STRUCTURE/FUNCTION OF THE CCK-B/GASTRIN RECEPTOR

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 3R01DK046767-10S2 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: GMA

Abstract: The cholecystokinin-B/gastrin receptor (CCK-BR) is a seven transmembrane domain peptide hormone receptor. Among its most important physiologic functions, the CCK-BR modulates acid secretion and mucosal proliferation in the stomach as well as anxiety and pain perception in the central nervous system. The potential clinical relevance of this receptor has generated considerable interest in understanding the molecular basis of ligand - CCK-BR interactions. Mutational analysis of the receptor, carried out in our laboratory, suggests he existence of a ligand binding pocket comprised of transmembrane domain residues. Non-peptide benzodiazepine-based ligands appear to occupy this putative pocket . Minor structural modifications either of these compounds or of the amino acids which comprise the receptor pocket , can influence whether these ligands act as agonists or antagonists. In addition to these findings with non-peptides, we have obtained evidence that affinity for the endogenous peptide agonist, gastrin, is conferred by an interaction between trans-membrane and extracellular domain amino acids. In the current application, we propose to further explore the molecular determinants of ligand binding to the CCK-BR, and of ligand-induced receptor activation, comparing peptide and non-peptide compounds. The relative roles of both the extracellular and transmembrane domains will be explored. Specific Aim 1 of this grant is directed toward defining CCK-BR amino acids which confer ligand affinity. Specific Aim 2 addresses which CCK-BR -ligand interactions influence the ability of the ligand to induce second messenger signaling. Specific Aim 3 will exploit the recent discoveries in our laboratory of a constitutively active CCK-BR together with a compound that functions as an inverse agonist and as such attenuates ligand-independent signaling. These novel tools will be utilized to explore the receptor-ligand interactions which result in inverse agonism. A combination of molecular (generation of chimeric and mutant receptors, transient expression of recombinant proteins) and pharmacologic methods (radioligand binding, second messenger signaling assays) will be utilized to address these objectives. The proposed studies of the CCK-BR will establish a framework for understanding how non-peptide ligands mimic the activity of endogenous peptides. This information should expand current knowledge of structure-function relationships of peptide hormone receptors in general and may therefore be useful in developing new therapeutic options for a wide range of diseases mediated by this class of proteins.

Keywords: cholecystokinin, gastrin, hormone receptor, neuropeptide receptor, protein structure function, G protein, chemical model, receptor binding, receptor coupling, receptor expression, second messenger, autoradiography, polymerase chain reaction, protein sequence, site directed mutagenesis, tissue /cell culture

Project start date: 1992-09-30

Project end date: 2005-05-31

3R01DK046767-10S2 (2004): $129866

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STRUCTURE/FUNCTION OF THE CCK-B/GASTRIN RECEPTOR

Alan S Kopin
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533

Grant 3R01DK046767-09S1 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: GMA

Abstract: The cholecystokinin-B/gastrin receptor (CCK-BR) is a seven transmembrane domain peptide hormone receptor. Among its most important physiologic functions, the CCK-BR modulates acid secretion and mucosal proliferation in the stomach as well as anxiety and pain perception in the central nervous system. The potential clinical relevance of this receptor has generated considerable interest in understanding the molecular basis of ligand - CCK-BR interactions. Mutational analysis of the receptor, carried out in our laboratory, suggests he existence of a ligand binding pocket comprised of transmembrane domain residues. Non-peptide benzodiazepine-based ligands appear to occupy this putative pocket . Minor structural modifications either of these compounds or of the amino acids which comprise the receptor pocket , can influence whether these ligands act as agonists or antagonists. In addition to these findings with non-peptides, we have obtained evidence that affinity for the endogenous peptide agonist, gastrin, is conferred by an interaction between trans-membrane and extracellular domain amino acids. In the current application, we propose to further explore the molecular determinants of ligand binding to the CCK-BR, and of ligand-induced receptor activation, comparing peptide and non-peptide compounds. The relative roles of both the extracellular and transmembrane domains will be explored. Specific Aim 1 of this grant is directed toward defining CCK-BR amino acids which confer ligand affinity. Specific Aim 2 addresses which CCK-BR -ligand interactions influence the ability of the ligand to induce second messenger signaling. Specific Aim 3 will exploit the recent discoveries in our laboratory of a constitutively active CCK-BR together with a compound that functions as an inverse agonist and as such attenuates ligand-independent signaling. These novel tools will be utilized to explore the receptor-ligand interactions which result in inverse agonism. A combination of molecular (generation of chimeric and mutant receptors, transient expression of recombinant proteins) and pharmacologic methods (radioligand binding, second messenger signaling assays) will be utilized to address these objectives. The proposed studies of the CCK-BR will establish a framework for understanding how non-peptide ligands mimic the activity of endogenous peptides. This information should expand current knowledge of structure-function relationships of peptide hormone receptors in general and may therefore be useful in developing new therapeutic options for a wide range of diseases mediated by this class of proteins

Keywords: cholecystokinin, gastrin, hormone receptor, neuropeptide receptor, protein structure function G protein, chemical model, receptor binding, receptor coupling, receptor expression, second messenger autoradiography, polymerase chain reaction, protein sequence, site directed mutagenesis, tissue /cell culture

Project start date: 1992-09-30

Project end date: 2003-05-31

3R01DK046767-09S1 (2001): $119655

3R01DK046767-08S1 (2000): $66463

STRUCTURE/FUNCTION RELATIONSHIPS OF THE CCK B RECEPTOR

Alan S Kopin
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533

Grant 3R01DK046767-05S1 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: NPNC

Project start date: 1992-09-30

Project end date: 1998-05-31

3R01DK046767-05S1 (1998): $141808

BIOSYNTHESIS OF SECRETIN

Alan S Kopin
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533

Grant 5K08DK001934-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: DDK

Abstract: The principal investigator´s training and research interests relate to the functions and regulation of the gastrointestinal endocrine system. is completing fellowship training in gastroenterology and is committed to a career of laboratory investigation in academic gastroenterology. The long term goal of this proposal is to elucidate the biological role of secretin, a gastrointestinal hormone which regulates pancreatic secretion. More specifically, the aims of this proposal are to define the secretin precursor protein, to determine the tissue distribution of secretin gene expression, and to determine how secretin gene expression is regulated. To accomplish these aims, full length cDNA´s encoding porcine and rat secretin will be cloned. From the nucleotide sequence of the cDNA´s encoding porcine and rat secretin will be cloned. From the nucleotide sequence of the cDNAs, the amino acid sequence of the secretin precursor will be deduced. From the sequence of the precursor peptide, it will be possible to predict all of the potential peptides that can be generated by post-translational cleavage. Probes generated from the cDNA, will be used to establish the tissues in which the secretin gene is expressed. In particular, these studies will address whether secretin is produced in brain. By generating a map of the secretin producing cells in the CNS using in situ hybridization, the function of secretin in the brain may be clarified. Secretin producing cells will be characterized further by combining in situ hybridization with immunocytochemistry to see if other peptides are coexpressed with secretin. The cDNA probes will be used to study the regulation of secretin mRNA levels by different signal transduction pathways in a secretin producing cell line. Finally, the role of established secretin secretagogues in regulation of secretin mRNA levels will be studied in vivo in rats. These studies will be carried out in the of the Division of Gastroenterology in space adjacent to Dr. Letter´s lab. The Division of Molecular Medicine is located one floor below. The expertise available in both the sponsor´s and cosponsor´s laboratories includes extensive experience in the molecular biology of gastrointestinal peptide hormones as well as familiarity with all the methodology proposed in this application. This will provide an ideal environment to facilitate ´s transition to independent investigator

Keywords: gene expression, hormone biosynthesis, protein structure function, secretin CNS neoplasm, biological signal transduction, genetic regulation, immunocytochemistry, messenger RNA, molecular cloning, nucleic acid probe, nucleic acid sequence, protein sequence in situ hybridization, laboratory rat, tissue /cell culture

Project start date: 1989-09-30

Project end date: 1994-08-31

5K08DK001934-05 (1993): $85666

STRUCTURE/FUNCTION OF THE CCK-B/GASTRIN RECEPTOR

Alan S Kopin
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533

Grant 2R01DK046767-06A1 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: GMA

Abstract: The cholecystokinin-B/gastrin receptor (CCK-BR) is a seven transmembrane domain peptide hormone receptor. Among its most important physiologic functions, the CCK-BR modulates acid secretion and mucosal proliferation in the stomach as well as anxiety and pain perception in the central nervous system. The potential clinical relevance of this receptor has generated considerable interest in understanding the molecular basis of ligand - CCK-BR interactions. Mutational analysis of the receptor, carried out in our laboratory, suggests he existence of a ligand binding pocket comprised of transmembrane domain residues. Non-peptide benzodiazepine-based ligands appear to occupy this putative pocket . Minor structural modifications either of these compounds or of the amino acids which comprise the receptor pocket , can influence whether these ligands act as agonists or antagonists. In addition to these findings with non-peptides, we have obtained evidence that affinity for the endogenous peptide agonist, gastrin, is conferred by an interaction between trans-membrane and extracellular domain amino acids. In the current application, we propose to further explore the molecular determinants of ligand binding to the CCK-BR, and of ligand-induced receptor activation, comparing peptide and non-peptide compounds. The relative roles of both the extracellular and transmembrane domains will be explored. Specific Aim 1 of this grant is directed toward defining CCK-BR amino acids which confer ligand affinity. Specific Aim 2 addresses which CCK-BR -ligand interactions influence the ability of the ligand to induce second messenger signaling. Specific Aim 3 will exploit the recent discoveries in our laboratory of a constitutively active CCK-BR together with a compound that functions as an inverse agonist and as such attenuates ligand-independent signaling. These novel tools will be utilized to explore the receptor-ligand interactions which result in inverse agonism. A combination of molecular (generation of chimeric and mutant receptors, transient expression of recombinant proteins) and pharmacologic methods (radioligand binding, second messenger signaling assays) will be utilized to address these objectives. The proposed studies of the CCK-BR will establish a framework for understanding how non-peptide ligands mimic the activity of endogenous peptides. This information should expand current knowledge of structure-function relationships of peptide hormone receptors in general and may therefore be useful in developing new therapeutic options for a wide range of diseases mediated by this class of proteins

Keywords: cholecystokinin, gastrin, hormone receptor, neuropeptide receptor, protein structure /function G protein, chemical model, receptor binding, receptor coupling, receptor expression, second messenger autoradiography, polymerase chain reaction, protein sequence, site directed mutagenesis, tissue /cell culture

Project start date: 1992-09-30

Project end date: 2003-05-31

2R01DK046767-06A1 (1998): $234676

STRUCTURE/FUNCTION OF THE CCK-B/GASTRIN RECEPTOR

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DK046767-10 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: GMA

Abstract: The cholecystokinin-B/gastrin receptor (CCK-BR) is a seven transmembrane domain peptide hormone receptor. Among its most important physiologic functions, the CCK-BR modulates acid secretion and mucosal proliferation in the stomach as well as anxiety and pain perception in the central nervous system. The potential clinical relevance of this receptor has generated considerable interest in understanding the molecular basis of ligand - CCK-BR interactions. Mutational analysis of the receptor, carried out in our laboratory, suggests he existence of a ligand binding pocket comprised of transmembrane domain residues. Non-peptide benzodiazepine-based ligands appear to occupy this putative pocket . Minor structural modifications either of these compounds or of the amino acids which comprise the receptor pocket , can influence whether these ligands act as agonists or antagonists. In addition to these findings with non-peptides, we have obtained evidence that affinity for the endogenous peptide agonist, gastrin, is conferred by an interaction between trans-membrane and extracellular domain amino acids. In the current application, we propose to further explore the molecular determinants of ligand binding to the CCK-BR, and of ligand-induced receptor activation, comparing peptide and non-peptide compounds. The relative roles of both the extracellular and transmembrane domains will be explored. Specific Aim 1 of this grant is directed toward defining CCK-BR amino acids which confer ligand affinity. Specific Aim 2 addresses which CCK-BR -ligand interactions influence the ability of the ligand to induce second messenger signaling. Specific Aim 3 will exploit the recent discoveries in our laboratory of a constitutively active CCK-BR together with a compound that functions as an inverse agonist and as such attenuates ligand-independent signaling. These novel tools will be utilized to explore the receptor-ligand interactions which result in inverse agonism. A combination of molecular (generation of chimeric and mutant receptors, transient expression of recombinant proteins) and pharmacologic methods (radioligand binding, second messenger signaling assays) will be utilized to address these objectives. The proposed studies of the CCK-BR will establish a framework for understanding how non-peptide ligands mimic the activity of endogenous peptides. This information should expand current knowledge of structure-function relationships of peptide hormone receptors in general and may therefore be useful in developing new therapeutic options for a wide range of diseases mediated by this class of proteins.

Keywords: cholecystokinin, gastrin, hormone receptor, neuropeptide receptor, protein structure function, G protein, chemical model, receptor binding, receptor coupling, receptor expression, second messenger, autoradiography, polymerase chain reaction, protein sequence, site directed mutagenesis, tissue /cell culture

Project start date: 1992-09-30

Project end date: 2003-12-31

5R01DK046767-10 (2002): $285656

5R01DK046767-09 (2001): $277337

5R01DK046767-08 (2000): $269262

5R01DK046767-07 (1999): $261971

STRUCTURE/FUNCTION RELATIONSHIPS OF THE CCK B RECEPTOR

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DK046767-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: NPNC

Abstract: The cholecystokinin (CCK) type B receptor in brain plays an important role in modulating anxiety and panic attacks. CCK-B receptor antagonists have been shown to block the neuronal response to CCK and have been proposed as a potentially efficacious new class of anti-anxiety medication. Isolation of a cloned CCK-B receptor and expression in a heterologous cell line, would expedite the development of this new class of drugs. We have isolated a cDNA encoding the first cloned member of the gastrin/CCK receptor family and have evidence that our receptor is highly homologous to the brain CCK-B receptor. Understanding the link between structure and function of the CCK-B receptor will allow more rational design of receptor antagonists. Determination of whether the "CCK-B receptor" (like the biogenic amine receptors) represents a family of related receptors, each with characteristic pharmacology, rather than a single receptor, would allow development of drugs targeted to a specific receptor subtype in brain. We have brought together a consortium of individuals with expertise in molecular biology, pharmacology, protein chemistry, and receptor characterization to undertake the isolation, expression, and characterization of the CCK-B and other related brain receptors. Collectively this group is experienced in all aspects of the proposed project and have been working together for the past year on the cloning and characterization of the gastrin receptor (PNAS, 1992, in press). The first objective is to isolate the CCK-B and related receptors from brain by low stringency hybridization and PCR with degenerate oligonucleotides. The CCK-B and related receptors will ten be pharmacologically characterized to determine ligand binding specificity and the second messenger pathways which couple to these receptors. The ligand binding sites will be mapped with photoaffinity labels which we have previously used in characterizing other members of this receptor family as well as with a series of new intrinsic photoaffinity probes which we will develop specifically for the CCK-B receptor.

Keywords: cholecystokinin, neuropeptide receptor, protein structure function, G protein, brain cell, chimeric protein, gene deletion mutation, neuropharmacology, oligonucleotide, receptor binding, receptor coupling, receptor expression, second messenger, affinity labeling, complementary DNA, human genetic material tag, human tissue, laboratory rabbit, molecular cloning, neoplastic cell culture for noncancer research, nucleic acid hybridization, polymerase chain reaction, site directed mutagenesis

Project start date: 1992-09-30

Project end date: 1997-09-29

5R01DK046767-03 (1994): $196663

STRUCTURE-FUNCTION RELATIONSHIPS OF THE CCK-B RECEPTOR

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DK046767-02 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: NPNC

Abstract: The cholecystokinin (CCK) type B receptor in brain plays an important role in modulating anxiety and panic attacks. CCK-B receptor antagonists have been shown to block the neuronal response to CCK and have been proposed as a potentially efficacious new class of anti-anxiety medication. Isolation of a cloned CCK-B receptor and expression in a heterologous cell line, would expedite the development of this new class of drugs. We have isolated a cDNA encoding the first cloned member of the gastrin/CCK receptor family and have evidence that our receptor is highly homologous to the brain CCK-B receptor. Understanding the link between structure and function of the CCK-B receptor will allow more rational design of receptor antagonists. Determination of whether the "CCK-B receptor" (like the biogenic amine receptors) represents a family of related receptors, each with characteristic pharmacology, rather than a single receptor, would allow development of drugs targeted to a specific receptor subtype in brain. We have brought together a consortium of individuals with expertise in molecular biology, pharmacology, protein chemistry, and receptor characterization to undertake the isolation, expression, and characterization of the CCK-B and other related brain receptors. Collectively this group is experienced in all aspects of the proposed project and have been working together for the past year on the cloning and characterization of the gastrin receptor (PNAS, 1992, in press). The first objective is to isolate the CCK-B and related receptors from brain by low stringency hybridization and PCR with degenerate oligonucleotides. The CCK-B and related receptors will ten be pharmacologically characterized to determine ligand binding specificity and the second messenger pathways which couple to these receptors. The ligand binding sites will be mapped with photoaffinity labels which we have previously used in characterizing other members of this receptor family as well as with a series of new intrinsic photoaffinity probes which we will develop specifically for the CCK-B receptor.

Keywords: cholecystokinin, neuropeptide receptor, protein structure function, G protein, brain cell, chimeric protein, gene deletion mutation, neuropharmacology, oligonucleotide, receptor binding, receptor coupling, receptor expression, second messenger, affinity labeling, complementary DNA, human genetic material tag, human tissue, laboratory rabbit, molecular cloning, neoplastic cell culture for noncancer research, nucleic acid hybridization, polymerase chain reaction, site directed mutagenesis

Project start date: 1992-09-30

Project end date: 1997-09-29

5R01DK046767-02 (1993): $142563

STRUCTURE/FUNCTION RELATIONSHIPS OF THE CCK B RECEPTOR

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DK046767-04 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: NPNC

Abstract: The cholecystokinin (CCK) type B receptor in brain plays an important role in modulating anxiety and panic attacks. CCK-B receptor antagonists have been shown to block the neuronal response to CCK and have been proposed as a potentially efficacious new class of anti-anxiety medication. Isolation of a cloned CCK-B receptor and expression in a heterologous cell line, would expedite the development of this new class of drugs. We have isolated a cDNA encoding the first cloned member of the gastrin/CCK receptor family and have evidence that our receptor is highly homologous to the brain CCK-B receptor. Understanding the link between structure and function of the CCK-B receptor will allow more rational design of receptor antagonists. Determination of whether the "CCK-B receptor" (like the biogenic amine receptors) represents a family of related receptors, each with characteristic pharmacology, rather than a single receptor, would allow development of drugs targeted to a specific receptor subtype in brain. We have brought together a consortium of individuals with expertise in molecular biology, pharmacology, protein chemistry, and receptor characterization to undertake the isolation, expression, and characterization of the CCK-B and other related brain receptors. Collectively this group is experienced in all aspects of the proposed project and have been working together for the past year on the cloning and characterization of the gastrin receptor (PNAS, 1992, in press). The first objective is to isolate the CCK-B and related receptors from brain by low stringency hybridization and PCR with degenerate oligonucleotides. The CCK-B and related receptors will ten be pharmacologically characterized to determine ligand binding specificity and the second messenger pathways which couple to these receptors. The ligand binding sites will be mapped with photoaffinity labels which we have previously used in characterizing other members of this receptor family as well as with a series of new intrinsic photoaffinity probes which we will develop specifically for the CCK-B receptor.

Project start date: 1992-09-30

Project end date: 1997-09-29

5R01DK046767-04 (1995): $204528

Evaluation Dopamine Receptors In Parkinson s

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R21NS043692-02 from National Institute Of Neurological Disorders And Stroke, IRG: ZRG1

Abstract: Parkinson s disease (PD) results from the degeneration of nigrostriatal dopaminergic neurons. This process ultimately leads to a progressive decrease in dopamine mediated striatal signaling which manifests as a clinical syndrome characterized by bradykinesia, rigidity, tremor, and gait abnormalities. Traditional therapy for PD has aimed at restoring dopamine levels in the striatum through administration of the dopamine precursor, L-dopa. With advanced disease, L-dopa leads to dyskinesias and periods of marked fluctuation in motor activity ( on-off effect ). Alleviation of these side effects has been a major challenge and has prompted a search for alternative strategies which can provide a more stable level of dopaminergic signaling. A previously unexplored option to restore striatal dopaminergic activity and at the same time to potentially avoid the consequences of long term L-dopa administration, is through the introduction of constitutively active dopamine receptors. The laboratory of the PI has extensive experience in generating receptors with ligand independent (or constitutive) activity through the introduction of activating point mutations. These receptors have the potential to maintain dopaminergic signaling even in the absence of dopamine and/or dopaminergic agonist drugs. The premise of this application is that constitutively active dopamine receptors can be identified using in vitro assays and expressed in the striatum of rats to enhance dopaminergic signaling over an extended time interval. The objective of Specific Aim 1 is to generate and pharmacologically characterize in vitro a series of constitutively active dopamine 1 and dopamine 2 receptors. Using recombinant adeno-associated virus, the functional consequences of striatal overexpression of constitutively active dopamine receptors will be explored in rats (Specific Aim 2). Circling behavior after unilateral viral administration will be used as an index of construct activity. The methodologies utilized will include molecular (generation of constitutively active mutant receptors, expression of recombinant proteins), pharmacologic (radioligand binding, second messenger signaling assays), and behavioral approaches (assessment of circling behavior). These experiments will provide additional insight into the role of dopaminergic receptors in the striatum as well as potentially take the first steps toward the development of a new therapeutic option for Parkinson s disease.

Keywords: Parkinson s disease, biological signal transduction, brain disorder chemotherapy, dopamine receptor, nonhuman therapy evaluation, receptor expression, adeno associated virus group, corpus striatum, mutant, protein structure function, recombinant protein, recombinant virus, second messenger, substantia nigra, behavior test, laboratory rat

Project start date: 2003-01-01

Project end date: 2004-12-31

5R21NS043692-02 (2004): $153900

Evaluation Of Dopamine Receptors For Parkinson s Disease

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 1R21NS043692-01A1 from National Institute Of Neurological Disorders And Stroke, IRG: ZRG1

Abstract: Parkinson s disease (PD) results from the degeneration of nigrostriatal dopaminergic neurons. This process ultimately leads to a progressive decrease in dopamine mediated striatal signaling which manifests as a clinical syndrome characterized by bradykinesia, rigidity, tremor, and gait abnormalities. Traditional therapy for PD has aimed at restoring dopamine levels in the striatum through administration of the dopamine precursor, L-dopa. With advanced disease, L-dopa leads to dyskinesias and periods of marked fluctuation in motor activity ( on-off effect ). Alleviation of these side effects has been a major challenge and has prompted a search for alternative strategies which can provide a more stable level of dopaminergic signaling. A previously unexplored option to restore striatal dopaminergic activity and at the same time to potentially avoid the consequences of long term L-dopa administration, is through the introduction of constitutively active dopamine receptors. The laboratory of the PI has extensive experience in generating receptors with ligand independent (or constitutive) activity through the introduction of activating point mutations. These receptors have the potential to maintain dopaminergic signaling even in the absence of dopamine and/or dopaminergic agonist drugs. The premise of this application is that constitutively active dopamine receptors can be identified using in vitro assays and expressed in the striatum of rats to enhance dopaminergic signaling over an extended time interval. The objective of Specific Aim 1 is to generate and pharmacologically characterize in vitro a series of constitutively active dopamine 1 and dopamine 2 receptors. Using recombinant adeno-associated virus, the functional consequences of striatal overexpression of constitutively active dopamine receptors will be explored in rats (Specific Aim 2). Circling behavior after unilateral viral administration will be used as an index of construct activity. The methodologies utilized will include molecular (generation of constitutively active mutant receptors, expression of recombinant proteins), pharmacologic (radioligand binding, second messenger signaling assays), and behavioral approaches (assessment of circling behavior). These experiments will provide additional insight into the role of dopaminergic receptors in the striatum as well as potentially take the first steps toward the development of a new therapeutic option for Parkinson s disease.

Keywords: Parkinson s disease, biological signal transduction, brain disorder chemotherapy, dopamine receptor, nonhuman therapy evaluation, receptor expression, adeno associated virus group, corpus striatum, mutant, protein structure function, recombinant protein, recombinant virus, second messenger, substantia nigra, behavior test, laboratory rat

Project start date: 2003-01-01

Project end date: 2004-12-31

1R21NS043692-01A1 (2003): $153900

GPCR Variants As Genetic Determinants Of Obesity

Alan S Kopin
Tufts Medical Center

Grant 5R01DK072497-04 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZDK1

Abstract: More than 26 percent of the U.S. population has a body mass index (BMI) in excess of 30 and thus qualifies as obese. Although heritability is a well-established factor in the development of obesity, the genes underlying this tendency have been difficult to identify. One of the few monogenic forms of obesity identified to date results from mutations GPCR, MC4R. In light of this precedent and the need to define more common explanations underlying genetic susceptibility to obesity, it is of particular note that pharmacologic and/or genetic evidence has implicated more than twenty GPCRs as modulators of food intake, body weight and/or the hedonic (i.e., rewarding) response to feeding. The vast majority of these receptors have multiple known non-synonymous (i.e., changes in coding sequence) variants. By analyzing the prevalence, pharmacologic function, and co-existence of GPCR polymorphisms in the Look AHEAD population we will test our central hypothesis that a significant portion of obesity in the general population is linked with abnormal GPCR function. We will first define the MC4R variants (known and novel) in the Look AHEAD population by sequence and haplotype analysis. Aim 1 will provide insight into the role of this receptor in a large cohort of obese diabetic subjects as well as address the controversy surrounding the extent to which a known common variant (VI031) is protective against obesity. In addition, study of the MC4R will establish a baseline on which to explore the importance of other factors (e.g., GPCR polymorphisms) as genetic determinants of body weight. Furthermore, the analysis of the MC4R will enable other Look AHEAD investigators to take this variable into account as a potentially confounding factor in defining clinical susceptibilities. In Aim 2, we will genotype non-synonymous coding region polymorphisms and haplotype markers in a series of twenty orexigenic, anorexigenic and hedonic GPCRs postulated to have an etiologic role in the development of obesity. These receptor variants have been identified from the NCBI SNP database and from the literature. In parallel, corresponding mutant recombinant GPCRs will be expressed in heterologous cell lines and pharmacologically assessed. Polymorphic receptors will be classified as gain of function, loss of function, or wild type. The correlation between functional abnormalities and phenotypic parameters will be assessed within the study population. In Aim 3, combinations of anorexigenic, orexigenic and hedonic receptor variants will be assessed as potential synergistic or additive factors underlying the polygenic basis of obesity and/or other Look AHEAD study endpoints

Keywords: cell surface receptor, gene mutation, genetic susceptibility, noninsulin dependent diabetes mellitus, obesity, protein structure function, single nucleotide polymorphism body composition, gene environment interaction, gene interaction, genotype, lifestyle, phenotype, recombinant protein cell line, gene expression profiling, human genetic material tag, human tissue, site directed mutagenesis

Project start date: 2005-09-15

Project end date: 2010-06-30

5R01DK072497-03 (2007): $758848

5R01DK072497-02 (2006): $761976

1R01DK072497-01 (2005): $726255

Genetic Analysis Of Feeding Behavior And Fat Deposition

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DK070155-02 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZDK1

Abstract: Important mechanisms underlying the control of feeding and fat deposition have been conserved between Drosophila melanogaster and human. This validates the fruit fly as a genetic model system for identifying and characterizing novel genes that mediate metabolic processes. In Preliminary Studies, we have demonstrated that serotonin (5-hydroxytryptamine, 5HT) acts as a potent inhibitor of fly feeding behavior through activation of one or more 5HT receptors. These findings provide a direct parallel with mammals, for which it has been established that the 5HT2C receptor is an important mediator of feeding behavior and a well recognized target for the treatment of obesity. Aim 1 of this application proposes to further explore the molecular mechanisms underlying the serotonergic control of feeding in Drosophila, utilizing a combination of existing mutants and RNA interference flies which are currently being generated in our laboratory. These strains will provide tools to investigate the extent to which different 5HT receptor subtypes as well as their tissue specific expression (e.g. CMS vs. intestine) underlie the serotonergic control of feeding. The highly sensitive food intake assay that we have established for assessing 5HT mediated food intake has also been utilized to begin a forward genetic screen (Aim 2). To date, 1315 strains of mutant Drosophila, each carrying a single precisely mapped insertion, have been screened for abnormalities in feeding. Twelve of the most promising candidates have been selected for detailed analysis (Aim 2A). To explore the role of the , candidate gene (and its corresponding mammalian homolog) in modulating feeding and/or fat deposition, a combination of approaches will be used, including biochemical (e.g. fatty acid profiling), histologic (e.g. scanning electron microscopy of fat tissue), and genetic (e.g. transgenic rescue) methodologies. Based on our initial success in identifying candidate genes, we propose to continue the forward genetic screen (Aim 2B). The goal of this subsequent effort is to define modifiers of (i) food intake under basal conditions (ii) food intake in the presence of 5HT (to identify modifiers of feeding within the 5HT and intersecting pathways) and (iii) fat deposition. To enhance the likelihood of finding a phenotype and of identifying a gene with physiologic relevance in humans, we will utilize bioinformatic tools to pre-select insertion strains for screening. The selection criteria will include (i) disruption of the transcription unit, and (ii) existence of a mammalian homolog corresponding to the targeted fly gene. To date, -2,500 insertion bearing strains which meet these criteria have been identified. Each of these strains will be assessed for alterations in feeding behavior and fat deposition. Outliers will be characterized as described above. We anticipate that the proposed studies will identify novel genes relevant to the pathophysiology of human obesity

Keywords: Diptera, behavior, fat, flying, gene, genetics, mutant, Carnivora, Drosophilidae, RNA interference, base, behavioral genetics, bioinformatics, complement, conditioning, fatty acid, food, gene targeting, genetic model, genetic screening, human, intestine, lipid, model, obesity, phenotype, protein, receptor, role, scanning electron microscopy, serotonin, success, tissue, tryptophan

Project start date: 2006-02-01

Project end date: 2010-01-31

5R01DK070155-02 (2007): $356114

1R01DK070155-01A1 (2006): $366750

Molecular Analysis Of Dopamine 2 Like Receptor Function

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DA020415-03 from National Institute On Drug Abuse, IRG: ZRG1

Abstract: Dopamine receptors in the central nervous system regulate locomotor function and as such are important therapeutic targets for neuropathologic conditions including Parkinson s disease. To better understand the processes that determine receptor-mediated function in mammals we will utilize Drosophila as a model organism. Flies are amenable to a unique spectrum of genetic approaches for which advanced research tools (e.g. microarrays, repository of mutant fly lines) are readily available. It is of note that there is a high degree of structural and functional conservation between the Drosophila and mammalian proteins that modulate dopaminergic neurotransmission. Our laboratory has recently extended these known parallels with the cloning and pharmacologic characterization of a Drosophila dopamine 2 like receptor (DD2R), the homolog of a well-established mammalian protein that is a key regulator of locomotor function. In vitro pharmacologic comparison between the fly and human D2 receptors reveals a similar profile of agonist (endogenous amines and selected synthetic ligands) induced signaling. At the same time, a subset of anti-Parkinsonian D2R drugs preferentially binds and activates the human (vs. the fly) receptor homolog. In Aim 1, receptor mutants will be generated in which divergent residues are exchanged between the human D2R and the DD2R. These constructs will be utilized to identify human amino acids that confer functional activity to clinically important agonists. In Aim 2, we propose to define the tissue specific expression and the pharmacologic profile of multiple DD2R splice variants that we have identified. As a rationale for these studies, it is well established that mammalian D2 receptor isoforms have distinct cellular distributions and modulate different functions in vivo. The assessment of DD2R splice variants will rely on a combination of approaches including quantitative PCR to assess relative transcript abundance, confocal microscopy with DD2R antibodies to assess expression profiles, and in vitro cell based assays to assess pharmacologic properties. To examine in vivo function, we have generated trangenic RNA interference (RNAi) flies with reduced expression of the DD2R. Characterization of these flies has revealed a highly significant decrease in locomotor function. In Aim 3, we propose to identify novel genes that are linked to dopaminergic signaling pathways, utilizing two complementary approaches (i) transcriptome analysis of the DD2R RNAi (vs. control) flies, and (ii) screening an existing collection of insertion bearing fly lines for alterations in locomotor activity/dopaminergic signaling. Once identified, these genes will be functionally assessed to determine their potential role in dopaminergic signaling.

Keywords: arthropod genetics, biochemical evolution, biological signal transduction, dopamine receptor, molecular biology, neural transmission, neurogenetics, receptor expression, RNA splicing, biological model, chimeric protein, dopamine agonist, gene mutation, neuropharmacology, protein isoform, psychomotor function, species difference, transposon /insertion element, Drosophilidae, RNA interference, cell line, confocal scanning microscopy, gene expression profiling, human genetic material tag, site directed mutagenesis

Project start date: 2005-09-28

Project end date: 2010-07-31

5R01DA020415-03 (2007): $154553

5R01DA020415-02 (2006): $159170

1R01DA020415-01A2 (2005): $163000

Genetic Analysis Of Feeding Behavior And Fat Deposition

Alan S Kopin
New England Medical Center Hospitals
750 Washington St
boston, Ma 021111533

Grant 5R01DK070155-03 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: ZDK1

Keywords: Diptera, behavior, fat, flying, gene, genetics, mutant Carnivora, Drosophilidae, RNA interference, base, behavioral genetics, bioinformatics, complement, conditioning, fatty acid, food, gene targeting, genetic model, genetic screening, human, intestine, lipid, model, obesity, phenotype, protein, receptor, role, scanning electron microscopy, serotonin, success, tissue, tryptophan

Project start date: 2006-02-01

Project end date: 2010-01-31

STRUCTURE/FUNCTION RELATIONSHIPS OF THE CCK B RECEPTOR

Alan S Kopin
New England Medical Center Hospitals 750 Washington St Boston, Ma 021111533

Grant 5R01DK046767-05 from National Institute Of Diabetes And Digestive And Kidney Diseases, IRG: NPNC

Project start date: 1992-09-30

Project end date: 1998-05-31

5R01DK046767-05 (1996): $212711