Functional expression and direct visualization of the human α 2B-adrenergic receptor and α 2B-AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

Abstract

The α 2B-adrenergic receptor (α 2B-AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the α 2B-AR. The receptor fusions α 2B-AR–eGFP and CAP–α 2B-AR expressed in CHO-K1 cells generated B max values of 176 and 122 pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, α 2B-AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.