2AR Gene Research Articles

Prenatal and postnatal polycyclic aromatic hydrocarbon exposure, airway hyperreactivity, and Beta-2 adrenergic receptor function in sensitized mouse offspring.

Despite data associating exposure to traffic-related polycyclic aromatic hydrocarbons (PAH) in asthma, mechanistic support has been limited. We hypothesized that both prenatal and early postnatal exposure to PAH would increase airway hyperreactivity (AHR) and that the resulting AHR may be insensitive to treatment with a β 2AR agonist drug, procaterol. Further, we hypothesized that these exposures would be associated with altered β 2AR gene expression and DNA methylation in mouse lungs. Mice were exposed prenatally or postnatally to a nebulized PAH mixture versus negative control aerosol 5 days a week. Double knockout β 2AR mice were exposed postnatally only. Prenatal exposure to PAH was associated with reduced β 2AR gene expression among nonsensitized mice offspring, but not increases in DNA methylation or AHR. Postnatal exposure to PAH was borderline associated with increased AHR among sensitized wildtype, but not knockout mice. In the first study that delivers PAH aerosols to mice in a relatively physiological manner, small effects on AHR and β 2AR gene expression, but not β 2AR agonist drug activity, were observed. If confirmed, the results may suggest that exposure to PAH, common ambient urban pollutants, affects β 2AR function, although the impact on the efficacy of β 2AR agonist drugs used in treating asthma remains uncertain.

Overexpression of β 2AR improves contractile function and cellular survival in rabbit cardiomyocytes under chronic hypoxia

Chronic hypoxia usually evokes sustained release of endogenous neurohormones, leading to β 2-adrenergic receptor (β 2AR) desensitization and downregulation of expression, which impacts cellular contractility. We investigated whether exogenous β 2AR could compensate for the functional deficiency of β 2AR in rabbit cardiomyocytes under chronic hypoxia, and whether this led to improved contractility and cellular survival. A surgical experimental model of cyanotic heart disease was established in rabbits. Adv.hβ 2AR was transfected into cardiomyocytes isolated from animals subjected to 6-week systemic hypoxia. The levels of cellular contractile function, protein expression of hβ 2AR, p-Akt, p-Erk, and caspase-3, and cellular survival pre- and post-Adv.hβ 2AR delivery were determined. In the cyanotic cells, decreased shortening and lengthening of TPC and R50 were evident. Cellular diastolic functioning showed greater deterioration compared to the systolic function ( P < 0.05). In cyanotic cells, the positive inotropic response to isoproterenol was decreased ( P < 0.01), low levels of cellular survival were found, protein levels of β 2AR, p-Akt, and p-Erk were downregulated, and protein levels of caspase-3 were upregulated. After Adv.hβ 2AR delivery, enhanced contractile function was achieved ( P < 0.01), TPC and R50 levels recovered up to 99% and 81.7% of the normal control levels, respectively ( P < 0.05), and cellular survival improved ( P < 0.01). Our results demonstrate that overexpression of the β 2AR gene in cardiomyocytes exposed to chronic hypoxia provides significant catecholamine-dependent inotropic support and cellular protection.

Characterization of genomic organization of the adenosine A 2A receptor gene by molecular and bioinformatics analyses

The adenosine A 2A receptor (A 2AR) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A 2AR gene expression, we have characterized the genomic organization of the mouse and human A 2AR genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5′ untranslated regions (5′-UTRs) of mouse A 2AR mRNA were identified by rapid amplification of 5′ cDNA end (5′ RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding “exon 1” (h1A, h1B, h1C, h1D, h1E and h1F) in the human A 2AR gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5′ oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A 2AR transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A 2AR gene by 5′ oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A 2AR gene expression. Finally, our analysis indicates that A 2AR mRNA and a novel transcript partially overlapping with the 3′ exon h3, but in opposite orientation to the A 2AR gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A 2AR genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5′-UTR. This raises the possibility of generating multiple tissue-specific A 2AR mRNA species by alternative promoters with varying regulatory susceptibility.

Association between β 2-adrenoceptor polymorphisms and asthma diagnosis among Mexican adults

Background Recent studies demonstrate that genetic variations in the human β 2-adrenergic receptor (β 2AR) structure at codons 16 and 27 alter receptor function in vitro and are associated with asthma severity and airway hyperresponsiveness but have not been linked to asthma diagnosis. The nature of the relation in a more homogeneous population is uncertain. Objective We determined frequencies of these polymorphisms to explore the association between β 2AR haplotypes and asthma diagnosis and phenotype. Methods This is a population-based, case-control study that involves a total sample of 907 unrelated Mexican Mestizos. Genotyping at β 2AR was identified by polymerase chain reaction–restriction fragment length polymorphism analysis. Multivariate logistic regression analysis was used to estimate the odds ratio (OR) of the association between β 2AR haplotype status and asthma diagnosis. Results A significant inverse association was found between subjects with Glu27 allele (OR, 0.5; 95% CI, 0.4 to 0.7) and Gly16-Glu27 alleles (OR, 0.5; 95% CI, 0.3 to 0.8) and asthma. Sex differences in this association were explored, given the complex relation between sex and asthma. Among men, a positive association was present between the “Gly16 allele without Glu27” (OR, 2.9; 95% CI, 1.26 to 6.8) and asthma. In contrast, a lower risk of asthma was found among women Gly16-Glu27 alleles (OR, 0.3; 95% CI, 0.2 to 0.6). Nocturnal asthma was associated with the Gly16 allele (OR, 1.8; 95% CI, 1.3 to 2.6). Conclusions Variation in the β 2AR gene is associated in the pathogenesis of asthma and acts as a disease modifier in nocturnal asthma.

In vivo timing of onset of transgene expression following adenoviral-mediated gene transfer

Recombinant adenoviruses are efficient gene transfer vehicles that could be used for treatment of acute diseases. However, the time required for adenoviruses to produce physiologically relevant levels of transgene in vivo is unknown. To address this question rat lungs were infected with an E1a −/E3a − adenovirus that contains an hCMV-driven human β 2-adrenergic receptor (β 2AR) cDNA. Human β 2AR message and protein expression were noted 2–4 h postinfection without evidence of pseudotransduction. β 2AR function (cAMP production) was increased at 6 h postinfection. To determine when β 2AR gene transfer affects downstream catecholamine-sensitive pathways, we measured lung Na,K-ATPase expression and alveolar fluid clearance (AFC). β 2AR gene transfer increased Na,K-ATPase number by 80% at 6 h, and AFC by 20% at 8 h postinfection. These data indicate that recombinant adenoviruses can produce physiologically significant levels of transgene within hours of infection and that they may be suitable for gene therapies for acute, rapidly progressive diseases.

Adaptive immunity in mice lacking the β 2-adrenergic receptor

The beta-2-adrenergic receptor (β 2AR) is expressed by most lymphocyte populations and binds the sympathetic neurotransmitter norepinephrine (NE). Stimulation of the β 2AR is reported to be the primary mechanism by which signals from the sympathetic nervous system influence both cell-mediated and humoral immunity. We report here that body/organ weights, lymphoid organ cell number/phenotype/histology, the contact sensitivity response, and the amount, avidity, and isotype of antibody resulting from a T cell-dependent antibody response in β 2AR deficient mice (β 2AR−/− mice) were all similar to measures made in β 2AR+/+ mice. Other members of the adrenergic receptor family did not appear to compensate for the absence in β 2AR expression. In contrast, β 2AR−/− B cells cultured in vitro were unable to respond to NE in a manner similar to β 2AR+/+ B cells. Thus, mice in which expression of the β 2AR gene is defective from early development to adulthood may no longer require that NE stimulate the β 2AR to maintain immune homeostasis, and this may be due to a non-adrenergic mechanism that provides compensation in vivo.

Identification of nuclear factor 1 (NF1) as a transcriptional modulator of rat A 2A adenosine receptor

By a combination of PCR and DNA walking technique, we isolated a 4.8-kb DNA fragment containing a 4.3 kb 5′-flanking region and a 0.5-kb 5′-untranslated region of the rat A 2A adenosine receptor (A 2A-R) gene. Various lengths of the 5′-flanking region of the A 2A-R gene were inserted into an expression vector and transfected into several different cell lines for promoter analysis. Our results reveal that a consensus NF1 element (designated as A 2A-R/NF1), located between bases −2846 and −2827 of the A 2A-R gene, functions as a repressor for A 2A-R promoters in the rat brain-derived type-2 astrocyte cell line (RBA2), which expresses no A 2A-R. Electrophoretic gel mobility shift assay (EMSA) revealed that two A 2A-R/NF1–protein complexes of RBA2 nuclear extract were formed. Supershift experiments using an anti-NF1 antibody suggest that NF1 proteins exist in both A 2A-R/NF1–protein complexes. Furthermore, mutations in the conserved NF1 binding site of this A 2A-R/NF1 element disturbed DNA–protein formation. Thus, NF1 proteins appear to mediate this cell line-specific suppression of A 2A-R promoters in RBA2 cells. The importance of NF1 proteins in regulating A 2A-R promoters was further confirmed in another cell line (Siha) which expresses no endogenous A 2A-R. Moreover, addition of the A 2A-R/NF1element upstream of an irrelevant thymidine kinase (TK) promoter suppressed its promoter activity in Siha cells, but not in RBA2 cells. Thus, the NF1-mediated inhibition of the A 2A-R promoter was promoter- and cell line-specific. In summary, we have defined a distal negative element (A 2A-R/NF1) that plays a functional role in modulating the expression of A 2A-R.

Glucocorticoid effects on the β-adrenergic receptor–adenylyl cyclase system of human airway epithelium

Background: Activation of the β 2-adrenergic receptor (β 2AR) system expressed by human airway epithelial cells elicits a variety of cyclic adenosine monophosphate (cAMP)-dependent processes that help determine airway caliber and the intensity of airway inflammation in asthma. Glucocorticoids, mainstays in the treatment of asthma, profoundly affect the expression and function of the β 2-adrenergic receptor-adenylyl cyclase (β 2AR-AC) system in a variety of cell types. However, the effects of glucocorticoids on the β 2AR-AC system expressed by human airway epithelial cells are unstudied. Objective: We examined the effects of dexamethasone (DEX) on β 2AR gene expression and the function of the β 2AR-AC system in cultured human airway epithelial cells. Methods: Studies were performed in normal airway epithelial cells and BEAS-2B cells. β 2AR gene expression was assessed from measurements of β-adrenergic receptor density, β 2AR mRNA, and the activity of a full-length β 2AR promoter-luciferase reporter construct. The function of the β 2AR-AC system was assessed from cAMP production in response to the β 2-agonist isoproterenol and the expression of the stimulatory G protein Gαs. Results: DEX had no effect on β-adrenergic receptor density or on the β 1/β 2 ratio over a wide range of concentrations and exposure times. However, DEX significantly but transiently enhanced β 2AR mRNA levels (~1.5-fold) and β 2AR promoter activity (~1.5-fold), indicating increased β 2AR gene transcription. DEX also dose-dependently enhanced cAMP responses to isoproterenol but not to forskolin, a direct activator of adenylyl cyclase. DEX-induced changes in cAMP production were associated with small (~15%) increases in Gαs expression. Conclusions: These data indicate that glucocorticoids only transiently enhance β 2AR gene transcription and fail to increase steady-state levels of β 2AR protein in human airway epithelial cells. Nonetheless, glucocorticoid-induced effects on the β 2AR-AC system of human airway epithelial cells contribute to the beneficial effects of corticosteroids in asthma by enhancing the functional response to β 2-agonists. (J Allergy Clin Immunol 2002;109:491-7.)

EXPRESSION OF FUNCTIONAL ß2-ADRENERGIC RECEPTORS IN A RAT AIRWAY EPITHELIAL CELL LINE (SPOC1) AND CELL DENSITY-DEPENDENT INDUCTION BY GLUCOCORTICOIDS

The beta 2-adrenergic receptor (beta 2AR) signal transduction system regulates many key functions of airway epithelium. In this study, we have pharmacologically characterized the beta 2AR and determined the impact of glucocorticoids on beta 2AR gene transcription in SPOC1 cells, a continuous cell line derived from the tracheal epithelium of rats. [125I]Cyanoiodopindolol assays demonstrated that binding to SPOC1 cell membranes was saturable (Bmax = 62.6 +/- 6 fmol/mg protein) and of high affinity (Kd = 6.3 +/- 0.8 pM). From competition experiments, the rank order of potency of agonists (isoproterenol > epinephrine >> norepinephrine) and the high affinity (Ki = 0.37 +/- 0.05 nM) of the beta 2-selective antagonist ICI 118,551 suggested the predominance of the beta 2AR subtype. Two isoforms of the alpha subunit of Gs (45 and 52 kDa) were identified by Western blot analysis. Isoproterenol-stimulated cyclic AMP levels increased in a dose-dependent manner, confirming that SPOC1 cell beta 2ARs are functionally coupled to adenylyl cyclase. The effect of glucocorticoids on beta 2AR expression was assessed in radioligand and transient transfection assays. Dexamethasone treatment of SPOC1 cells increased both beta 2AR protein and beta 2AR-luciferase fusion gene expression 1.6- to 3.1-fold, with the greatest increase demonstrated in cells cultured at low density compared to cells grown at high density.

Genetic linkage to the serotonin transporter protein and 5HT 2A receptor genes excluded in generalized social phobia

Social phobia, particularly the generalized form, is strongly familial and frequently comorbid with major depression, panic disorder, and obsessive–compulsive disorder. It has also recently been shown to be responsive to selective serotonin reuptake inhibitors. We conducted a study to determine if generalized social phobia is genetically linked to either of two candidate genes: the serotonin transporter protein (5HTT) gene, or the 5HT 2A receptor (5HT 2AR) gene. Rates of social phobia (using several phenotype definitions) were ascertained and blood samples obtained from consenting first-degree family members of generalized social phobic probands. 5HT 2AR and 5HTT genotyping was performed using the polymerase chain reaction (PCR). Linkage was tested using LINKAGE and GENEHUNTER software. No evidence of linkage was found; power analysis indicated that failure to find linkage was unlikely due to inadequate statistical power. These findings reasonably exclude linkage between generalized social phobia and the 5HTT or 5HT 2AR genes in these samples, although modifier effects cannot be ruled out. Other 5HT receptor subtypes or indirect modulatory effects of 5HT on other neurotransmitter systems may be involved.

Genetic Polymorphisms of the beta2-Adrenergic Receptor in the Severity of Bronchial Asthma

BACKGROUND: Genetic and environmental factors are known to affect the incidence and severity of asthma. Stimulation of beta2-Adrenergic Receptor (beta 2AR) results in smooth muscle relaxation, leading to decrease in resistance of airflow. The gene encoding the beta 2AR has recently been seguenced. The beta 2AR genotype at the polymorphic lociof codons 16, 27, 34, and 164 was known to cause changes in the amino acids. The relationships between the structure of the beta 2AR and its functions are being elucidated. PURPOSE: The gene encoding the beta 2AR was carried out to assess the frequency of polymorphisms in bronchial asthma, to determine wheather these polymorphisms have any relation to the severity, or nocturnal symptoms in bronchial asthma. METHOD: The subjects studied were 103 patients with bronchial asthma, which consisted of 30 mild episodic, 32 mild persistent, 17 moderate, and 24 severe asthma patients. The polymorphisms of the beta 2AR gene were detected by mutated allele specific amplification (MASA) method at the codons 16, 27, 34, and 164. RESULTS: The most frequent polymorphism was arginine 16 to glycine. The other two polymorphisms, valine 34 to methionine and glutamine 27 to glutamic acid occured in 11 and 6 patients respectively. The polymorphism of threonine 164 to isoleucine was not found in our enrolled patients. The homozygous polymorphism of beta 2AR gene was found in only arginine 16 to glycine (12.6%). The heterozygous polymorphisms of beta 2AR gene were in arginine 16 to glycine, valine 34 to methionine, and glutamine 27 to glutamic acid, as 65.1%, 10.7%, and 5.8% respectively in asthma patients. The presence of agrginine 16 to glycine heterozygous or/and homozygous polymorphism was associated in severe asthma (p=0.015), but there was no association between the other three polymorphisms and the severity of asthma. The frequency of the 182AR gene polymorphisms was no relation in nocturnal asthma as compared with non-nocturnal asthma. CONCLUSION: The arginine 16 to glycine polymorphism of the beta 2AR gene is the most frequently found in asthma patients and association with severe asthma. But there was no association between the polymorphism of the beta 2AR gene and nocturnal asthma.

DNA elements and protein factors involved in the transcription of the beta 2-adrenergic receptor gene in rat liver. The negative regulatory role of C/EBP alpha.

Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.

The human β2-adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

We have developed a rapid and efficient expression system to study the human β 2 adrenergic receptor (hu β 2AR) in the fission yeast Schizosaccharomyces pombe. This was achieved by cloning the hu β 2AR gene, modified by replacement of the 5′ untranslated and a small part of the N-terminal coding sequence (first 14 amino acids) with the corresponding region of the yeast Saccharomyces cerevisiae STE2 (α-factor receptor) gene. The gene was then placed under the control of a S. pombe constitutive promoter for alcohol dehydrogenase (adh). Hu β 2AR expression was assessed by immunoblot analysis of the chimeric protein with an anti-STE2 serum raised against a dodecapeptide homologous to the N-terminal amino acids of STE2 and ligand binding was assayed using [ 125I]cyanopindolol. We demonstrate here that the chimeric receptor expressed in S. pombe exhibits the same characteristic ligand specificity and affinity as that of the authentic hu β 2AR. This system constitutes a convenient alternative to existing methods for studying seven transmembrane domain receptors due to its simplicity and high reproducibility.

Ethanol differentially increases alpha 2-adrenergic and muscarinic acetylcholine receptor gene expression in NG108-15 cells.

Modulation of alpha 2-adrenergic and opioid neurotransmission may contribute to ethanol intoxication, tolerance, and physical dependence. We showed previously that ethanol increased the expression of functional delta-opioid receptors in NG108-15 cells (Charness, M. E., Querimit, L. A., and Diamond, I. (1986) J. Biol. Chem. 261, 3164-3169). Here we report that long-term (2 days) treatment of NG108-15 cells with ethanol increased the binding of the alpha 2-adrenergic receptor (alpha 2AR) antagonist [3H]rauwolscine and the muscarinic acetylcholine receptor (mAChR) antagonist [3H]quinuclidinyl benzilate by 2.8- and 1.4-fold, respectively. Increased receptor expression was associated with a proportionate increase in the potency of oxymetazoline and carbachol in inhibiting cAMP accumulation. Ethanol did not change the expression of G alpha i2 and reduced levels of G alpha s. Pertussis toxin pretreatment did not prevent the ethanol-induced increase in alpha 2AR, mAChR, and delta-opioid receptor expression. Ethanol caused a large (3.6-fold), dose-dependent increase in the abundance of alpha 2BAR mRNA (rat cDNA probe RNG, 4.1-kb transcript). Ethanol-induced increases in alpha 2BAR and alpha 2CAR (rat probe RG10, 2.5-kb transcript) mRNAs were first detected after 6 h of exposure to 100 mM ethanol, became maximal after 24 h, and persisted for up to 5 days. In contrast, ethanol caused only a small (1.3-fold) increase in the abundance of hm4 mAChR mRNA and did not change levels of G alpha i2 and G alpha s mRNAs. Our data indicate that clinically attainable concentrations of ethanol regulate alpha 2AR gene expression within the time frame of a single session of drinking.

The immune response evokes up- and down-modulation of beta 2-adrenergic receptor messenger RNA concentration in the male rat thymus.

Important alterations of noradrenergic activity are known to occur in specific brain regions and in different lymphoid tissues during the course of an immune response. Our recent characterization of the beta 2-adrenergic receptor (beta 2AR)-cAMP system of the rat thymus gland, the identification of a thymic beta 2AR gene expression, and the marked modulation of receptor mRNA concentration after castration and replacement with estrogen prompted us to study the ability of products of immune axis activation to modulate beta 2AR number, distribution, and expression in the male rat thymus. Moreover, the effect of adrenergic stimulation of adenylyl cyclase activity on thymus gland membrane preparations was measured. The beta 2AR present in the rat thymus undergoes marked changes in both number and distribution during the course of an immune response. At 3 days after antigenic challenge (injection of BSA in complete Freund's adjuvant), a sharp decrease of receptor number coupled with a significant loss of the autoradiographic reaction in the medullary compartment of the rat thymus gland were observed. These effects were followed by a significant increase in receptor density and number without changes in receptor affinity at 7 and 15 days post immunization, corresponding to the pick of the immune response. Parallel alterations in adenylyl cyclase activity were measured. Northern blot analysis, using a human beta 2AR cDNA as a probe, revealed dramatic alterations of the beta 2AR mRNA in the thymus, characterized by an approximately 75% decrease of mRNA level 3 days after immunization, and by a progressive increase at 7 and 15 days, with beta 2AR mRNA concentration rising to levels even higher than those found in control animals. These results suggest that the immune response evokes marked alterations of the thymic beta 2AR-cAMP signaling pathway. Moreover, antigenic stimulation triggers a down- and up-modulation of beta 2AR gene expression. Although it is presently unknown whether factor(s) released by immune axis activation act at the level of gene transcription to modulate adrenergic receptor function in the rat thymus, such down- and up-regulation of beta 2AR mRNA may play a role in the dynamic regulation of the immune response.

Mutational analysis of ligand binding activity of beta 2 adrenergic receptor expressed in Escherichia coli.

A novel in situ screening procedure was used to identify neutral mutations in the human beta 2 adrenergic receptor (beta 2AR). The coding region of the human beta 2AR gene was subcloned under transcriptional control of an inducible T7 promoter and used to transform Escherichia coli. Colonies expressing the beta 2AR bound the radiolabeled antagonist [125I]iodocyanopindolol and could be identified by autoradiography after transfer to nitrocellulose filters. The region of the beta 2AR between residues 76 and 83, in the second transmembrane helix, was mutagenized by a saturation mutagenesis technique, so that virtually all of the beta 2AR genes contained at least one mutation. Colonies retaining ligand binding activity were isolated using the in situ screen. Sequence analysis of the active mutant receptor genes allowed the identification of individual amino acid side chains which are essential for either ligand binding or structural integrity of the beta 2AR receptor.

CAMP stimulates transcription of the beta 2-adrenergic receptor gene in response to short-term agonist exposure.

In addition to conveying cellular responses to an effector molecule, receptors are often themselves regulated by their effectors. We have demonstrated that epinephrine modulates both the rate of transcription of the beta 2-adrenergic receptor (beta 2AR) gene and the steady-state level of beta 2AR mRNA in DDT1MF-2 cells. Short-term (30 min) exposure to epinephrine (100 nM) stimulates the rate of beta 2AR gene transcription, resulting in a 3- to 4-fold increase in steady-state beta 2AR mRNA levels. These effects are mimicked by 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) or foskolin but not by phorbol esters. The half-life of the beta 2AR mRNA after addition of actinomycin D (46.7 +/- 10.2 min; mean +/- SEM; n = 5) remained unchanged after 30 min of epinephrine treatment (46.8 +/- 10.6 min; mean +/- SEM; n = 4), indicating that a change in transcription rate is the predominant factor responsible for the increase of beta 2AR mRNA. Whereas brief exposure to epinephrine or Bt2cAMP does not significantly affect the total number of cellular beta 2ARs (assessed by ligand binding), continued exposure results in a gradual decline in beta 2AR number to approximately 20% (epinephrine) or approximately 45% (Bt2cAMP) of the levels in control cells by 24 hr. Similar decreases in agonist-stimulated adenylyl cyclase activity are observed. This loss of receptors with prolonged agonist exposure is accompanied by a 50% reduction in beta 2AR mRNA. Transfection of the beta 2AR promoter region cloned onto a reporter gene (bacterial chloramphenicol acetyltransferase) allowed demonstration of a 2- to 4-fold induction of transcription by agents that elevate cAMP levels, such as forskolin or phosphodiesterase inhibitors. These results establish the presence of elements within the proximal promoter region of the beta 2AR gene responsible for the transcriptional enhancing activity of cAMP and demonstrate that beta 2AR gene expression is regulated by a type of feedback mechanism involving the second messenger cAMP.

Regulation of the β 2-adrenergic receptor and its mRNA in the rat ventral prostate by testosterone

To investigate the regulation of β 2AR expression in rat ventral prostate the effects of castration and testosterone replacement on the β 2AR were studied by ligand binding and Northern blot analysis. Orchidectomy depressed β 2AR number by 50% within 4 days and testosterone administration to 4-day castrates produced a rapid and complete recovery of β 2AR number within 24 h. In contrast to receptor number, β 2AR mRNA levels did not change relative to total RNA following castration. However, during the testosterone replacement period β 2AR mRNA levels rose transiently, reaching a maximum (3.5-fold) between 8 and 12 h, and this increase in mRNA preceded the recovery in β 2AR number in the membrane. Regulation of β 2AR gene expression by testosterone in the ventral prostate is thus complex and probably involves both transcriptional and post-transcriptional components.

Isoproterenol response following transfection of the mouse beta 2-adrenergic receptor gene into Y1 cells.

The beta 2-adrenergic receptor (beta 2AR) gene was isolated from a mouse genomic library, sequenced and shown to share 93% identity with the hamster beta 2AR cDNA at the amino acid level. This mouse beta 2AR genomic clone was transfected into the Y1 mouse adrenal cortex tumor cell line. Northern blot and S1 nuclease analysis showed that the beta 2AR-transfected cells expressed an mRNA of the appropriate size to encode the receptor. Membrane receptor number and affinities for various beta-adrenergic agonists demonstrated that the transfected clone encoded a beta 2AR protein product. Incubation of the transfected Y1 cells, which do not normally possess beta 2AR, with the beta 2AR agonist, isoproterenol, resulted in an increase in the rate of steroid secretion by these cells as well as a rapid change in cell morphology. This response was fully blocked by the beta 2AR antagonist, propranolol. Prolonged incubation of the cells with isoproterenol resulted in agonist insensitivity and an 80% reduction in membrane receptor number.