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Effects of the ?-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of ?2-adrenergic receptors with N-terminal polymorphisms.

ABSTRACT: The ?2-AR (?2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of ?2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of ?2-AR variants following ?-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human ?2-AR (designated ?2-AR-RE, ?2-AR-GE, ?2-AR-RQ and ?2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 ?M isoprenaline, isoforms with Arg16 (?2-AR-RE and ?2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (?2-AR-GE and ?2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in ?2-AR-RE relative to ?2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of ?2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of ?2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the ?-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.

SUBMITTER: Koryakina Y

PROVIDER: S-EPMC4018189 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Effects of the β-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of β2-adrenergic receptors with N-terminal polymorphisms.

Koryakina Yulia Y Jones Stacie M SM Cornett Lawrence E LE Seely Kathryn K Brents Lisa L Prather Paul L PL Kofman Alexander A Kurten Richard C RC

Cell biology international 20120101 12

The β2-AR (β2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of β2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2-AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2-AR (designated β2-AR-R ...[more]

PMID: 22938397

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Project description:Evidence suggests that variation in the length of the poly-C repeat in the 3' untranslated region (3'UTR) of the ?2-adrenergic receptor gene (ADRB2) may contribute to interindividual variation in ?-agonist response. However, methodology in previous studies limited the assessment of the effect of sequence variation in the context of poly-C repeat length. The objectives of this study were to design a novel genotyping method to fully characterize sequence variation in the ADRB2 3'UTR poly-C repeat in asthma patients treated with inhaled corticosteroid and long-acting ?2-adrenergic agonist (ICS/LABA) combination therapy, and to analyze the effect of the poly-C repeat polymorphism on clinical response.In 2,250 asthma patients randomized to treatment with budesonide/formoterol or fluticasone/salmeterol in a six-month study (AstraZeneca study code: SD-039-0735), sequence diversity in the ADRB2 poly-C repeat region was determined using a novel sequencing-based genotyping method. The relationship between the poly-C repeat polymorphism and the incidence of severe asthma exacerbations, and changes in pulmonary function and asthma symptoms from baseline to the average during the treatment period, were analyzed.Poly-C repeat genotypes were assigned in 97% (2,192/2,250) of patients. Of the 13 different poly-C repeat alleles identified, six alleles occurred at a frequency of >5% in one or more population in this study. The repeat length of these six common alleles ranged from 10 to 14 nucleotides. Twelve poly-C repeat genotypes were observed at a frequency of >1%. No evidence of an association between poly-C repeat genotype and the incidence of severe asthma exacerbations was observed. Patients' pulmonary function measurements improved and asthma symptoms declined when treated with ICS/LABA combination therapy regardless of poly-C repeat genotype.The extensive sequence diversity present in the poly-C repeat region of the ADRB2 3'UTR did not predict therapeutic response to ICS/LABA therapy.

Project description:At the cell surface, ?ARs and endothelin receptors can regulate nitric oxide (NO) production. ?-adrenergic receptors (?ARs) and type B endothelin receptors (ETB) are present in cardiac nuclear membranes and regulate transcription. The present study investigated the role of the NO pathway in the regulation of gene transcription by these nuclear G protein-coupled receptors. Nitric oxide production and transcription initiation were measured in nuclei isolated from the adult rat heart. The cell-permeable fluorescent dye 4,5-diaminofluorescein diacetate (DAF2 DA) was used to provide a direct assessment of nitric oxide release. Both isoproterenol and endothelin increased NO production in isolated nuclei. Furthermore, a ?3AR-selective agonist, BRL 37344, increased NO synthesis whereas the ?1AR-selective agonist xamoterol did not. Isoproterenol increased, whereas ET-1 reduced, de novo transcription. The NO synthase inhibitor l-NAME prevented isoproterenol from increasing either NO production or de novo transcription. l-NAME also blocked ET-1-induced NO-production but did not alter the suppression of transcription initiation by ET-1. Inhibition of the cGMP-dependent protein kinase (PKG) using KT5823 also blocked the ability of isoproterenol to increase transcription initiation. Furthermore, immunoblotting revealed eNOS, but not nNOS, in isolated nuclei. Finally, caged, cell-permeable isoproterenol and endothelin-1 analogs were used to selectively activate intracellular ?-adrenergic and endothelin receptors in intact adult cardiomyocytes. Intracellular release of caged ET-1 or isoproterenol analogs increased NO production in intact adult cardiomyocytes. Hence, activation of the NO synthase/guanylyl cyclase/PKG pathway is necessary for nuclear ?3ARs to increase de novo transcription. Furthermore, we have demonstrated the potential utility of caged receptor ligands in selectively modulating signaling via endogenous intracellular G protein-coupled receptors.

Dataset Information

Effects of the ?-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of ?2-adrenergic receptors with N-terminal polymorphisms.

Publications

Effects of the β-agonist, isoprenaline, on the down-regulation, functional responsiveness and trafficking of β2-adrenergic receptors with N-terminal polymorphisms.

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