High-Throughput Analysis of Ligand-Induced Internalization of β₂-Adrenoceptors Using the Coiled-Coil Tag–Probe Method

Receptor internalization is a useful indicator of the activity of ligands. The N-terminus of the β₂-adrenergic receptor expressed on the cell surface was labeled with fluorophores using a novel coiled-coil labeling system. Endocytosis of the receptors was automatically detected using a fluorescence image analyzer by evaluating (1) translocation of the receptor from cell-surface to intracellular regions and (2) acidification in endosomes. Both parameters increased upon agonist stimulation in a dose-dependent manner. The extent of endocytosis was significantly dependent on the agonist used, indicating the presence of a biased signaling for endocytosis. The receptor antagonists can also be screened by competitive inhibition of agonist-induced endocytosis. The image analysis approach has proven to be useful for high-throughput characterization and screening of GPCR ligands

Stoichiometric Analysis of Oligomerization of Membrane Proteins on Living Cells Using Coiled-Coil Labeling and Spectral Imaging

Many membrane proteins are proposed to work as oligomers; however, the conclusion is sometimes controversial, as for β₂-adorenergic receptor (β₂AR), which is one of the best-studied family A G-protein-coupled receptors. This is due to the lack of methods for easy and precise detection of the oligomeric state of membrane proteins on living cells. Here, we show that a combination of the coiled-coil tag–probe labeling method and spectral imaging enable a stoichiometric analysis of the oligomeric state of membrane proteins on living cells using monomeric, dimeric, and tetrameric standard membrane proteins. Using this method, we found that β₂ARs do not form constitutive homooligomers, while they exhibit their functions such as the cyclic adenosine 5'-monophosphate (cAMP) signaling and internalization upon agonist stimulation

Cholesterol-Induced Lipophobic Interaction between Transmembrane Helices Using Ensemble and Single-Molecule Fluorescence Resonance Energy Transfer

The solvent environment regulates the conformational dynamics and functions of solvated proteins. In cell membranes, cholesterol, a major eukaryotic lipid, can markedly modulate protein dynamics. To investigate the nonspecific effects of cholesterol on the dynamics and stability of helical membrane proteins, we monitored association–dissociation dynamics on the antiparallel dimer formation of two simple transmembrane helices (AA­L­A­L­AA)₃ with single-molecule fluorescence resonance energy transfer (FRET) using Cy3B- and Cy5-labeled helices in lipid vesicles (time resolution of 17 ms). The incorporation of 30 mol % cholesterol into phosphatidylcholine bilayers significantly stabilized the helix dimer with average lifetimes of 450–170 ms in 20–35 °C. Ensemble FRET measurements performed at 15–55 °C confirmed the cholesterol-induced stabilization of the dimer (at 25 °C, ΔΔ<i>G</i>_a = −9 kJ mol^–1 and ΔΔ<i>H</i>_a = −60 kJ mol^–1), most of which originated from “lipophobic” interactions by reducing helix–lipid contacts and the lateral pressure in the hydrocarbon core region. The temperature dependence of the dissociation process (activation energy of 48 kJ) was explained by the Kramers-type frictional barrier in membranes without assuming an enthalpically unfavorable transition state. In addition to these observations, cholesterol-induced tilting of the helices, a positive Δ<i>C</i>_<i>p</i>(a), and slower dimer formation compared with the random collision rate were consistent with a hypothetical model in which cholesterol stabilizes the helix dimer into an hourglass shape to relieve the lateral pressure. Thus, the liposomal single-molecule approach highlighted the significance of the cholesterol-induced basal force for interhelical interactions, which will aid discussions of complex protein–membrane systems

Paradoxical Downregulation of CXC Chemokine Receptor 4 Induced by Polyphemusin II-Derived Antagonists

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor implicated in cell entry of T-cell line-tropic HIV-1 strains. CXCR4 and its ligand stromal cell derived factor-1 (SDF-1)/CXCL12 play pivotal parts in many physiological processes and pathogenetic conditions (e.g., immune cell-homing and cancer metastasis). We previously developed the potent CXCR4 antagonist T140 from structure–activity relationship studies of the antimicrobial peptide polyphemusin II. T140 and its derivatives have been exploited in biological and biomedical studies for the SDF-1/CXCR4 axis. We investigated receptor localization upon ligand stimulation using fluorescent SDF-1 and T140 derivatives as well as a specific labeling technique for cellular-membrane CXCR4. Fluorescent T140 derivatives induced translocation of CXCR4 into the perinuclear region as observed by treatment with fluorescent SDF-1. T140 derivative-mediated internalization of CXCR4 was also monitored by the coiled-coil tag-probe system. These findings demonstrated that the CXCR4 antagonistic activity and anti-HIV activity of T140 derivatives were derived (at least in part) from antagonist-mediated receptor internalization