition to these MD psychiatrists, we recruit MD/PhD psychiatrists with a background in basic science research, PhD psychologists, MD neurologists, and PhD biologists with a special interest in schizophrenia. We adapt the training to suit their background and interests. Seven stipends are requested to support 2 to 3 fellows per year, with most fellows projected to receive support for a third year of training. This program is conducted at the New York State Psychiatric Institute (NYSPI) and at the Health Sciences Division of Columbia University, by faculty members of Columbia. With a total of over eighty million dollars annually in federal grants, a research institute with 64 beds (including a 12 bed designated schizophrenia inpatient research unit funded by NYS at NYSPI), a foundation-funded schizophrenia center grant, a Hughes Institute, and 5 MHCRCs, there exists in the Columbia Department of Psychiatry the research personnel and clinical facilities to continue to execute this proposed training program, which has had excellent success in its training efforts thus far. Schizophrenia is a devastating illness and a major public health problem. It is estimated that 0.8% of the population develops this disorder, and that one quarter of all hospital beds are occupied by such patients. The illness typically diminishes or destroys the patients' capabilities for productive work. Though treatment advances have been made, most patients remain severely incapacitated. In addition to the personal terror, confusion and misery which it produces, there is a tremendous stress on family members and immense costs to society.
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1 |
2009 — 2013 |
Javitch, Jonathan A Weinstein, Harel [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Functional Properties of Protein Segments in Receptors and Transporters @ Weill Medical Coll of Cornell Univ
DESCRIPTION (provided by applicant): The proposed research aims to enhance the structure-based understanding of molecular mechanisms in cell signaling at the membrane, focusing on functionally important structural elements of specific membrane proteins and their interaction partners in signaling mechanisms of drugs of abuse. A primary target are the structures of protein segments (termed loops) connecting the transmembrane (TM) segments in TM proteins, in the context of specific mechanistic hypotheses about the role these loops have in signaling protein-protein interactions. The motivation is that loops have been shown to contribute to the regulation of function of these proteins, e.g., in ligand recognition, signal transduction, substrate transport, making this project a fundamental and essential part of the ongoing effort to understand signaling mechanisms at the molecular level from a true structural and dynamic perspective; it will add invaluable information and tools without which this effort cannot succeed. Reliable loop structure calculation will be integrated into rigorous modeling of G Protein Coupled Receptor (GPCR) and Neurotransmitter transporters (NTs), in the context of specific biological problems and informed by the relevant experimental data. The proposed studies apply 3D modeling and computational analysis of the dynamics and energetics of the structural elements, with experimental probing and validation of the computational inferences in experimental assays of function. The combined computational/experimental approach is a paradigm for structure-function studies of a wide range of molecular mechanisms of cell function. The Specific Aims probe first the loops for which a specific functional role has been suggested in the regulation and action of GPCRs and NTs, to understand their specific contributions to the functional mechanisms, and to the responses elicited by drugs of abuse: Specific Aim 1: To calculate the structures of the loops that complete the models of specific GPCRs, including Dopamine D2 and D4 receptors and 5HT receptors to reveal mechanisms of both direct and indirect involvement in ligand binding, oligomerization, and interactions with G proteins and arrestin. A broadening of the scope to a class C GPCR (CRF1) is also planned. Specific Aim 2: To establish a protocol for the prediction of structural properties of loops in the neurotransmitter transporter (NT) proteins and apply the protocol to study computationally the involvement of key loops in the modulation of function by transporters including DAT, which is the target of cocaine and amphetamine. Specific Aim 3: To validate experimentally the findings of the computational probing of loop structure, and interactions with specific proteins that modulate GPCR and NT activity, to reveal and probe the functional involvement of the loops, and to integrate the structural and dynamic models into the mechanism of the complete system. BRET, scintillation proximity assays, and a variety of 1D and 2D mutagenesis studies will be used to study loop structure and protein and/or ligand interaction of dopamine receptors and transporters, serotonin receptors, and CRF1 - a class C GPCR.
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0.924 |
2009 — 2013 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Mechanisms of Bacterial Homologs of Neurotransmitter:Sodium Symporters @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): Neurotransmitter:sodium symporters (NSS) couple the accumulation of substrate to the movement of sodium ions down their concentration gradient across the plasma membrane, and as such constitute key elements in cellular signaling and homeostasis. NSS include the transporters for dopamine, serotonin and norepinephrine-targets for amphetamine, cocaine, and antidepressant drugs-as well as the transporters for GABA and glycine, which are targeted for treatment of epilepsy and schizophrenia. In 2005 the Gouaux group solved at 1.65 [unreadable] the structure of LeuT, a bacterial NSS homolog, crystallized with 1 Leu and 2 Na+ bound in an occluded binding pocket (referred to as primary substrate binding (S1) site). The structure provided no easy clues to the pathway of substrate to the S1 site from the extracellular or the intracellular side. An unexpected second substrate binding (S2) site located in the extracellular vestibule was identified during the previous project period;binding and flux experiments showed that the two binding sites can be occupied simultaneously. Substrate in the S2 site allosterically triggers intracellular release of Na+ and substrate from the S1 site, thereby functioning as a "symport effector." Because tricyclic antidepressants (TCA) bind differently to this S2 site, they do not promote substrate release from the S1 site and thus act as symport uncouplers to inhibit transport. Identifying the conformational changes associated with transport and the permeation pathways that are formed within the transporter are long term goals of this project critical to understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. To achieve this goal, an integrated approach has been developed based on active collaborations with investigators whose expertise in computational modeling (Harel Weinstein), membrane protein crystallography (Poul Nissen), and single-molecule fluorescence spectroscopy (Scott Blanchard) enables the combined multidisciplinary approach described in this application. The following specific aims are proposed: 1) To use our novel discoveries regarding the specificity and modulation of S2 binding, by detergents, mutations, and ionic substitution, to develop conditions that enable us to understand the regulation of LeuT properties by the S2 binding site and to solve a structure of LeuT with substrate bound to the S2 site. This will provide atomic resolution data to inform our mechanistic hypothesis as to the essential role in transport of substrate binding to this site. 2) To characterize the mechanism of substrate transport in terms of specific conformational changes in the transporter that propagate the allosteric signal triggered by substrate binding to the S2-site towards the intracellular gate of the transporter and allow inward release of substrate. 3) To establish the relevance of our structural and functional findings in bacterial transporters to understanding the function of SERT and DAT. We will: a) demonstrate the essential functional role of the S2 site in these human transporters, and b) use a Cl-- dependent LeuT mutant to determine the structure of the Cl- binding site and thus to explicate the functional role of Cl- in SERT and DAT. PUBLIC HEALTH RELEVANCE: Neurotransmitter transporters are the target of psychostimulant drugs such as cocaine and amphetamine and are targets for antidepressants as well as for new drugs in development for the treatment of epilepsy and schizophrenia. Identifying the conformational dynamics of transport, the permeation pathways within the transporter, and the role of substrate and inhibitor binding are critical for understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. The powerful approaches we have established will allow us to reach this understanding and address drug action and guidelines for therapy design anchored in solid structural and functional information.
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1 |
2010 — 2021 |
Javitch, Jonathan A |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training in Schizophrenia and Psychotic Disorders: From Animal Models to Patients @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): This is an application for renewal of grant T32-MH018870, Schizophrenia Research Fellowship, which has been funded continuously since 1988. The primary goal of this proposal is to train postdoctoral (MD, MD/PhD and PhD) fellows for careers as independent researchers in Schizophrenia. An intensive three-year program is outlined in which fellows will learn how to identify key research questions, formulate hypotheses, and design and execute experiments that effectively test those hypotheses. In the course of training, fellows will acquire a range of skills relevant to research methodology, including expertise in experimental design and statistical analysis relevant to basic, translational and clinical research programs. Graduating fellows will be able to present clearly an entire project in both written and oral form as evidenced by publications and presentations. The success of the training program is best judged by the remarkable accomplishments of our former trainees. In the past 10 years, 27 fellows have been selected to enter the training program, 55% MDs, 30% MD/PhDs and 15% PhDs. 59% of our trainees have been women. Prospective fellows apply specifically to this program, and the competition for training slots remains intense. 26% of fellows have been recruited from the Columbia Psychiatry Residency program. The graduation rate of the fellowship is 100% over the last 10 years (21/21). Currently, there are six fellows: one fourth-year (special waiver related to a complicated pregnancy and maternity leave), one third-year, two second-year and 2 in the first year of training; four new fellows have been accepted to begin before the end of 2009. Of the 21 fellows who have graduated the program in the past 10 years, 11 (52%) have received K awards and two K award submissions are presently under review from current fellows. Two graduates (10%) have already received R01s with another two receiving R21s (total of 19% for both ROIs and R21s), and several others have applications pending. In total, 18 (86%) graduates have received substantial independent funding including ROIs, K awards, R03 awards, R21 awards, NARSAD awards, and grants from the Office of Naval Research and other funding agencies. Of the 21 fellows who graduated the program, 16 (76%) are in full-time academic research positions. No training program can maintain a successful track record if it remains stagnant; it must adapt to changes in circumstances and the growing opportunities in terms of translational research in Psychiatry. The training in translational research has been markedly strengthened by enhancement of the didactic teaching program and through the strategic addition of faculty mentors in this area. The greatest strengths of this training program have always been Columbia's faculty and the research environment within the Department of Psychiatry and beyond. This remains the case today, and the core of the training program has been strengthened by the changes in the current application.
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1 |
2011 — 2015 |
Javitch, Jonathan A |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
P3 - Regulation of Dopamine Transporter Function @ Weill Medical Coll of Cornell Univ
The dopamine transporter (DAT) mediates the inactivation of released dopamine (DA) through its reuptake. The long-term goals of this research are to understand how DA accumulation and efflux are regulated by post-translatlonal modification of DAT, by association of DAT with other proteins, and by localization of DAT to specific membrane microdomains. During the previous project period we demonstrated that phosphorylation ofthe DAT N-terminus is essential for AMPH-induced DA efflux, CaMKIIa binds to the distal C-termlnus of DAT, and CaMKIIa phosphorylates serines In the distal N-terminus of DAT in vitro. The CaMKll Inhibitor KN93 reduces AMPH-induced DA efflux in cells as well as in vivo in mouse striatum. In order to develop a model system for mechanistic examination, we have established a behavioral assay for AMPH-induced DAT-mediated DA efflux in Drosophila melanogaster larvae. In larvae, inhibition of CaMKll only in DA neurons inhibits the AMPH-induced behavior, whereas expression of constitutively active CaMKll enhances AMPH-induced behavior. We have shown that the membrane raft-associated protein Flotillini (Floti) is necessary for localization of DAT in membrane rafts. Floti knockdown blunts AMPH-induced DA efflux in mouse DA neurons in primary culture and AMPH-induced behavior in D. melanogaster larvae. The precise mechanisms by which Floti modulates DAT localization and function remain unknown. Our working hypothesis is that Floti traffics DAT to a membrane raft compartment containing the necessary signaling machinery to phosphorylate the DAT N-terminus and thereby allow AMPH-induced DA efflux. We propose to: 1) characterize the relationship between Floti, DAT, and DAT-interacting proteins in membrane rafts, 2) determine the role of Floti in AMPH-induced DA efflux and behavio, and 3) determine the role of N-terminal phosphorylation of DAT and its raft localization in AMPH-induced DA efflux and behavior.These aims will be pursued in heterologous cells, in intact behaving D. melanogaster larvae, and in genetically modified mice, in a collaborative and synergistic interaction with the other PPG projects and the electrophysiological expertise of the core.
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0.924 |
2013 — 2014 |
Javitch, Jonathan A |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Single Molecule Imaging of Beta2 Adrenergic Receptor Activation @ Columbia University Health Sciences
DESCRIPTION (provided by applicant): An important advance in pharmacological understanding is that of functional selectivity, whereby different downstream effectors of a single G protein-coupled receptor (GPCR) can be differentially activated by distinct ligands. Interesting examples of functional selectivity have been identified in a number of different GPCRs, including dopamine receptors and opiate receptors, two key targets of drugs of abuse. Despite enormous recent advances in the structural biology of GPCRs, the detailed structural and kinetic mechanisms by which drug binding modulates receptor activity, and particularly why different drugs can lead to different effects at the same receptor, remain poorly understood. We propose a series of single-molecule fluorescence resonance energy transfer (smFRET) experiments on the beta2 adrenergic receptor (B2AR) to delineate the mechanistic basis for its differential activation by different ligands. SmFRET makes it possible to follow the movements of an individual molecule over time, providing a means of making direct measurements of the rates and amplitudes of transient changes in protein conformation during function. Data of this kind hold the promise of providing critical information for elucidating functional mechanisms, including the detection of static and dynamic heterogeneities and transiently populated, non-accumulating intermediates, mechanisms that are masked by ensemble methods. We propose the following specific aims: 1) To determine the impact of a range of ligands on the equilibrium distribution of the conformational states of the B2AR and on the transition rates between these states, as determined by smFRET. 2) To probe structural dynamics within the heterotrimeric G?s?? complex bound to agonist-activated B2AR using smFRET and to compare a spectrum of agonists to provide insights into their signaling differences. The platform of technologies we develop will help us to understand the distinctive molecular properties of different B2AR agonists and to formulate hypotheses for designing drugs with optimized properties. Additionally, these methods will be broadly applicable to studies of other GPCRs that are critical therapeutic targets in numerous diseases.
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1 |
2014 — 2017 |
Javitch, Jonathan A Lambert, Nevin Alan |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Single-Molecule and Ensemble Imaging of Gpcr-G Protein Complexes in Live Cells @ Georgia Regents University
DESCRIPTION (provided by applicant): G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors, and are the targets of a substantial fraction of all prescribed ad abused drugs. GPCRs change cellular physiology primarily by activating intracellular heterotrimeric GTP-binding proteins (G proteins). The steps involved in G protein activation include ligand binding, receptor activation, and ultimately the formation of a coupled complex between a ligand, an active receptor and an inactive G protein (LR*GGDP), leading directly to G protein activation. It has long been hypothesized that inactive GPCRs and G proteins associate with each other prior to receptor activation in precoupled or preassembled RGGDP complexes. However, the existence and relevance of these complexes in cells have been difficult to document, largely because methods to study transient interactions between membrane proteins have not been available. We recently succeeded in detecting preassembled complexes between GPCRs and Gq heterotrimers in living cells, and in demonstrating their physiological significance. However, these studies left a number of important questions unanswered, in part because they relied exclusively on ensemble measurements. One critical question is the lifetime of inactive-state preassembled receptor-G protein (RGGDP) complexes. Ensemble experiments suggest that the lifetime of a preassembled RGGDP complex is long compared to the active-state, coupled complex, but neither of these lifetimes can be determined using existing methods. Long-lived preassembled RGGDP complexes would allow receptors to self-scaffold G proteins, i.e. maintain a high local concentration of heterotrimers ready for activation. Here we propose to study RGGDP complexes using quantitative ensemble and single-molecule imaging in living cells. Ensemble imaging will allow us to determine if preassembly serves as a self-scaffolding mechanism for several different GPCRs and G protein heterotrimers. Single-molecule imaging will allow us to directly observe the formation and dissociation of inactive-state preassembled RGGDP complexes. We will thus be able to quantitatively assess the lifetimes of the macromolecular complexes important for G protein signaling. The impact of these experiments on the immediate field will be to determine if preassembly is a significant step along the pathway to G protein activation, or alternatively if itis a rare side-reaction. With respect to macromolecular interactions in general, the impact of this project will include refinement of ensemble and single-molecule imaging technology to detect membrane protein interactions in living cells, including expression systems, dyes, labeling, immobilization, imaging and analysis strategies.
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0.957 |
2015 — 2017 |
Javitch, Jonathan A |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Bridge 3: the Transport Cycle in Neurotransmitter Uptake Systems
Secondary active transporters use the energy from electrochemical ion gradients and/or substrate gradients to mediate concentrative substrate translocation across biological membranes of all organisms. With advances in membrane protein structural biology, it has become clear that a large number of secondary active symporters and exchangers, which belong to distant families without discernable sequence identity, nonetheless, share common structural features that classify them into a single structural family, known as the LeuT-fold. This fold is characterized by 10 transmembrane helices (TMs) organized into two inverted structural repeats each containing 5 TMs. In Bridge 3 we seek to understand commonalities as well as divergence in the functional mechanisms of the LeuT-fold proteins, with a focus on the general rules of protein structural dynamics that underlie function, in the context of differences in their driving mechanism and the conformational changes associated with substrate translocation. To achieve these goals we build on the synergistic approach we have established with the study of LeuT, employing iterative computational, functional and spectroscopic methods. The working hypothesis of this Bridge is that the discovery and interpretation of mechanistic differences between LeuT and other LeuT-fold transporters depends on revealing dynamic properties enabled by local structural differences. We will use a new generation of quantitative computational approaches, in parallel with binding and flux studies, and with EPR and single-molecule fluorescence studies to measure distance between pairs of probes in different conformational states as well as the dynamics of the associated movements. The work will take advantage of specific established collaborations among the team members, and with the Computational Modeling, Spectroscopy and Instrumentation, and Protein Expression Cores. ApcT, a member of the APC family that also includes product/precursor exchangers, shares the LeuT-fold but has been reported to be a H+-dependent amino acid transporter. Interestingly, ApcT has the side chain ?-amino group of Lys158 occupying what is the Na2 site in LeuT, and it has been suggested that protonation and deprotonation of this Lys, like binding and unbinding of Na2 in LeuT, drives transport. In contrast to the profound mechanistic differences between ApcT and LeuT, the Drosophila dopamine transporter (dDAT) is closely related to LeuT in overall structure and function but differentiated by the presence of large amino and carboxy termini, which have been shown to critically modulate transporter function in the eukaryotic transporters. To understand similarities and differences in functional mechanisms for these compared LeuT- fold transporters we propose the following Specific Aims: 1) To determine how substrates, H+ and Na+ coordinate dynamics and conformational changes in the transport cycle of ApcT as compared with LeuT. 2) To integrate CW and DEER measurements in exploring the role of the amino terminus in modulating the conformational dynamics of dDAT.
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0.955 |
2017 — 2021 |
Javitch, Jonathan A |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Probing Mechanisms of Amphetamine Action At Plasma Membrane and Vesicular Transporters in Vitro and in Vivo @ Columbia University Health Sciences
Amphetamines (AMPHs) are potent psychostimulants that are widely used and abused, with profound medical and societal impact. They are known to cause mobilization of cytoplasmic dopamine (DA) to the cell exterior via DA transporter (DAT)-mediated efflux, yet the mechanisms that mediate these actions remain poorly defined and are a focus of this proposal. Using heterologous expression systems and a Drosophila behavioral model, we have shown that AMPH-induced DA efflux and consequent behaviors, but not DA uptake, are dependent on N-terminal phosphorylation of DAT. Our team has also made critical advances in understanding the molecular mechanisms of substrate uptake by studying the bacterial transporter LeuT as a prototype, using state-of-the-art single-molecule approaches and computational analyses. Although the N-terminal region is essentially absent in LeuT and was truncated in the Drosophila DAT (dDAT) structures, our team has reported a computational model of the N terminus of the human DAT (hDAT) from ab initio structure prediction in combination with extensive atomistic molecular dynamics simulations. The analysis shows the N terminus to be highly dynamic, to contain secondary structure elements, and to interact with lipid membranes through electrostatic interactions. Here we aim to probe these structural elements to gain insight into the physiology of DAT and its regulation by AMPHs, using our team's synergistic behavioral, biochemical, biophysical, and computational tools. In parallel studies we aim to explore the mechanisms that regulate AMPH-induced release of DA from synaptic vesicles into the cytoplasm. Using multiphoton imaging of living Drosophila brain we have shown that at pharmacologically relevant concentrations, AMPHs must be actively transported both by DAT and by the vesicular monoamine transporter VMAT in order to diminish the vesicular pH gradient and redistribute vesicular contents. Still, how these events lead to redistribution of DA to the cytoplasm remains unknown. Recent data suggest that VMAT N-terminal phosphorylation is essential for AMPH-induced DA efflux from vesicles, and we propose to explore this hypothesis mechanistically and test it in vivo. Our established multi-scale approach integrates biochemistry and biophysics of purified proteins, single-molecule FRET and computational analysis, with cell-based assays, Drosophila brain imaging, analysis of in vivo phosphorylation, and behavioral studies in living flies to probe the role of DAT and VMAT in the actions of AMPHs in the appropriate physiological and structural contexts, in the following SPECIFIC AIMs: AIM 1. To elucidate the role of membrane interactions in modulating phosphorylation of the N terminus of DAT and its ability to mediate AMPH-induced DA efflux and behaviors. AIM 2. To determine how N-terminal phosphorylation alters DAT function and dynamics. AIM 3. To determine the role of VMAT and its putative N-terminal phosphorylation in AMPH-induced DA efflux from synaptic vesicles in vivo and in vitro. This work will provide a clear validation of novel targets for medications that block AMPH action through mechanisms that do not alter DA uptake.
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1 |