Horace H. Loh - US grants

In this proposal, two independent but related projects will be combined which describe some main research activities in my laboratory. 1) Molecular characterization of opioid receptors and 2) determination of the neurochemical mechanisms of tolerance and dependence. The first project will utilize a mu type opioid receptor which we recently purified to homogeneity, and will include a) reconstitution of opioid binding and opioid-mediated function into a membrane environment; b) raising monoclonal and polyclonal antibodies to the receptor, and using them to map the receptor's distribution in brain, determine the role of different portions of the receptor in binding and function, and for cloning the receptor; c) molecular characterization of opioid binding to the purified receptor, including tests of negative cooperativity, thermodynamic analysis of agonist and antagonist binding, effects of ions, guanine nucleotides and lipids on binding, and tests for interconversion of mu receptors to other types; and d) cloning the receptor by synthesis of oligodeoxynucleotide probes, isolation of receptor mRNA, and insertion into a cloning vector. Studies of opioid tolerance/dependence will employ NG108-15 hybrid cells model which exhibit opioid binding, opioid-mediated function (inhibition of adenylate cyclase), and a tolerance-like adaptation process during chronic treatment. In previous work, our lab has shown that chronic opioid agonist treatment induces three distinct adaptation processes in these cells: 1) receptor desensitization, or uncoupling from adenylate cyclase; 2) receptor down-regulation, or disappearance from cell surface; and 3) an increase in adenylate cyclase activity following withdrawal or antagonism of chronic of chronic agonist. We propose to study each of these processes in detail and determine their relevance to tolerance/dependence in mammalian brain. We will try to show that a) desensitization results from a covalent change in the receptor; b) during down-regulation, receptors move along a pathway similar to that traversed by other down-regulated receptors; we will also determine the kinetics of internalization, and the signal initiating it; and c) study the involvement of Ca++ and Ca++-binding proteins in the increase in adenylate cyclase activity. To make the NG cells model more similar to these in brain, we will induce differentiation in them and compare chronic effects of these cells with those in undifferentiated cells. Finally, we will characterize opioid receptor down-regulation in brain, which we have recently reported.

Progress in purifying the opiate receptor, a goal essential to understanding both its chemical nature and function, has been slow. Current evidence suggests that this may be due to the fact that the opiate receptor is a complex, multi-component system, containing differently selective binding sites, regulatory molecules, effector system, closely associated with a proper membrane lipid environment. Solubilization of membrane would be expected to dissociate these components, whose integration may be necessary for full expression of opiate binding as well as biologic activity. We accordingly propose to isolate different receptor components and test the ability of combinations of them to exhibit opiate binding, and, when reconstituted back into the membrane, opiate-mediated function, (e.g., inhibition of adenylate cyclase). The reconstitution step serves as an ultimate test of the integrity of the solubilized receptor and because of its multicomponent nature, may be necessary for full expression of binding activity. In isolation of opiate receptors, our approaches involve detergent solubilization, isolation of binding components by affinity chromatography, and receptor reconstitution. The latter emphasis on both reconstitution of binding and functional activities. Reconstitution methods used include dialysis, sonication and fusion with genetic mutants of S49 lymphoma cells. We will also use a newer approach, solubilizing with the natural detergent lysophosphatidylcholine and reconstituting the membrane by its enzymatic conversion to phosphatidylcholine. This method for the reconstitution of brain membrane, developed in our laboratory, by itself should be very important in the studies of general membrane structure and function. To make our approach thorough, we will also attempt to isolate receptor molecules through the use of monoclonal antibodies, using partially purified receptor to develop the antibodies.

We are continuing our studies of a simple model system, NG108-15 neuroblastoma-glioma hybrid cells, to study the molecular basis of opioid tolerance/dependence. We have recently developed several new conceptual and experimental approaches, with which we have identified several molecules specifically affected by chronic opioid treatment of NG108-15 cells, and that therefore are likely to play some role in tolerance mechanisms of these cells. We propose to characterize these molecules structurally and to determine their functional significance. In the first approach, subtraction hybridization, mRNA is isolated from control and down-regulated cells, and cDNA-mRNA hybridization used to identify molecules specifically reduced or eliminated in the latter. Using this approach, we have recently identified, cloned and sequenced two closely-related mRNA's, NGD5A and NGD5B, that are down-regulated by chronic treatment with opioid but not muscarinic agonist, in a naloxone-reversible fashion. We propose a) to characterize this down-regulation with respect to time course and agonist/antagonist specificity; b) stably transfect and express NGD5 cDNA in neuro 2A cells, which do not contain opioid receptors; c) express NGD5A in E. coli, prepare antibodies to the protein, and test them for their effect, as well as determine the cellular location of the NGD5 product and follow changes in levels of the NGD5 product during chronic opioid treatment; d) prepare NGD5 genomic DNA in NG108-15 cells; e) compare NGD5 and any other down-regulated molecules identified by subtraction hybridization to opioid receptors that are purified directly from NG108-15 cells, using a detergent solubilization and affinity chromatography, cross-linking of 125I-beta-endorphin, and affinity and photoaffinity labelling. Finally, using a second novel approach, we will determine the effect of stable transfection of NGD5 antisense cDNA in NG108-15 cells on NGD5 mRNA levels, opioid binding and opioid inhibition of adenylate cyclase, and on chronic opioid effects. Our third approach is based on our recent purification of an opioid binding protein from bovine brain. Antibodies to this protein not only block binding to mu, delta and kappa opioid receptors in brain, but react with two distinct proteins in NG108-15 cells, of 58 and 39 kD; the 39 kD band is also down-regulated by chronic treatment of NG cells with opioid agonist. We will determine the location of these species on NG108-15 cells, follow the kinetics of 39 kD down-regulation, and purify the 58 and 39 kD proteins using an affinity column constructed from the antibody, followed by sequencing and cloning. Antisense cDNA to the sequences will be prepared, and tested for its effect on opioid binding, inhibition of cyclase, and chronic effects of opioids. Finally, we will compare the sequence and other structural characteristics of the 58 and 39 kD proteins to opioid receptors that are purified directly from NG108-15 cells, as in the first approach.

In order to answer one of the many questions on the multiple opioid receptors, i.e. whether or not these receptors represent different gene products, different splicing of the same gene, or post- transnational modification of the products, it is the goal of the current proposal to clone for the kappa-opioid receptor. Kappa- opioid receptor will be cloned from either the guinea pig cerebellum or human placenta library by the cDNA expression method. The library will be enriched in kappa-opioid receptor clones with 3 sets of probes: (a) restriction enzyme fragments of the mu- and delta-opioid receptor clones; (b) subtraction probes synthesized from hybridizing mRNAs from tissues containing low level of kappa- opioid receptor from that containing high level of kappa-opioid receptor, i.e. chronic kappa agonist (U50-488) or antagonist (MR2266) treatment will be used to alter the kappa-opioid receptor level and hence mRNA levels; and (c) the oligodeoxynucleotides sequences of the putative transmembrane regions V, VI and VII of the cloned 8-adrenergic receptor. The cDNA clones which hybridize with the first two sets of probes or with all three sets of probes will be expressed in eukaryotes previously devoid of opioid receptor activities. Clones which induced kappa-opioid receptor binding activity will be subcloned into Sp6 vectors for sense and anti-sense RNA synthesis. The RNAs thus synthesized will be injected into frog oocytes and the ability of kappa agonist to regulate the Ca+2 channels will be used to substantiate the identity of the clones. Antibodies will be developed against the deduced peptide sequence and will be used to immunoprecipitate ligand-kappa-opioid receptor complex and/or used to inhibit the kappa-opioid receptor binding activities in brain membranes. Such antibodies will be used also in immunocytochemical analysis of kappa-opioid receptor distribution and compared with the reported distribution of putative kappa binding sites. The identity of the kappa-opioid receptor clone will be substantiated further by the isolation and sequencing of the 125I-B-endorphin-receptor complexes when labelling was carried out in the presence of mu- and delta- opioid ligands. The gene structure and the nucleotide sequence of the kappa-opioid receptor clone will be determined and compared with that of mu- and delta-opioid receptor clones. Deletion and nucleotide insertion mutation studies will be carried out to investigate the structural requirement for kappa-opioid receptor activities.

New developments in understanding the mechanisms of actions of biological messengers and their receptors require research carried out on many different levels of the organism, and making use of a wide range of scientific disciplines and methodologies. This program project proposes to carry out such a wide ranging study, with its primary, though not exclusive, focus on the opioid system. The various individual proposals in this project study opioid and other receptors from levels ranging from their molecular structure to their biochemical regulation and consequences to their biological functions. Approaches used range from the pharmacological (in vivo and in vitro manipulation), biochemical (receptor characterization and purification, receptor-channel coupling), chemical (structure-activity studies), immunological (mono- and polyclonal antibodies), molecular biological (gene expression and regulation of receptors or ligands) and neuroanatomical (characterization of storage site and release of neuropeptides). Studies of receptor structure will be carried out by Conti-Tronconi, Loh and Portoghese. Conti-Tronconi will prepare monoclonal antibodies to specific amino acid sequences of the nicotinic receptor, and use these antibodies to map agonist and antagonist binding sites, intra- and extra- membrane portions of the receptor, and tissue distribution of these receptors. Loh will apply a somewhat similar approach to a recently- cloned putative delta opioid receptor. Portoghese will further characterize the binding of opioid azines to brain membranes, which his previous research has determined involves conversion of the azine to a hydrazone, which then reacts with a neighboring phosphatide in the membrane. Law and Takemori will study opioid receptor regulation, and Eide will study regulation of neuropeptides. Law will study the regulation of the putative delta opioid receptor gene, which he and his collaborators recently cloned from NG108-15 neuroblastoma-glioma hybrid cells. Takemori will test the ability of selective irreversible mu and delta antagonists to block the development of tolerance. Elde will study processes involved in the storage and release of neuropeptides. Receptor function will be studied by Raftery, Holtzman and Lee. Raftery will study the cholinergic receptor as a sodium pump. Holtzman will further characterize the role of thiol:potein disulfide oxidoreductase in hormone action, which he recently found was activated by glucagon. Lee will use mono and polyclonal antibodies to explore the role of dynorphin in inducing analgesia and/or modulating morphine-induced analgesia, in both the brain and the spinal cord.

In this proposal, two independent but related projects will be combined which describe some main research activities in my laboratory. 1) Molecular characterization of opioid receptors and 2) determination of the neurochemical mechanisms of tolerance and dependence. The first project will utilize a mu type opioid receptor which we recently purified to homogeneity, and will include a) reconstitution of opioid binding and opioid-mediated function into a membrane environment; b) raising monoclonal and polyclonal antibodies to the receptor, and using them to map the receptor's distribution in brain, determine the role of different portions of the receptor in binding and function, and for cloning the receptor; c) molecular characterization of opioid binding to the purified receptor, including tests of negative cooperativity, thermodynamic analysis of agonist and antagonist binding, effects of ions, guanine nucleotides and lipids on binding, and tests for interconversion of mu receptors to other types; and d) cloning the receptor by synthesis of oligodeoxynucleotide probes, isolation of receptor mRNA, and insertion into a cloning vector. Studies of opioid tolerance/dependence will employ NG108-15 hybrid cells model which exhibit opioid binding, opioid-mediated function (inhibition of adenylate cyclase), and a tolerance-like adaptation process during chronic treatment. In previous work, our lab has shown that chronic opioid agonist treatment induces three distinct adaptation processes in these cells: 1) receptor desensitization, or uncoupling from adenylate cyclase; 2) receptor down-regulation, or disappearance from cell surface; and 3) an increase in adenylate cyclase activity following withdrawal or antagonism of chronic of chronic agonist. We propose to study each of these processes in detail and determine their relevance to tolerance/dependence in mammalian brain. We will try to show that a) desensitization results from a covalent change in the receptor; b) during down-regulation, receptors move along a pathway similar to that traversed by other down-regulated receptors; we will also determine the kinetics of internalization, and the signal initiating it; and c) study the involvement of Ca++ and Ca++-binding proteins in the increase in adenylate cyclase activity. To make the NG cells model more similar to these in brain, we will induce differentiation in them and compare chronic effects of these cells with those in undifferentiated cells. Finally, we will characterize opioid receptor down-regulation in brain, which we have recently reported.

New developments in understanding the mechanisms of actions of biological messengers and their receptors require research carried out on many different levels of the organism, and making use of a wide range of scientific disciplines and methodologies. This program project proposes to carry out such a wide ranging study, with its primary, though not exclusive, focus on the opioid system. The various individual proposals in this project study opioid and other receptors from levels ranging from their molecular structure to their biochemical regulation and consequences to their biological functions. Approaches used range from the pharmacological (in vivo and in vitro manipulation), biochemical (receptor characterization and purification, receptor-channel coupling), chemical (structure-activity studies), immunological (mono- and polyclonal antibodies), molecular biological (gene expression and regulation of receptors or ligands) and neuroanatomical (characterization of storage site and release of neuropeptides). Studies of receptor structure will be carried out by Conti-Tronconi, Loh and Portoghese. Conti-Tronconi will prepare monoclonal antibodies to specific amino acid sequences of the nicotinic receptor, and use these antibodies to map agonist and antagonist binding sites, intra- and extra- membrane portions of the receptor, and tissue distribution of these receptors. Loh will apply a somewhat similar approach to a recently- cloned putative delta opioid receptor. Portoghese will further characterize the binding of opioid azines to brain membranes, which his previous research has determined involves conversion of the azine to a hydrazone, which then reacts with a neighboring phosphatide in the membrane. Law and Takemori will study opioid receptor regulation, and Eide will study regulation of neuropeptides. Law will study the regulation of the putative delta opioid receptor gene, which he and his collaborators recently cloned from NG108-15 neuroblastoma-glioma hybrid cells. Takemori will test the ability of selective irreversible mu and delta antagonists to block the development of tolerance. Elde will study processes involved in the storage and release of neuropeptides. Receptor function will be studied by Raftery, Holtzman and Lee. Raftery will study the cholinergic receptor as a sodium pump. Holtzman will further characterize the role of thiol:potein disulfide oxidoreductase in hormone action, which he recently found was activated by glucagon. Lee will use mono and polyclonal antibodies to explore the role of dynorphin in inducing analgesia and/or modulating morphine-induced analgesia, in both the brain and the spinal cord.

We propose to characterize structurally and functionally the mu type opioid receptor, which we have recently purified to homogeneity in our laboratory. Specifically, we will: (1) reconstitute the receptor into a membrane environment, and attempt to demonstrate restoration of both opioid binding and opioid-receptor mediated function; (2) raise monoclonal and polyclonal antibodies to the receptor, and use them to (a) map the receptor's distribution in the brain; (b) determine the role of different portions of the receptor on binding and function; (c) construct an immunoaffinity column for purifying large quantities of the receptor; and (d) clone the receptor (see also aim #4, below); (3) to run several critical tests of receptor models and to study the molecular mechanisms of opiate binding to its receptor, including (a) determining whether negative cooperativity occurs during opiate binding; (b) use thermodynamic analysis of opiate binding, to determine the molecular mechanism of agoinst and antagonist interactions; (c) determining the regulatory effects of ions, GTP, and lipids on opioid-receptor interactions; (d) determining whether the mu type opioid receptor can inter- convert to other types, such as delta or kappa; and (e) determining by physical methods whether receptor conformational changes occur during opiate binding; and (4) clone the opiate receptor, by synthesis of oligodeoxynucleotide probes corresponding to the N- terminal sequence of the receptor, use of these to isolate opioid receptor mRNA, and insertion of the corresponding DNA into a cloning vector. In addition, the availability of receptor antibody should provide an alternative means for receptor cloning (see specific aim #2, above). These studies will make it possible to clarify the role opioid receptors play in analgesia and other clinically important effects, and to determine the biochemical processes by which these effects are produced.

We are continuing our studies of a simple model system, NG108-15 neuroblastoma-glioma hybrid cells, to study the molecular basis of opioid tolerance/dependence. We have recently developed several new conceptual and experimental approaches, with which we have identified several molecules specifically affected by chronic opioid treatment of NG108-15 cells, and that therefore are likely to play some role in tolerance mechanisms of these cells. We propose to characterize these molecules structurally and to determine their functional significance. In the first approach, subtraction hybridization, mRNA is isolated from control and down-regulated cells, and cDNA-mRNA hybridization used to identify molecules specifically reduced or eliminated in the latter. Using this approach, we have recently identified, cloned and sequenced two closely-related mRNA's, NGD5A and NGD5B, that are down-regulated by chronic treatment with opioid but not muscarinic agonist, in a naloxone-reversible fashion. We propose a) to characterize this down-regulation with respect to time course and agonist/antagonist specificity; b) stably transfect and express NGD5 cDNA in neuro 2A cells, which do not contain opioid receptors; c) express NGD5A in E. coli, prepare antibodies to the protein, and test them for their effect, as well as determine the cellular location of the NGD5 product and follow changes in levels of the NGD5 product during chronic opioid treatment; d) prepare NGD5 genomic DNA in NG108-15 cells; e) compare NGD5 and any other down-regulated molecules identified by subtraction hybridization to opioid receptors that are purified directly from NG108-15 cells, using a detergent solubilization and affinity chromatography, cross-linking of 125I-beta-endorphin, and affinity and photoaffinity labelling. Finally, using a second novel approach, we will determine the effect of stable transfection of NGD5 antisense cDNA in NG108-15 cells on NGD5 mRNA levels, opioid binding and opioid inhibition of adenylate cyclase, and on chronic opioid effects. Our third approach is based on our recent purification of an opioid binding protein from bovine brain. Antibodies to this protein not only block binding to mu, delta and kappa opioid receptors in brain, but react with two distinct proteins in NG108-15 cells, of 58 and 39 kD; the 39 kD band is also down-regulated by chronic treatment of NG cells with opioid agonist. We will determine the location of these species on NG108-15 cells, follow the kinetics of 39 kD down-regulation, and purify the 58 and 39 kD proteins using an affinity column constructed from the antibody, followed by sequencing and cloning. Antisense cDNA to the sequences will be prepared, and tested for its effect on opioid binding, inhibition of cyclase, and chronic effects of opioids. Finally, we will compare the sequence and other structural characteristics of the 58 and 39 kD proteins to opioid receptors that are purified directly from NG108-15 cells, as in the first approach.

The purpose of this program project is to study the mechanisms of action of several drugs of abuse at multiple levels of the organism. These levels include the molecular, cellular, physiological/anatomical and behavioral, and the drugs studied include opioids, caffeine, and cocaine. While some of the individual projects are dedicated to elucidating in further detail the classical effects of these drugs, other projects seek to define and characterize novel effects, such as may be exerted on the immune system and on circadian rhythms. The University of Minnesota Medical School is an ideal environment for this multidisciplinary effort to understand drug abuse, because of the broad range of interests of the participating faculty and a strong institutional commitment. All the component PI's have committed themselves to work together through this program grant, and through monthly seminars and annual retreats, thus a great deal more communication and collaboration can be developed.

This program project renewal application continues our multidisciplinary approaches to studying drugs of abuse. Dr. Law will determine the gene structure of the delta opioid receptor (DOR), including positions and sizes of introns and the nature of regulator elements. In the hope of understanding the molecular mechanism of opioid tolerance, Drs. Loh and Law will engineer deletions/substitutions in critical regions of DOR, including the C- terminus and third cytoplasmic loop, to determine the role of specific residues in this region and their phosphorylation state on regulation of the receptor activities and receptor level in response to chronic opioid agonist treatment. Reagents such as receptor antibodies and epitope tagged receptor will be developed for these studies. In the final renewal proposal, Dr. Conti-Tronconi will quantitate acetylcholine receptor in several non-neuronal tissues known to be affected by nicotine abuse, including upper respiratory tract, smooth muscle cells and endothelium of blood vessels. She will characterize these AChRs with respect to subunit composition, demonstrate the presence of ACh metabolizing enzymes, determine whether they mediate such functions as cell adhesion, motility and differentiation, and investigate the effects of nicotine abuse on these molecules.

Molecular mechanism of opioid tolerance and dependence is a subject under intense investigations. Models have been developed so as to facilitate the understanding of these opioid pharmacological effects. Neuroblastoma x glioma NG108-15 cells is one such model. Previous studies with this model have suggested that the homogeneous population of delta-opioid receptor in this cell line is under stringent cellular regulation. Chronic opioid agonist treatment resulted in a loss in receptor's responses due to receptor desensitization and receptor down-regulation. At least in receptor down-regulation parallel observations with other opioid receptor types were obtained in animals chronically administered opioid receptor selective agonists. Thus, understanding of molecular basis for receptor desensitization and down-regulation could illuminate the problem of tolerance. Previous efforts have been hampered by lack of opioid receptor reagents, such as receptor specific antibodies. Now with recent cloning of delta-opioid followed by mu- and kappa-opioid receptor, receptor specific antibodies could now be developed. Therefore, in current studies, we propose to develop to delta-opioid receptor specific antibodies by immunizing rabbits with peptides synthesized according to deduced primary sequence of cloned delta-opioid receptor, and immunizing rabbits with receptor proteins expressed in and purified from E. coli. Identities of antibodies produced will be established by comparing western analysis of membrane isolated from CHO cells, CHO stably transfected with delta-opioid receptor cDNAs, and NG108-15 cells. Immunocytochemistry will be performed with brain slices and these antibodies in order to utilize known delta-opioid receptor distribution to characterize these antibodies. The abilities of these antibodies to inhibit opioid receptor binding, to immunoprecipitate delta-opioid receptor will be established. The hypothesis of receptor phosphorylation as mechanisms for receptor desensitization will be investigated using these receptor specific antibodies to examine the phosphorylation states of receptor during agonist treatment. Delta-opioid receptor will be separated from other phosphoproteins using these antibodies. Abilities of known protein kinases to phosphorylate delta-opioid receptor will be examined. Degree phosphorylation and sites of phosphorylation will be examined by peptide mapping of immunoprecipitated or immunoaffinity purified receptors. Effect of receptor phosphorylation will be investigated also by mutation analysis of cloned delta-opioid receptor. Delta-opioid receptor clone will be mutated by site-directed mutagenesis or deletion mutagenesis, with the focus on serine and threonine moieties in the cytosolic domains of the receptor molecule. Effect of these mutations on phosphorylation, receptor desensitization and receptor down-regulation will be evaluated by transient expression in COS7 cells and stable transfection in CHO cells of wide type and mutant delta-opioid receptor. Through all mutation studies and phosphorylation studies, we will establish the role of phosphorylation in cellular adaptation to chronic presence of opioid agonists.

Opioid receptors belong the superfamily of G protein-coupled receptors (GPCRs). Similar to a majority of this superfamily members, prolonged activation of the opioid receptors resulted in a loss of response, mainly due to receptor desensitization. There is overwhelming evidence to suggest that the phosphorylation of GPCRs is the general mechanism for receptor desensitization. In the case of opioid receptor, phosphorylation of the mu- nd delta-opioid receptor upon agonist activation have been reported. Though there is some peripheral indication of a relationship between delta-opioid receptor phosphorylation and receptor desensitization, detailed correlation has not been established. In our studies with mu- opioid receptor phosphorylation, we could demonstrate that receptor phosphorylation occurred within minutes of agonist binding, while the ability of agonist to inhibit adenylyl cyclase was not blunted until hours after agonist addition. Therefore, we decided to investigate thoroughly the relationship between delta-opioid receptor phosphorylation and desensitization. We will utilize the polyclonal antibodies specific against the delta-opioid receptor and hemagglutinin (HA) epitope tagged receptor we have developed in our studies. We will correlate the degree of delta opioid receptor phosphorylation/dephosphorylation to the ability of agonist in inhibiting the forskolin-stimulated adenylyl cyclase activity. We will investigate the effect of various protein kinases' inhibitors on receptors phosphorylation and desensitization. We will pin-point the phosphorylation sites on the delta-opioid receptor which are involved in receptor desensitization. This will be accomplish by the receptor truncational and mutational analysis. The attenuation of receptor phosphorylation with the removal of putative phosphorylation sites, SER and Thr, and the subsequent effect on agonist-induced receptor desensitization will be determined. In order to eliminate any misconclusion, effect on mutation of Ser/Thr of interest to Asp will be evaluated and the amino acid sequencing of the receptor domains involved in phosphorylation will be carried out. Finally, the protein kinases which re involved in opioid receptor phosphorylation will be identified by the transient expression of these kinases in HEK293 cells which stably expressing the delta-opioid receptor. The effect of the over-expression of these kinases,or the dominant mutants of GRK on receptor phosphorylation and desensitization will be determined. The probably presence of a specific kinases for delta-opioid receptor will be investigated by the in vitro phosphorylation and peptide mapping of the phosphorylated purified receptor carried out with endogenous and exogenous protein kinases.

In order to synchronize the two exiting program project grants, e propose to establish a Drug Abuse Research Center in Basic Molecular and Cell Biology at University of Minnesota which utilizes the expertise of the core faculty in the Department of Pharmacology and more effective promotes service and interaction with other drug abuse researchers in this community. During the past 8 years, we have proven that two P01 grants have worked successfully and coherently as a single program, with Horace H. Loh as the P.I. Thus, in the current proposal, we have combined various components from these two program projects to establish the proposed Drug Abuse Research Center. The main objectives of the proposed center are: (a) foster interdisciplinary approaches in drug abuse research among the investigators; (b) to serve as an "activity" center to coordinate and to promote all academic and scholarly activities in drug abuse research in the Minneapolis metropolitan area; (c) to serve as a national resource for molecular and cellular studies in the mechanisms of drug abuse either by providing expertise, reagents, clonal cell lines or transgenic/knockout animals to the drug abuse community locally and nationally; and (d) to serve as training center for young scientists here in Minnesota. Within the proposed center, there will be an Administration Core, a Molecular/Cellular and Genetic Core, an Antibodies Production Core and a Bioimaging Core which will support the activities of the scientific components. There are total 8 scientific components with 5 components which deal directly with the different aspects of the drug opiate, while the other three deals with other drugs of abuse, marijuana, nicotine and caffeine. All these 8 components deal with the investigation of the basic molecular and cell biological mechanisms of these drugs of abuse. The different approaches used: transgenic animals, molecular biological mutational analysis of proteins and genes, second messengers regulations, electrophysiological measurements and transcription control studies will provide a fertile ground for training of young scientists, interaction among investigators nd developing resource for the drug abuse research community locally and nationally.

DESCRIPTION (provided by applicant): Basic Research Center on Molecular and Cell Biology of Drug Abuse (BRCMCDA) was established in 1998 with the objectives to (1) to foster interdisciplinary approaches in drug addiction research; (2) to serve as an "activity" center to coordinate and to promote all academic and scholarly activities on drug addiction research at University of Minnesota; (3) to serve as a national resource for molecular and cell biology of drug addiction research: and (4) to serve as training center for young scientists here at University of Minnesota. The goal of the Center is to promote molecular and cell biology approaches for drug addiction research, hence a thematic program is absent in the 8 scientific components. Instead, multidisciplinary approaches in the programs present fertile ground for training and interaction among scientists. The Center's activities include sponsored seminars and biannual symposium focus on the state-of-the-art techniques and breakthroughs in the field of drug addiction research. The Center has continued to raise the visibility of drug addiction research at University of Minnesota and has served as national resources in supplying investigators nationally and internationally reagents and genetically altered mice for their research programs. We wish to build on the past success and continue the missions of BRCMCBDA in promoting drug addiction research and training at University of Minnesota. Thus the proposed structure of BRCMCBDA remains to consist of 8 scientific components supported by an administrative core. The principal investigators involved in these components have demonstrated records of past collaboration and are committed to the mission of the Center. However, in order to establish a formal recruiting mechanism, a "seed grants" program will be implemented to create an opportunity for investigators not associated with the Center to test their hypotheses or new techniques in drug addiction research. Such program not only will enhance the existing research programs within the Center, but also will establish the bases for future components of the Center and grants application in drug addiction research. Hence, together with the other programs of the Center, BRCMCBDA will continue to be a research training center and national resource on the understanding of the molecular mechanism of drug addiction.

[unreadable] DESCRIPTION (provided by applicant): This is a competitive renewal application of NIDA grant DA000564-34. Since opioid receptors have been identified to be members of the rhodopsin subfamily of GPCRs, the general mechanism for GPCR desensitization has been applied to account for in vivo opiate tolerance. In this mechanism, opioid receptors are phosphorylated by GRKs in the presence of agonists and beta-arrestin is recruited to the receptor vicinity resulting in the blunting of the signals. Such mechanism is supported by studies with beta-arrestin knockout mice in which morphine tolerance was attenuated. However, a direct correlation between receptor phosphorylation and desensitization could not be demonstrated. Nevertheless, it remains our central hypothesis that covalent modification of opioid receptor and the signaling complex, or receptosome, during chronic agonist treatment is the key for the eventual manifestation of opiate tolerance and dependence. Therefore, in the proposed studies, we will continue our on-going studies to investigate the role of MOR phosphorylation on morphine tolerance response. We will determine the protein kinases involved in the phosphorylation of the consensus motif TXXXPS within the receptor. We will re-determine the agonist-dependent MOR phosphorylation sites by mass spectrometry analyses of purified receptor. We will validate our observations in clonal cell models with primary neuronal cultures. We will investigate the role of receptor phosphorylation in morphine tolerance by the use of GRK null mice together with inhibitors of protein kinases involved in MOR phosphorylation. The role of receptor phosphorylation in morphine tolerance will also be investigated by restoration of morphine acute and chronic responses in MOR null mice with adenovirus carrying the wild type and phosphorylation receptor mutants. In addition to receptor desensitization, our preliminary data also suggested that Src activation during chronic morphine treatment could be the basis for adenylyl cyclase (AC) superactivation. Since beta-arrestin has been implicated in Src activation, receptor phosphorylation could be the trigger for such event. Thus, we will examine the mechanism for and the role of beta-arrestin in Src activation. We will investigate the consequence of Src activation on AC superactivation, and will identify the cellular targets of Src with mass spectrometry analysis. The role of Src activation in the in vivo morphine dependence and withdrawal responses will be established by the use of Src inhibitor PP2 and inducible siRNA approach. All these studies will allow us to elucidate the molecular components involved in opiate tolerance and dependence. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This is a resubmission of my renewal application of DA001583-30. In the last review, it received a score of 156 (12.9%) and was not funded. First of all I would like to thank the reviewers for their thorough review and overall very positive assessment of the original application. There were only two points raised in the review, one questioning why not study human gene and the second on physiological relevance of our study on the regulation of MOR receptors. Both are clarified and addressed in the revision. This renewal will continue our study on the regulatory mechanisms underlying the expression of the Mu opioid receptor (MOR) in neurons. Studies during the current award period focused on transcriptional events that provided essential information for our understanding of the genetic basis of how this important drug receptor is regulated. This renewal will focus on regulatory events involving higher-order structures of chromatin (chromatin remodeling) and epigenetic effects. Further, our preliminary studies identified several potentially novel factors that acted beyond transcriptional control, and it is hypothesized that the expression of the functional product of MOR gene, i.e. the mu receptor, is tightly regulated at both transcriptional and post-transcriptional levels. The renewal application is to address this principal hypothesis in three aims. Aim 1 will continue ongoing studies of MOR gene transcriptional regulation with chromatin as the focus. Firstly, the chromatin structure and nucleosomal arrangement of MOR gene, as well as methylation-induced epigenetic control, will be defined using various biochemical and molecular biology based methods. Secondly, the dynamic behavior of previously identified transcription factors on the MOR promoter will be examined in its endogenous genomic context using primarily chromatin immunoprecipitation. Aim 2 will extend studies to the post-transcriptional events, primarily translational control of MOR protein expression. Three major regulatory events will be addressed, including the roles of 5'-untranslated regions (UTR), the 3'-UTR and the polyadenylation signal. Aim 3 will identify external factors involved in the ultimate regulation of MOR protein expression, including vitamin A hormone (retinoic acid) and other signals that possibly modulate translation of MOR mRNA in neurons. Our immediate goal in this renewal application is to determine the mechanism of action of genetic, epigenetic and extracellular signals that act, in combination, to regulate the ultimate expression of appropriate amounts of MOR receptor protein in neurons. Our long-term goal is to be able to delineate signal transduction pathways that affect the manifestation of MOR gene in the context of whole animals, and possibly to contribute to the understanding of genetic and molecular basis of problems related to the use of morphine in humans. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The Basic Research Center on Molecular and Cell Biology of Drug Addiction (MCBDA) was established 10 years ago with the objectives to (1) to foster interdisciplinary approaches in drug addiction research;(2) to serve as an "activity" center to coordinate and to promote all academic and scholarly activities on drug addiction research at University of Minnesota;(3) to serve as a national resource for molecular and cell biology of drug addiction research: and (4) to serve as training center for young scientists here at University of Minnesota. The Center was founded with 8 scientific components that use multidisciplinary approaches in their investigation of molecular mechanisms of drug addiction. Center activities have included sponsored seminars and biannual symposium, which have raised the visibility of drug addiction research at the University of Minnesota and provided opportunities for interactions among Center members and nonmembers. The Center has served as a national and international source of reagents, plasmid constructs, viruses and genetically altered mice for drug addiction research. The Center has proven to be an excellent training ground for investigators at the University of Minnesota, nationally and internationally. Through the Seed Grants Program initiated during last funding period, we have provided opportunities for investigators to explore innovative approaches for drug addiction research. The seed grants also provided opportunities to recruit new investigators into drug addiction field. We wish to build on the past success and continue the missions of the Center in the next funding period. Our research theme will focus on the molecular and cell biology of opioid actions and addiction. In order to further foster the synergistic interactions among the scientific components, we will re-establish the Molecular Cellular and Genetic Core. Thus, the proposed structure of the Center will consist of 5 scientific components supported by an administrative core and a service core. Adhering to our goal to recruit and foster development of young investigators in drug addiction research, we will continue our successful Seed Grant Program and also will initiate a new Pilot Project Program for the recruitment and development of young investigators who could contribute to the mission of the Center. The principle investigators of the scientific components have demonstrated records of past collaboration and are committed to the mission of the Center. We will raise the visibility of drug addiction by participating in the existing community outreach programs, and initiate contacts with the clinicians at the University of Minnesota Medical School. Together with the other Centers'programs, the Center will continue to be a research training center and national resource on the understanding of the molecular and cellular mechanism of drug addiction. CENTER CHARACTERISTICS

The molecular mechanism for morphine tolerance has not been firmly established yet. The current model of [j-opioid receptor (MOR) desensitization via the &-Arrestin pathway cannot account for the numerous observations that other neurotransmitter receptor activities, such as NMDA, could contribute to morphine tolerance. The activity of other opioid receptors, such as the 6-opioid receptor (DOR), could be implicated in morphine tolerance development also. Since morphine can activate and desensitize DOR during prolonged treatment, our working hypothesis is that the post-signaling events occurring within the DOR-containing neurons during morphine treatment contribute to tolerance development. Our working hypothesis also is that morphine has post-signaling events distinct from those of other opioid agonists. In order to demonstrate these hypotheses, agonist-dependent signaling events will be established for morphine activation of DOR. In our studies with MOR signaling, we have demonstrated that morphine differs from other agonists in its pathway to activate ERK1/2. Agonists such as etorphine activate ERK1/2 via the B-Arrestin-dependent pathway, while morphine activates ERK1/2 via the PKC-dependent pathway. This divergent activation results in differential translocation of the activated ERK1/2 and the transcripts produced. Therefore, the signaling pathway and the post-signaling events of morphine in cell models expressing DOR will be established. The possible involvement of the PKC-dependent pathway on morphine-mediated DOR activation of ERK1/2 will be studied. The specific PKC subtypes involved will be defined. The reasons for the differences among agonists in selecting a pathway will be investigated by monitoring protein-protein interactions using a novel protease assay system. Parallel studies will be conducted with primary neuronal cultures. The blockade of specific PKC subtypes in DOR-expressing neurons on in vivo morphine tolerance development will be explored. By selectively inactivating the morphine signaling pathway, and subsequently its post-signaling events in DOR-containing neurons, possible blockade of morphine tolerance without altering morphine activities in MOR containing neuron could be accomplished.

Our Center, the Basic Research Center on Molecular and Cell Biology of Drug Addiction (MCBDA, www.MCBDA.ahc.umn.edu), was established 10 years ago with the mission of developing treatments for drug addiction by understanding the mechanisms of drug actions, via basic research. We had 4 objectives when we established the Center: (1) to foster interdisciplinary approaches in drug addiction research;2) to serve as an "activity" Center in coordinating and promoting all academic and scholarly activities on drug addiction research at University of Minnesota;3) to serve as a national resource for molecular and cell biology of drug addiction research;and 4) to serve as training Center for young scientists at the University of Minnesota. Thus far, we have been successful in accomplishing many aspects of these objectives. With 8 scientific components and an Administrative Core constituting the structure of Center during the last funding period, and due to the diversity and breath in the research interests of the faculty involved, the Center has established itself as a productive training ground for young scientists interested in drug addiction research. As summarized in our discussion of the Seed Grant Program in the Training and Education section (page 55), the Center has funded some innovative research projects submitted by young scientists. Two of the Seed Grant awardees, Dr. Dezhi Liao (Department of Neuroscience) and Dr. Kirill Martemyanov (Department of Pharmacology), used the results generated with Center support to successfully compete for R01 research awards aimed at: 1) the study of opioid regulation of dendritic spine stability (DA020582, Opioid Receptors in Excitatory Synapses) and, 2) the role of RGS9-2 and its anchoring protein R7BP on drug addiction (DA021743, Molecular Basis of RGS Protein Function in the Striatum). The Center was also successful during the last funding period in providing opportunities for interactions among investigators and for training pre- and post-doctoral fellows. Despite the limited budget, the Center continued to sponsor seminars and organized a biannual symposium to raise the visibility of drug addiction research at the University of Minnesota. Two Center members, Dr. P.Y. Law and Dr. Li-Na Wei, co-chaired the programming committee of the International Narcotic Research Conference held July 9-14, 2006 in St. Paul, Minnesota. These organized activities of the Center, in addition to the weekly research group meetings that are open to all Center members, have generated sustained interest in drug addiction research and opportunities for interactions. Through such activities, Dr. Liao, an expert on AMPA receptor transport and synaptic plasticity, became interested in drug addiction research. Synergism among the various approaches used by Center investigators has facilitated the research progress of individual investigators. This is best reflected by the $5.3 million of national funding obtained during the last fiscal year by the principle investigators associated with the Center and the 20 manuscripts co-authored by the Center's principle investigators during the last funding period. In addition, 2 Center members are recipients of NIDA K05 senior scientist awards (Dr. H.H. Loh and Dr. P.Y. Law), 2 are recipients of NIDA K02 career development awards (Dr. Li-Na Wei and Dr. Sabita Roy) and 1 is a recipient of a NIDA research merit award (Dr. Stanley Thayer). The Center has been and will continue to be national and international source of reagents for drug addiction research. Individual investigators have provided reagents, plasmid constructs, and genetically-altered mice in the Center, and the Administrative Core has assisted in disseminating these materials to intereted investigators, both nationally and internationally. A list of reagents supplied during the last funding period is provided in the subsequent progress report. We remain committed to our goal of attracting young scientists to drug addiction research. One way in which the Center can accomplish this goal, while operating within our budget, is to rotate principle investigators. In the 2002 competitive renewal of our Center, Dr. P.Y. Law did not head an individual scientific component so that we could recruit Dr. Kevin Wickman to our Center. Dr. Law then expanded his original Center component project and successfully competed for an R01 award to pursue his receptor trafficking studies (DA016674, Neuronal Regulation of Opioid Receptor Trafficking). In the last submission, three of the original members of the Center (Dr. Bianca Conti-Fine, Dr. Robert Hide and Dr. Tim Walseth) were replaced with three young scientists (Dr. Kirill Martemyanov, Dr. Jonathan Marchant and Dr. Van Zeng) who were recruited to the Center via the Seed Grant Program. Given the diversity of the proposed research projects, it is understandable that a cohesive scientific theme across the Center was not apparent. Since we wish to remain true to the original goal of the Center, i.e., to foster young scientists in drug addiction research, we have re-organized the Center in this re-submission to focus on the molecular and cell biology of opiate action and addiction. There is no debate on the severity of the problem of opiate addiction. To address and develop treatments for such a severe problem, research on the molecular and cellular mechanisms of drug addiction, and on neural systems and behavior, must be carried out in conjunction with one another. The molecular and cellular analyses cannot focus simply on one aspect of drug addiction. The process of drug signaling that leads to tolerance, dependence, and addiction exhibited by animals must be investigated. The approach cannot be limited simply to gene transcription, but also must include investigations on the actions of gene products that could modify the drug signaling process and neural transmission. Thus, multi-disciplinary approaches that integrate molecular, biochemical, electrophysiological, neuroimmunological and behavioral studies must be applied to the opiate addiction problem. Such an integrated approach could facilitate the rapid implementation of the basic research data into probable treatment paradigms. With this in mind, our proposed Center has several strong points. One clear strength of the Center is the proven track records of Center participants in applying their basic research observations to probable treatments of opiate addiction. An excellent example is the use of clonidine to suppress opiate withdrawal signs in animals, which has been translated into clinical treatment paradigms. Another example is the discovery, during receptor-structure analyses studies, of an opioid receptor mutation that can be activated by opiate alkaloid antagonists. This receptor mutant has been issued a USA patent for the treatment of chronic pain without the tolerance and addiction associated with opiate analgesics. These clinical applications are products of our projects on the molecular and cell biology of opiate action and addiction. Another strength of the Center is the multidisciplinary nature of the proposed research. Well-established faculty who are committed to solving problems of opiate addiction and action head the scientific components of the Center. They are highly-trained experts in transcriptional regulation, biochemical and molecular aspects of receptor signaling, electrophysiology, neuropharmacology, neuroimmunology, and behavioral studies. They have proven records of collaboration, and have worked together synergistically for over a decade. The unique feature of our Center faculty is that collectively, they can investigate opiate addiction from the molecule to the whole animal. As such, our Center faculty provides ample and diverse training opportunities to foster the development of young scientists in the molecular and cell biology of opiate action and addiction.

DESCRIPTION (provided by applicant): Cloning and gene knockout studies of the mouse mu opioid receptor (MOR) have established the functional role for MOR in mediating the pharmacological effects of morphine. A proper control for the expression of MOR, from transcription to post-transcription, is crucial to the effects of morphine. Our goal is to understad the regulation of MOR expression in a physiological context, which is not possible by studying the human h-OPRM gene; therefore, we primarily have employed a mouse model by which we are able to systematically dissect the molecular mechanisms. Our progress in the previous funding cycles enabled us to construct a relatively comprehensive scheme about the hierarchy of various regulatory steps that direct and control temporally and spatially specific synthesis of MOR protein, from transcription to post-transcriptional events. Recent data revealed gene-environment interaction of the mouse m-oprm gene transcription and critical regulation in MOR mRNA translation; therefore this renewal grant will now specifically address epigenetic regulation and translational control. We propose a central hypothesis that missteps in these two levels of m-oprm gene regulation can have certain pharmacological implications. We further hypothesize that, at the molecular level, environmental and cell-autonomous factors work in concert to guide the m-oprm gene's adaptation to specific conditions in order to enhance the MOR-producing neurons' genome capacity (i.e., epigenetic regulation of m-oprm gene transcription), and ensure proper control of MOR translation in specific cells (i.e., functional plasticity). We propose two specific aims to test these hypotheses. Aim 1 focuses on mechanisms underlying epigenetic regulation of the mouse m-oprm gene including a) differential chromatin remodeling processes of its distal (DP) and proximal (PP) promoters, b) possible machineries responsible for its chromatin remodeling, and c) its epigenetic regulation in normal and morphine-tolerant mouse brains. Aim 2 focuses on MOR protein synthesis and relevance to morphine tolerance with regard to a) microRNAs, b) RNA binding proteins of the 3'-UTR of MOR mRNA, and c) RNA binding proteins of the 5'-UTR of MOR mRNA, as well as the potential role of extracellular factors. Through these studies, we will begin to examine the translational potential of our studies by asking whether and how any of these molecular mechanisms may be relevant to certain pharmacological problems such as morphine tolerance.

DESCRIPTION (provided by applicant): Opioids are the most efficacious compounds in the treatment of moderate to severe pain. However, with chronic use, many adverse effects including tolerance and dependence development will result. Differential tolerance development between the analgesic and respiration depression responses decreases the therapeutic index of opioids during chronic administration, which is a major concern. In order to overcome this obstacle, the holy grail of opioid research has been the development of an ideal analgesic, i.e., one that has minimal side effects, including tolerance and dependence development. Instead of developing specific orthosteric ligands that will activate a single receptor regardless of the oligomeric state of the receptor, we have pursued a novel approach to develop an opioid receptor mutant that can be activated by the opioid antagonist. This approach was based on our accidental discovery that mutation of Ser196 residue in the 4th transmembrane domain of mu-opioid receptor (OPRM1) results in the ability of opiate alkaloid antagonists such as naloxone and naltrexone to activate the receptor, without altering ligand afinity or agonist activity. This antagonist activity was demonstrated in vivo with a S196A knock-in mouse line and also with the adenoassociated virus- mediated delivery of the mutant receptor to various sites of the pain pathway. Importantly, chronic administration of naloxone in activating this mutant receptor does not result in tolerance development. The success of the OPRM1 mutant leads us to hypothesize that there must be allosteric modulators that can convert OPRM1 into conformations similar to that converted by the S196A mutation. We term such modulators as antagonist to agonist modulators or AAMs. The proof of concept for the existence of AAMs activity was established by our recent identification of 10 probable hits in our screens of 50,000 compounds in a library using a cell-based assay. Encouraged by these observations, we propose to (1) continue our screens of a chemical compound library for the existing of additional AAM activities; (2) validate the AAM activity in the identified hits with other OPRM1 activity measurements, such as inhibition of adenylyl cyclase activity, induction of K+ current via activation of GIRK channels, and activation of ERK1/2. Studies to demonstrate that the hits are actual allosteric modifiers of OPRM1 will be carried out also; and (3) to test for in vivo AAM activity by measuring the antinociceptive activity of the OPRM1 antagonist naloxone in the presence of such hits. The current proposed studies will be the initial steps in our development of allosteric modulators for OPRM1, and will validate our hypothesis that there is a new class of allosteric modulators, AAMs. AAMs will represent a novel class of drug molecules that can limit tolerance development during chronic opioid administration, thereby maintaining the therapeutic index of the opioid analgesic treatment paradigm.