Common ADRB2 Haplotypes Derived from 26 Polymorphic Sites Direct β2-Adrenergic Receptor Expression and Regulation Phenotypes

Alfredo Panebra,Molly M Malone,Demar R G Pitter,Gregory A Hawkins,Stephen B Liggett,Scott T Weiss,Wayne C Wang,Arnold Schwartz

doi:10.1371/journal.pone.0011819

Abstract

BackgroundThe β2-adrenergic receptor (β2AR) is expressed on numerous cell-types including airway smooth muscle cells and cardiomyocytes. Drugs (agonists or antagonists) acting at these receptors for treatment of asthma, chronic obstructive pulmonary disease, and heart failure show substantial interindividual variability in response. The ADRB2 gene is polymorphic in noncoding and coding regions, but virtually all ADRB2 association studies have utilized the two common nonsynonymous coding SNPs, often reaching discrepant conclusions.Methodology/Principal FindingsWe constructed the 8 common ADRB2 haplotypes derived from 26 polymorphisms in the promoter, 5′UTR, coding, and 3′UTR of the intronless ADRB2 gene. These were cloned into an expression construct lacking a vector-based promoter, so that β2AR expression was driven by its promoter, and steady state expression could be modified by polymorphisms throughout ADRB2 within a haplotype. “Whole-gene” transfections were performed with COS-7 cells and revealed 4 haplotypes with increased cell surface β2AR protein expression compared to the others. Agonist-promoted downregulation of β2AR protein expression was also haplotype-dependent, and was found to be increased for 2 haplotypes. A phylogenetic tree of the haplotypes was derived and annotated by cellular phenotypes, revealing a pattern potentially driven by expression.Conclusions/SignificanceThus for obstructive lung disease, the initial bronchodilator response from intermittent administration of β-agonist may be influenced by certain β2AR haplotypes (expression phenotypes), while other haplotypes may influence tachyphylaxis during the response to chronic therapy (downregulation phenotypes). An ideal clinical outcome of high expression and less downregulation was found for two haplotypes. Haplotypes may also affect heart failure antagonist therapy, where β2AR increase inotropy and are anti-apoptotic. The haplotype-specific expression and regulation phenotypes found in this transfection-based system suggest that the density of genetic information in the form of these haplotypes, or haplotype-clusters with similar phenotypes can potentially provide greater discrimination of phenotype in human disease and pharmacogenomic association studies.

Highlights

The b2-adrenergic receptor (b2AR) is a member of the G-protein coupled receptor superfamily (GPCR), and is expressed on virtually all cell-types [1]
Of particular interest for therapeutic purposes have been b2AR expressed on smooth muscle cells of the airway which promote bronchodilatation and those expressed on cardiomyocytes which promote cardiac contractility. bagonists acting on airway smooth muscle b2AR, and non-selective bblockers acting on b2- and b1AR on cardiomyocytes, are standard treatments of obstructive lung disease and heart failure, respectively
PLoS ONE | www.plosone.org (SNPs, Table 1) are at nucleic acid 46 resulting in amino acid position 16 being either Arg or Gly, and at nucleic acid 79 resulting in amino acid position 27 being Glu or Gln [4]. These polymorphisms are localized to the extracellular amino-terminus, and have a subtle effect on agonist-promoted downregulation of receptor expression when studied in a cell-based system where transfections of vectors containing only the open reading frames were carried out [7]

Summary

Introduction

The b2-adrenergic receptor (b2AR) is a member of the G-protein coupled receptor superfamily (GPCR), and is expressed on virtually all cell-types [1]. These polymorphisms are localized to the extracellular amino-terminus, and have a subtle effect on agonist-promoted downregulation of receptor expression when studied in a cell-based system where transfections of vectors containing only the open reading frames were carried out [7]. The constructs utilized for transfection consisted of 5,580 bp of ADRB2 (23685 to +1895) which represents contiguous promoter, 59UTR, coding and 39UTR sequence up to the poly-A termination site.

Results

Conclusion