Introduction

As the largest receptor superfamily in the human genome (1), G protein-coupled receptors (GPCRs) represent a class of macromolecules of great importance in both academic science and pharmacological intervention. Substantial bottlenecks remain in precise mechanistic understanding of transmembrane signaling events and new advancements in methodologies are of paramount importance. The Human Genome Project has identified over 800 GPCR genes (1), the largest fraction of which are orphan receptors with neither an assigned function nor a determined ligand set (1,2,3). Mechanistic understanding of trans-membrane signaling and recognition of ligands by GPCRs demands robust functional and structural characterization. The investigation of critical questions regarding affinity shifts, cross-talk signaling (transactivation), and domain-swapped dimerization (4,5,6) would be greatly aided by the ability to control the oligomeric state of the target receptor. Additional advantages would be offered by an aqueous assay system wherein the integral membrane protein would be housed in a native membrane bilayer environment offering enhanced stability and full functionality. Various methods utilizing detergents (7,8,9), vesicles (10,11,12), and whole-cell preparations (13,14,15,16,17) have attempted to ameliorate difficulties in handling GPCRs. However, this repertoire of assay systems cannot easily address questions related to the effects of receptor oligomerization due to the difficulty of controlling both the oligomeric state of the receptor and, in whole cell preparations, difficulty in defining the complete composition of the reconstituted system.

The β₂-adrenergic receptor (β₂AR) was the first GPCR cloned (18) and represents a GPCR that has been extensively investigated as a major pharmaceutical target for the control of asthma and hypertension (7)(19,20,21,22). β₂AR has a preference for the stimulatory subset of G proteins (G_s), and while known to interact with some of the other G protein families, such as the inhibitory G_i and G_q (23,24), the major function of the β₂AR remains the agonist-induced activation of the stimulatory G protein system and the downstream effectors thereof.

Recently we developed the Nanodisc system as a new methodology for the functional reconstitution of integral membrane proteins into soluble nanoscale lipid bilayers (25,26,27,28,29). Nanodiscs utilize an encircling membrane scaffold protein (MSP) to form a discoidal structure nominally 10 nm in diameter with a 7 nm diameter lipid bilayer core (26). These structures can incorporate single molecules of integral membrane protein targets through simple chemical self-assembly. The resultant nanostructures represent a highly stable and homogeneous population with an aqueous solubility in the millimolar range. Recent work utilized the Nanodisc system for the successful reconstitution of membrane proteins from both purified and crude membrane preparations that included human cytochrome P450 3A4 (27), insect cytochrome P450 6B1 (28), and bacteriorhodopsin (25). In this report we describe the application of this technology to the more difficult problem of isolating GPCRs in a soluble and functional state and demonstrate antagonist and agonist binding as well as G protein coupling of human adrenergic receptor in a stable nanoscale detergent-free lipid bilayer environment.

Materials and Methods

Expression and Purification of Human β₂AR

A DNA sequence encoding the β₂AR was cloned into pCEP4 vector from Invitrogen (Carlsbad, CA, USA) and expressed in HEK 293 cells grown in the Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 250 µg/mL hygromysin B, and 50 µg/mL gentamicin (Mediatech, Herndon, VA, USA). Cells were harvested at a density ≥10⁶ cells/mL, suspended in 100 mM sodium phosphate, pH 7.5, 10% (w/v) glycerol, 0.1 mM EDTA, Complete Protease Inhibitor (Roche Applied Science, Indianapolis, IN, USA), 1 mM phenyl methyl sulfonyl fluoride (PMSF), and broken in Avestin cell disruptor (Avestin, Ottawa, Canada). Membranes were isolated by centrifugation at 100,000× g for 1 h and suspended in 25 mM sodium phosphate, pH 7.5, 10% (w/v) glycerol, 150 mM NaCl, 0.1 mM EDTA, 0.1 mM PMSF, complete protease inhibitor cocktail (Roche Applied Science). Solubilization of the receptor was initiated by addition of the dodecyl-β-D-maltoside (DβM) to 1% concentration to the membrane suspension. After 1 h incubation at 4°C, NaCl was added to a final concentration 0.5 M, and insolubilized material was removed by centrifugation at 100,000× g for 1 h. The supernatant was loaded onto a Ni-NTA column (Qiagen, Valencia, CA, USA) equilibrated with 25 mM sodium phosphate, pH 7.5, 10% (w/v) glycerol, 0.5 M NaCl, 8 mM imidazole, 0.1% DβM (buffer A). After extensive washing of the column with buffer A, the receptor was eluted by 100 mM imidazole in buffer A. Fractions containing β₂AR were pooled and loaded onto a column with Anti-FLAG® resin (Sigma-Aldrich, St. Louis, MO, USA) equilibrated with 25 mM sodium phosphate, pH 7.5, 10% (w/v) glycerol, 150 mM NaCl, 0.1% DβM (buffer B). The column was extensively washed with buffer B, and the β₂AR was eluted with 0.1 mg/mL of the FLAG peptide in buffer B. Fractions containing homogeneous β₂AR were immediately frozen in liquid nitrogen and stored at -80°C. This affinity purification has yielded efficient purification of β₂AR to >95% homogeneity. N-terminal sequence analysis confirmed that purified protein represents β₂AR.