Many membrane proteins are responsible for signaling and ionic transport necessary to maintain biological functions in vivo. Recently， not only conformational changes but also oligomerization have been proposed to regulate protein activation. Thus， the study of membrane protein oligomerization is crucial for new drug development. The existing destructive methodologies such as immunoprecipitation， however， are not suitable to determine oligomeric states precisely because of the artificial aggregation of proteins after detergent solubilization. In the present study， the coiled-coil tag-probe labeling method and spectral imaging were first combined to establish a new methodology based on fluorescence res... More
Many membrane proteins are responsible for signaling and ionic transport necessary to maintain biological functions in vivo. Recently， not only conformational changes but also oligomerization have been proposed to regulate protein activation. Thus， the study of membrane protein oligomerization is crucial for new drug development. The existing destructive methodologies such as immunoprecipitation， however， are not suitable to determine oligomeric states precisely because of the artificial aggregation of proteins after detergent solubilization. In the present study， the coiled-coil tag-probe labeling method and spectral imaging were first combined to establish a new methodology based on fluorescence resonance energy transfer (FRET) for stoichiometric analysis of the oligomeric states of membrane proteins on living cells. After validating the method for mono-， di-， and tetrameric standard membrane proteins， the oligomeric state of β₂-adrenergic receptors (β₂ARs) was examined to clarify its functional significance. It was found that β2ARs could transduce cyclic adenosine 5'-monophosphate (cAMP) signals and internalize them upon treatment with ligands without showing any FRET signals. Thus， β₂ARs do not form constitutive homooligomers， and homooligomerization is not necessary for the receptor function of β₂ARs. Finally， the oligomeric state of full-length M2 proton-selective channels of influenza A virus was investigated. Although the results of X-ray crystallography and NMR studies using fragment peptides suggested that M2 stably forms a tetrameric channel， the full-length M2 proteins formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH， indicating that the minimal functional unit of the M2 channel is a dimer.
