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1.
Pharmacogenomics J ; 13(2): 130-6, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22212731

ABSTRACT

A pro-asthmatic culture milieu and ß2-agonist (isoproterenol) were previously shown to regulate the expression of select transcription factors (TFs) within human airway epithelial and smooth muscle cells. This study tests 1116 single-nucleotide polymorphisms (SNPs) across 98 of these TF genes for association with bronchodilator response (BDR) in asthma patients. Genotyping was conducted using the Illumina HumanHap550v3 Beadchip in 403 non-Hispanic White asthmatic children and their parents. SNPs were evaluated for association with BDR using family and population-based analyses. Forty-two SNPs providing P-values <0.1 in both analyses were then genotyped in three adult asthma trials. One SNP 5' of the thyroid hormone receptor-ß gene was associated with BDR in the childhood population and two adult populations (P-value=0.0012). This investigation identified a novel locus for inter-individual variability in BDR and represents a translation of a cellular drug-response study to potential personalization of clinical asthma management.


Subject(s)
Asthma/genetics , Epithelial Cells/metabolism , Myocytes, Smooth Muscle/metabolism , Thyroid Hormone Receptors beta/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Asthma/drug therapy , Asthma/pathology , Biomarkers, Pharmacological/metabolism , Bronchodilator Agents/administration & dosage , Bronchodilator Agents/adverse effects , Child , Child, Preschool , Epithelial Cells/pathology , Female , Gene Expression Regulation/drug effects , Genetic Association Studies , Haplotypes , Humans , Male , Middle Aged , Myocytes, Smooth Muscle/pathology , Polymorphism, Single Nucleotide , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Clin Pharmacol Ther ; 89(3): 366-78, 2011 Mar.
Article in English | MEDLINE | ID: mdl-21289619

ABSTRACT

G-protein-coupled receptors (GPCRs) are the targets for many drugs, and genetic variation in coding and noncoding regions is apparent in many such receptors. In this superfamily, adrenergic receptors (ARs) were among the first in which single-nucleotide polymorphisms (SNPs) were discovered, and studies including in vitro mutagenesis, genetically modified mouse models, human ex vivo and in vitro studies and pharmacogenetic association studies were conducted. The signal transduction in these receptors includes amplification steps, desensitization, crosstalk, and redundancies, enabling potential mitigation of the size of the clinical effect for a single variant in a single gene. Nevertheless, convincing evidence has emerged that several variants have an impact on therapy, with certain caveats as to how the results are to be interpreted. Here we review these results for selected ARs and associated regulatory kinases relative to the pharmacogenomics of ß-blocker treatment for hypertension and heart failure. We emphasize the linking of clinical results to molecular mechanisms, discuss study design limitations, and offer some recommendations for future directions.


Subject(s)
Adrenergic beta-Antagonists/pharmacology , Pharmacogenetics , Receptors, Adrenergic/drug effects , Animals , Genetic Variation , Heart Failure/drug therapy , Heart Failure/genetics , Humans , Hypertension/drug therapy , Hypertension/genetics , Mice , Mutagenesis , Polymorphism, Single Nucleotide , Receptors, Adrenergic/genetics , Signal Transduction/genetics
4.
Arch Mal Coeur Vaiss ; 99(6): 616-20, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16878723

ABSTRACT

Currently it is generally accepted that an individual's genetic makeup can modify the efficacy of drug treatment or the risk of adverse reactions. Although not a new concept, the availability of human genome sequence and rapid genotyping at variable loci in drug targets or metabolizing genes has provided new opportunities for the field termed "pharmacogenetics". Somewhat surprisingly, multiple studies have shown the existence of common variants (polymorphisms) in members of the G-protein coupled receptor superfamily, which constitute around 50% of all the targets of currently prescribed drugs. The beta1-adrenergic receptors (beta1ARs) are interesting candidates for pharmacogenetic studies in two complex cardiovascular disease, heart failure and hypertension, since they mediate the effects of catecholamines in the sympathetic nervous system. These receptors are involved in the progression and treatment (beta-blockers therapy) of both diseases, and have polymorphisms that show altered function or regulation as compared to their allelic counterparts in recombinant expression systems and genetically modified mice. These results have prompted prospective and retrospective clinical studies examining whether polymorphisms of these genes are risk factors, disease modifiers, or predictors of b-blocker response in heart failure and hypertension. To date, it appears that beta1AR variants are very likely one genetic component that defines responsiveness to beta-blockers in heart failure and hypertension. Altogether, results are promising, but discrepancies between studies require resolution before these polymorphisms can be utilized in practice. With the goal of personalizing therapy based on an individual's genetic makeup, additional adequately powered, multiethnic, multi-drug studies will be needed.


Subject(s)
Adrenergic beta-Antagonists/therapeutic use , Heart Failure/drug therapy , Hypertension/drug therapy , Receptors, Adrenergic, beta-1/genetics , Heart Failure/genetics , Humans , Hypertension/genetics , Polymorphism, Genetic , Risk Factors
5.
Pharmacogenomics J ; 6(5): 311-26, 2006.
Article in English | MEDLINE | ID: mdl-16568148

ABSTRACT

Asthma affects approximately 300 million individuals worldwide. Medications comprise a substantial portion of asthma expenditures. Despite the availability of three primary therapeutic classes of medications, there are a significant number of nonresponders to therapy. Available data, as well as previous pharmacogenetic studies, suggest that genetics may contribute as much as 60-80% to the interindividual variability in treatment response. In this methodologic review, after providing a broad overview of the asthma pharmacogenetics literature to date, we describe the application of a novel family-based screening algorithm to the analysis of pharmacogenetic data and highlight our approach to identifying and verifying loci influencing asthma treatment response. This approach seeks to address issues related to multiple comparisons, statistical power, population stratification, and failure to replicate from which previous population-based or case-control pharmacogenetic association studies may suffer. Identification of such replicable loci is the next step towards the goal of 'individualized therapy' for asthma.


Subject(s)
Anti-Asthmatic Agents/therapeutic use , Asthma/drug therapy , Asthma/genetics , Pharmacogenetics , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Agonists/therapeutic use , Algorithms , Animals , Anti-Asthmatic Agents/pharmacology , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Asthma/metabolism , Glucocorticoids/pharmacology , Glucocorticoids/therapeutic use , Humans , Leukotriene Antagonists/pharmacology , Leukotriene Antagonists/therapeutic use , Phenotype , Polymorphism, Single Nucleotide , Practice Guidelines as Topic , Randomized Controlled Trials as Topic , Receptors, Adrenergic, beta/drug effects , Receptors, Adrenergic, beta/genetics , Receptors, Adrenergic, beta/metabolism , Receptors, Corticotropin-Releasing Hormone/drug effects , Receptors, Corticotropin-Releasing Hormone/genetics , Receptors, Corticotropin-Releasing Hormone/metabolism , Treatment Outcome
6.
Biochemistry ; 40(49): 15031-7, 2001 Dec 11.
Article in English | MEDLINE | ID: mdl-11732925

ABSTRACT

alpha(2)-adrenergic receptors (alpha(2)AR) couple to multiple effectors including adenylyl cyclase and phospholipase C. We hypothesized that signaling selectivity to these effectors is dynamically directed by kinase-sensitive domains within the third intracellular loop of the receptor. Substitution of Ala for Ser232, which is in the N-terminal region of this loop in the alpha(2A)AR, resulted in a receptor that was markedly uncoupled ( approximately 82% impairment) from stimulation of inositol phosphate accumulation while the capacity to inhibit adenylyl cyclase remained relatively intact. In S232A alpha(2A)AR transfected cell membranes, agonist-promoted [(35)S]GTPgammaS binding was reduced by approximately 50%. Coexpression of modified G proteins rendered insensitive to pertussis toxin revealed that the S232A receptor was uncoupled from both G(i) and G(o). S232 is a potential PKC phosphorylation site, and whole cell phosphorylation studies showed that the mutant had depressed phosphorylation compared to wild type (1.3- vs 2.1-fold/basal). Consistent with S232 directing coupling to phospholipase C, PMA exposure resulted in approximately 67% desensitization of agonist-promoted inositol phosphate accumulation without significantly affecting inhibition of adenylyl cyclase. The dominant effect of mutation or phosphorylation at this site on inositol phosphate as compared to cAMP signaling was found to most likely be due to the low efficiency of signal transduction via phospholipase C vs adenylyl cyclase. Taken together, these results indicate that S232 acts as a selective, PKC-sensitive, modulator of effector coupling of the alpha(2A)AR to inositol phosphate stimulation. This represents one mechanism by which cells route stimuli directed to multifunctional receptors to selected effectors so as to attain finely targeted signaling.


Subject(s)
Protein Kinase C/metabolism , Receptors, Adrenergic, alpha-2/metabolism , Signal Transduction , Adenylate Cyclase Toxin , Adenylyl Cyclase Inhibitors , Adenylyl Cyclases/metabolism , Adrenergic alpha-Agonists/pharmacology , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , Cyclic AMP/antagonists & inhibitors , Cyclic AMP/metabolism , Epinephrine/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Hemostatics/pharmacology , Humans , Inositol Phosphates/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Pertussis Toxin , Phosphorylation , Receptors, Adrenergic, alpha-2/chemistry , Receptors, Adrenergic, alpha-2/genetics , Sequence Alignment , Thrombin/pharmacology , Virulence Factors, Bordetella/pharmacology
7.
Trends Pharmacol Sci ; 22(9): 471-7, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11543875

ABSTRACT

For each alpha(2)-adrenoceptor subtype (alpha(2A), alpha(2B) and alpha(2C)), sequence variations within the coding region of each gene have been identified in humans. These result in substitutions or deletions of amino acids in the third intracellular loops of each receptor. This article summarizes the genetics and molecular biology of alpha(2)-adrenoceptor polymorphisms, including the consequences of each polymorphism on receptor signaling, as determined in transfected cells. These effects include alterations in G-protein coupling, desensitization and G-protein receptor kinase-mediated phosphorylation. Studies so far provide the mechanistic basis for future studies to investigate genetic risk factors and pharmacogenetics in pathophysiological conditions linked to alpha(2)-adrenoceptor function.


Subject(s)
Molecular Biology , Receptors, Adrenergic, alpha-2/genetics , Base Sequence , Humans , Molecular Sequence Data , Pharmacogenetics , Polymorphism, Genetic
8.
Am J Physiol Lung Cell Mol Physiol ; 281(4): L895-903, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11557593

ABSTRACT

Clearance of edema fluid from the alveolar space can be enhanced by endogenous and exogenous beta-agonists. To selectively delineate the effects of alveolar type II (ATII) cell beta(2)-adrenergic receptors (beta(2)-ARs) on alveolar fluid clearance (AFC), we generated transgenic (TG) mice that overexpressed the human beta(2)-AR under control of the rat surfactant protein C promoter. In situ hybridization showed that transgene expression was consistent with the distribution of ATII cells. TG mice expressed 4.8-fold greater beta(2)-ARs than nontransgenic (NTG) mice (939 +/- 113 vs. 194 +/- 18 fmol/mg protein; P < 0.001). Basal AFC in TG mice was approximately 40% greater than that in untreated NTG mice (15 +/- 1.4 vs. 10.9 +/- 0.6%; P < 0.005) and approached that of NTG mice treated with the beta-agonist formoterol (19.8 +/- 2.2%; P = not significant). Adrenalectomy decreased basal AFC in TG mice to 9.7 +/- 0.5% but had no effect on NTG mice (11.5 +/- 1.0%). Na(+)-K(+)-ATPase alpha(1)-isoform expression was unchanged, whereas alpha(2)-isoform expression was approximately 80% greater in the TG mice. These findings show that beta(2)-AR overexpression can be an effective means to increase AFC in the absence of exogenous agonists and that AFC can be stimulated by activation of beta(2)-ARs specifically expressed on ATII cells.


Subject(s)
Pulmonary Alveoli/metabolism , Pulmonary Edema/metabolism , Pulmonary Edema/physiopathology , Receptors, Adrenergic, beta-2/genetics , Adenylyl Cyclases/metabolism , Animals , Blotting, Southern , Body Fluids/metabolism , Female , GTP-Binding Proteins/metabolism , Gene Expression/physiology , Humans , Male , Mice , Mice, Transgenic , Pulmonary Alveoli/growth & development , RNA, Messenger/analysis , Receptors, Adrenergic, beta-2/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Transgenes/genetics
9.
Eur J Pharmacol ; 421(3): 141-7, 2001 Jun 15.
Article in English | MEDLINE | ID: mdl-11516429

ABSTRACT

beta(2)-adrenoceptors (beta(2)AR) are polymorphic at amino acid 164 (Thr or Ile) of the fourth transmembrane domain. In transfected fibroblasts, six agonists commonly used in the treatment of bronchospasm were studied. Isoproterenol, albuterol, metaproterenol, terbutaline, formoterol, and salmeterol displayed decreased binding affinities (K(i)s were 1.2-3.0-fold higher) and a significant degree of impaired maximal stimulation of adenylyl cyclase ( approximately 40%), was observed with all agonists for the Ile164 receptor. The ratios of signal transduction efficiencies (Tau function, Ile164/Thr164) varied from a low of 0.17 for terbutaline to 0.49 for salmeterol. In addition, Ile164 bound salmeterol at the exosite, as delineated in perfusion washout studies, at a decreased level (31+/-4.8% vs. 49+/-4.4% retained salmeterol, respectively, P=0.02). In cAMP production studies under perfusion conditions, this decreased exosite binding caused a approximately 50% decrease in the duration of action of salmeterol at Ile164 (t(1/2)=21.0+/-3.6 vs. 46.8+/-4.1 min for Thr164, P=0.001). The durations of action for isoproterenol and formoterol under similar perfusion conditions were not different between the two receptors. These in vitro results indicate the Ile164 polymorphic receptor represents a pharmacogenetic locus for the most commonly utilized agonists in the treatment of asthma with a unique phenotype for salmeterol.


Subject(s)
Adrenergic beta-Agonists/metabolism , Albuterol/analogs & derivatives , Albuterol/metabolism , Pindolol/analogs & derivatives , Receptors, Adrenergic, beta-2/metabolism , Adrenergic beta-Agonists/pharmacology , Albuterol/pharmacology , Amino Acid Substitution , Animals , Binding Sites , Binding, Competitive/drug effects , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Genotype , Humans , Iodine Radioisotopes , Isoproterenol/metabolism , Isoproterenol/pharmacology , Metaproterenol/metabolism , Metaproterenol/pharmacology , Pindolol/metabolism , Polymorphism, Genetic , Radioligand Assay , Receptors, Adrenergic, beta-2/genetics , Salmeterol Xinafoate , Terbutaline/metabolism , Terbutaline/pharmacology
10.
J Biol Chem ; 276(34): 31596-601, 2001 Aug 24.
Article in English | MEDLINE | ID: mdl-11402033

ABSTRACT

Depressed G-protein-coupled receptor (GPCR) signaling has been implicated as a component of the pathophysiology of a number of complex diseases including heart failure and asthma, and augmentation or restoration of signaling by various means has been shown to improve organ function. Because some properties of native GPCRs are disadvantageous for ectopic therapeutic expression, we utilized the beta(2)-adrenergic receptor (beta(2)AR) as a scaffold to construct a highly modified therapeutic receptor-effector complex (TREC) suitable for gene therapy. Altogether, 19 modifications were made to the receptor. The ligand-binding site was re-engineered in TM-3 so that a beta-hydroxylmethyl side chain acts as a proton donor for the binding of a novel ligand. In addition, sites critical for agonist-promoted down-regulation in the amino terminus and for phosphorylation by GPCR kinases, and protein kinases A and C, in the third intracellular loop and the carboxyl terminus of the receptor were altered. These modifications of the receptor resulted in depressed agonist-stimulated adenylyl cyclase activity (26.8 +/- 2.1 versus 41.4 +/- 8 pmol/min/mg for wild-type beta(2)AR). This was fully restored by fusing the carboxyl terminus of the modified receptor to G alpha(s) (43.3 +/- 2.7 pmol/min/mg). The fully modified fused receptor was not activated by beta-agonists but rather by a nonbiogenic amine agonist that itself failed to activate the wild-type beta(2)AR. This two-way selectivity thus provides targeted activation based on physiologic status. Furthermore, the TREC did not display tachyphylaxis to prolonged agonist exposure (desensitization was 1 +/- 5% versus 55 +/- 4% for wild-type beta(2)AR). Thus, despite extensive alterations in regions of conformational lability, the beta(2)AR can be tailored to have optimal signaling characteristics for gene therapy. As a general paradigm, TRECs for enhancement of other G-protein signaling appear to be feasible for modification of other pathologic states.


Subject(s)
Genetic Therapy , Receptors, Adrenergic, beta-2/metabolism , Animals , Cricetinae , Cricetulus , Genetic Engineering , Humans , Phosphorylation , Radioligand Assay , Receptors, Adrenergic, beta-2/genetics
12.
Int Arch Allergy Immunol ; 124(1-3): 183-6, 2001.
Article in English | MEDLINE | ID: mdl-11306963

ABSTRACT

BACKGROUND: Regular use of inhaled beta-adrenergic agonists may have adverse effects in some asthma patients. Polymorphisms of the beta(2)-adrenergic receptor (beta(2)-AR) can affect its regulation; however, results of smaller studies of the effects of such polymorphisms on response to beta-agonist therapy have been inconsistent. METHODS: We examined the possible effects of polymorphisms at codons 16 (beta(2)-AR-16) and 27 (beta(2)-AR-27) on response to albuterol by genotyping 190 asthmatics who had participated in a trial of regular versus as-needed albuterol use. RESULTS: During the 16-week treatment period, patients homozygous for arginine (Arg/Arg) at beta(2)-AR-16 who used albuterol regularly had a small decline in morning peak expiratory flow (AM PEF). This effect was magnified during a 4-week run-out period, when all patients returned to as-needed albuterol only. By the end of the study, Arg/Arg subjects who had used albuterol regularly had an AM PEF 30.5 +/- 12.1 liters/min lower (p = 0.012) than Arg/Arg patients who had used albuterol as needed only. Subjects homozygous for glycine at beta(2)-AR-16 showed no such decline. Evening PEF also declined in the Arg/Arg regular but not in as-need albuterol users. No significant differences between regular and as-needed treatment were associated with polymorphisms at beta(2)-AR-27. CONCLUSIONS: Polymorphisms of the beta(2)-AR may influence airway responses to regular inhaled beta-agonist treatment.


Subject(s)
Adrenergic beta-Agonists/therapeutic use , Albuterol/therapeutic use , Asthma/drug therapy , Asthma/genetics , Polymorphism, Genetic , Receptors, Adrenergic, beta-2/genetics , Adolescent , Adult , Child , Cohort Studies , Female , Genotype , Humans , Male , Peak Expiratory Flow Rate/drug effects , Time Factors
14.
Circulation ; 103(6): 889-96, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11171800

ABSTRACT

BACKGROUND: Relieving the inhibition of sarcoplasmic reticular function by phospholamban is a major target of beta-adrenergic stimulation. Chronic beta-adrenergic receptor activity has been suggested to be detrimental, on the basis of transgenic overexpression of the receptor or its signaling effectors. However, it is not known whether physiological levels of sympathetic tone, in the absence of preexisting heart failure, are similarly detrimental. METHODS AND RESULTS: Transgenic mice overexpressing phospholamban at 4-fold normal levels were generated, and at 3 months, they exhibited mildly depressed ventricular contractility without heart failure. As expected, transgenic cardiomyocyte mechanics and calcium kinetics were depressed, but isoproterenol reversed the inhibitory effects of phospholamban on these parameters. In vivo cardiac function was substantially depressed by propranolol administration, suggesting enhanced sympathetic tone. Indeed, plasma norepinephrine levels and the phosphorylation status of phospholamban were elevated, reflecting increased adrenergic drive in transgenic hearts. On aging, the chronic enhancement of adrenergic tone was associated with a desensitization of adenylyl cyclase (which intensified the inhibitory effects of phospholamban), the development of overt heart failure, and a premature mortality. CONCLUSIONS: The unique interaction between phospholamban and increased adrenergic drive, elucidated herein, provides the first evidence that compensatory increases in catecholamine stimulation can, even in the absence of preexisting heart failure, be a primary causative factor in the development of cardiomyopathy and early mortality.


Subject(s)
Aging , Calcium-Binding Proteins/metabolism , Cardiomyopathies/etiology , Receptors, Adrenergic, beta/metabolism , Adenylyl Cyclases/metabolism , Adrenergic beta-Antagonists/pharmacology , Animals , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Cardiomyopathies/blood , Cardiomyopathies/mortality , Echocardiography , Heart Failure/etiology , Heart Failure/mortality , Isoproterenol/pharmacology , Mice , Mice, Transgenic , Myocardium/metabolism , Myocardium/pathology , Norepinephrine/blood , Phosphorylation , Propranolol/pharmacology , Ventricular Function, Left
15.
J Biol Chem ; 276(7): 4917-22, 2001 Feb 16.
Article in English | MEDLINE | ID: mdl-11056163

ABSTRACT

A polymorphic variant of the human alpha(2B)-adrenergic receptor (alpha(2B)AR), which consists of a deletion of three glutamic acids (residues 301-303) in the third intracellular loop was found to be common in Caucasians (31%) and to a lesser extent in African-Americans (12%). The consequences of this deletion were assessed by expressing wild-type and the Del301-303 receptors in Chinese hamster ovary and COS cells. Ligand binding was not affected, although a small decrease in coupling efficiency to the inhibition of adenylyl cyclase was observed with the mutant. The deletion occurs within a stretch of acidic residues that is thought to establish the milieu for agonist-promoted phosphorylation and desensitization of the receptor by G protein-coupled receptor kinases (GRKs). Agonist-promoted phosphorylation studies carried out in cells coexpressing the alpha(2B)ARs and GRK2 revealed that the Del301-303 receptor displayed approximately 56% of wild-type phosphorylation. Furthermore, the depressed phosphorylation imposed by the deletion was found to result in a complete loss of short term agonist-promoted receptor desensitization. Thus the major phenotype of the Del301-303 alpha(2B)AR is one of impaired phosphorylation and desensitization by GRKs, and thus the polymorphisms renders the receptor incapable of modulation by this key mechanism of dynamic regulation.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/physiology , Polymorphism, Genetic , Receptors, Adrenergic, alpha-2/genetics , Receptors, Adrenergic, alpha-2/metabolism , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , CHO Cells , COS Cells , Cricetinae , Down-Regulation , Gene Frequency , Molecular Sequence Data , Norepinephrine/pharmacology , Phosphorylation , Sequence Deletion , Transfection , beta-Adrenergic Receptor Kinases
17.
Pharmacology ; 61(3): 167-73, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10971202

ABSTRACT

beta(1)- and beta(2)-adrenergic receptors are G protein-coupled receptors expressed throughout the body and serve as receptors for the catecholamines epinephrine and norepinephrine. They are targets for therapeutive agonists and/or antagonists in treatment of heart failure and asthma. Nonsynonymous coding and promoter polymorphisms of both receptors have been identified in the general population. These have been mimicked in transfected cell systems and transgenic mice, and show altered expression, ligand binding, coupling, or regulation phenotypes. Clinical studies to date have revealed that some of these polymorphisms have a significant disease modifying effect or alter the response to treatment. These are some of the first G protein coupled receptor polymorphisms to undergo extensive in vitro study and clinical validation; there are likely to be polymorphisms of other receptors of the superfamily that will have clinical relevance as well.


Subject(s)
Pharmacogenetics , Receptors, Adrenergic, beta-1/genetics , Receptors, Adrenergic, beta-2/genetics , Animals , Humans , Polymorphism, Genetic/genetics , Receptors, Adrenergic, beta-1/drug effects , Receptors, Adrenergic, beta-2/drug effects
18.
Proc Natl Acad Sci U S A ; 97(19): 10483-8, 2000 Sep 12.
Article in English | MEDLINE | ID: mdl-10984540

ABSTRACT

The human beta(2)-adrenergic receptor gene has multiple single-nucleotide polymorphisms (SNPs), but the relevance of chromosomally phased SNPs (haplotypes) is not known. The phylogeny and the in vitro and in vivo consequences of variations in the 5' upstream and ORF were delineated in a multiethnic reference population and an asthmatic cohort. Thirteen SNPs were found organized into 12 haplotypes out of the theoretically possible 8,192 combinations. Deep divergence in the distribution of some haplotypes was noted in Caucasian, African-American, Asian, and Hispanic-Latino ethnic groups with >20-fold differences among the frequencies of the four major haplotypes. The relevance of the five most common beta(2)-adrenergic receptor haplotype pairs was determined in vivo by assessing the bronchodilator response to beta agonist in asthmatics. Mean responses by haplotype pair varied by >2-fold, and response was significantly related to the haplotype pair (P = 0.007) but not to individual SNPs. Expression vectors representing two of the haplotypes differing at eight of the SNP loci and associated with divergent in vivo responsiveness to agonist were used to transfect HEK293 cells. beta(2)-adrenergic receptor mRNA levels and receptor density in cells transfected with the haplotype associated with the greater physiologic response were approximately 50% greater than those transfected with the lower response haplotype. The results indicate that the unique interactions of multiple SNPs within a haplotype ultimately can affect biologic and therapeutic phenotype and that individual SNPs may have poor predictive power as pharmacogenetic loci.


Subject(s)
Haplotypes , Promoter Regions, Genetic , Receptors, Adrenergic, beta-2/genetics , Base Sequence , Cell Line, Transformed , DNA/genetics , Genotype , Humans , Phylogeny , Polymorphism, Single Nucleotide
19.
J Biol Chem ; 275(49): 38518-23, 2000 Dec 08.
Article in English | MEDLINE | ID: mdl-10948191

ABSTRACT

alpha(2A)-Adrenergic receptors (alpha(2A)AR) are presynaptic autoinhibitory receptors of noradrenergic neurons in the central and peripheral sympathetic nervous systems, which act to dynamically regulate neurotransmitter release. Signaling through the G(i)/G(o) family of G-proteins, the receptor subserves numerous homeostatic and central nervous system functions. A single nucleotide polymorphism of this receptor, which results in an Asn to Lys substitution at amino acid 251 of the third intracellular loop, was identified in the human population. The frequency of Lys-251 was 10-fold greater in African-Americans than in Caucasians, but was not associated with essential hypertension. To determine the consequences of this substitution, wild-type and Lys-251 receptors were expressed in CHO and COS-7 cells. Expression, ligand binding, and basal receptor function were unaffected by the substitution. However, agonist-promoted [(35)S]GTPgammaS binding was approximately 40% greater with the Lys-251 receptor. This enhanced agonist function was observed with catecholamines, azepines, and imidazolines albeit to different degrees. In studies of agonist-promoted functional coupling to G(i), the polymorphic receptor displayed enhanced inhibition of adenylyl cyclase (60 +/- 4. 4 versus 46 +/- 4.1% inhibition) and markedly enhanced stimulation of MAP kinase (57 +/- 9 versus 15- +/- 2-fold increase over basal) compared with wild-type alpha(2A)AR. The potency of epinephrine in stimulating inositol phosphate accumulation was increased approximately 4 fold with the Lys-251 receptor. Unlike previously described variants of G-protein-coupled receptors, where the minor species causes either a loss of function or increased non-agonist function, Lys-251 alpha(2A)AR represents a new class of polymorphism whose phenotype is a gain of agonist-promoted function.


Subject(s)
Asparagine , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Lysine , Polymorphism, Genetic , Receptors, Adrenergic, alpha-2/genetics , Receptors, Adrenergic, alpha-2/metabolism , Adenylyl Cyclase Inhibitors , Adrenergic alpha-2 Receptor Agonists , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , CHO Cells , Cattle , Cell Membrane/physiology , Cricetinae , Epinephrine/pharmacology , Genetic Variation , Guinea Pigs , Humans , Inositol Phosphates/metabolism , Mice , Models, Molecular , Molecular Sequence Data , Oxymetazoline/pharmacology , Protein Structure, Secondary , Rats , Receptors, Adrenergic, alpha-2/chemistry , Recombinant Proteins/agonists , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Swine , Transfection
20.
Am J Physiol Lung Cell Mol Physiol ; 279(2): L379-89, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10926562

ABSTRACT

Airway epithelial cells express beta(2)-adrenergic receptors (beta(2)-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of beta(2)-ARs to airway epithelium of transgenic (CCSP-beta(2)-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that beta(2)-AR density in CCSP-beta(2)-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED(200)) was greater for CCSP-beta(2)-AR than for NTG mice (345 +/- 34 vs. 157 +/- 14 mg/ml; P < 0.01). CCSP-beta(2)-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline (P < 0.05) but remained unchanged in the CCSP-beta(2)-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED(200) for ozone-exposed CCSP-beta(2)-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell beta(2)-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of beta-agonists results from activation of receptors on both epithelial and smooth muscle cells.


Subject(s)
Bronchoconstriction/genetics , Receptors, Adrenergic, beta-2/biosynthesis , Receptors, Adrenergic, beta-2/genetics , Respiratory Mucosa/metabolism , Transgenes/genetics , Uteroglobin , Adrenergic beta-Agonists/pharmacology , Adrenergic beta-Antagonists/pharmacology , Animals , Animals, Genetically Modified/genetics , Bronchoalveolar Lavage Fluid/chemistry , Bronchoconstriction/drug effects , Dinoprostone/analysis , Gene Expression , Humans , Lung/cytology , Lung/metabolism , Mice , Mice, Transgenic , Muscarinic Agonists/pharmacology , Nitric Oxide/analysis , Ozone/pharmacology , Plethysmography, Whole Body , Promoter Regions, Genetic , Proteins/genetics , RNA, Messenger/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Respiratory Mucosa/cytology , Signal Transduction/genetics
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