Allele-Specific Binding of Airway Nuclear Extracts to Polymorphic β₂-Adrenergic Receptor 5′ Sequence

Abstract

Like other intronless G protein-coupled receptor genes, the ␤ 2 -adrenergic receptor (␤ 2 AR) has minimal genetic space for population variability, and has attained such via multiple coding and noncoding polymorphisms. Yet most clinical studies use the two nonsynonymous polymorphisms of the coding region for association analysis despite low levels of linkage disequilibrium with some promoter and 5UTR polymorphisms. To assess the potential for allele-specific transcription factor binding to ␤ 2 AR 5-flanking sequence, 3-biotin-labeled oligonucleotide duplexes were synthesized. Each was centered on variable sites representing major or minor alleles found in the human population with frequencies of 5% or greater (20 polymorphic sites). Electrophoretic mobility shift assays were performed using human airway smooth muscle or airway epithelial cell nuclear extracts. Many of these polymorphisms resulted in an alteration in binding, and both major allele and minor allele dominance were observed. For example, in airway smooth muscle nuclear extracts, 10 polymorphisms decreased and 2 increased binding, whereas 5 showed no differences. Concordance between airway smooth muscle and epithelial cell nuclear extract binding to polymorphic alleles was found in only ‫ف‬ 50% of cases. There was no tendency for the rare variants to be more likely to have altered nuclear extract binding compared to the more common variants. Taken together, these results provide potential mechanisms by which ␤ 2 AR 5-flanking polymorphisms affect obstructive lung phenotypes.Keywords: asthma; ␤-agonist; polymorphismThe ␤ 2 -adrenergic receptor (␤ 2 AR) gene is highly polymorphic in the human population within both the coding and noncoding regions. Within the coding block, two nonsynonymous polymorphisms are common, occurring at nucleotides 46 and 79 relative to the ATG start site (1). These result in Arg or Gly at amino acid 16 and Glu or Gln at position 27, with three of the four possible two-site haplotypes being prevalent in normal cohorts as well as those with asthma and chronic obstructive lung disease. An infrequent, nonsynonymous polymorphism at nucleotide 491 results in a substitution of Ile for Thr at amino acid 164 (1). These polymorphisms have been studied in recombinant systems whereby constructs (without cognate 5ЈUTR or promoter) were used to stably or transiently transfect model cells, such as fibroblasts (2, 3) and COS cells (4). These studies revealed several phenotypes of the polymorphic receptors, including those related to agonist-promoted receptor trafficking and receptor coupling to G s (see Refs. 5 and 6 for review). Given the interindividual variability in the response to ␤-agonists in asthma, of which ‫ف‬ 50% has been attributed to genetic variation (7), these polymor- CLINICAL RELEVANCEThe variability in the response to ␤-agonists in obstructive lung disease is not fully understood. We show that polymorphisms in the promoter 5Ј-flanking region of the receptor alter transcription factor binding. phisms of the ␤ 2 AR have been...

Allele- and parent-of-origin-specific effects on expression of the KCNJ11 gene: A candidate for meat tenderness in cattle

“…Therefore, it is presumably in linkage disequilibrium with one or more SNPs that lie within the promoter region, or another cis-acting regulatory element affecting TF binding (Tao et al, 2006;Panebra et al, 2007;2010;Murani et al, 2013). Examples of these variations are given by SNPs used to analyze ASE of the beta-2 adrenergic receptor (ADRB2) gene in swine muscle (Muráni et al, 2009) and humans (Panebra et al, 2007), for which variation in allelic expression seems to be caused by multiple cis-regulatory polymorphisms in both species (Panebra et al, 2007;2010;Murani et al, 2013). However, in the present study, none of the TFBSs found for the KCNJ11 gene were consistent with any SNPs already described in the literature.…”

SPINK5andADRB2haplotypes are risk factors for asthma in Mexican pediatric patients

“…Numerous evidences show that non-synonymous SNPs could drive differences in the transcriptional activation, protein expression and cellular localization of the mutant protein and have a potential pathological effect [58,59]. For instance, Panebra et al 2007 demonstrated that non-synonymous SNPs showing varying levels of LD with ADRB2 5 0 UTR SNPs (far upstream position) have a negative effect in obstructive lung phenotypes through altered transcription factor binding [59].…”

Heterogeneity of transcription factor expression and regulation in human airway epithelial and smooth muscle cells

“…The fourth condition was concomitant treatments with isoproterenol and the aforementioned agents of the asthma condition. Nuclear extracts were prepared by using reagents from the NE-PER extraction system (Pierce, Rockford, IL) as previously described (19). Briefly, attached cells were placed on ice, and the medium was aspirated and washed three times with cold PBS.…”