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

Abstract

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.

Segregation of Family A G Protein–Coupled Receptor Protomers in the Plasma Membrane

“…The reason(s) for these discrepant results are not immediately apparent. Similar unresolved discrepancies exist between different fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies of b 2 AR oligomerization (Mercier et al, 2002;James et al, 2006;Salahpour and Masri, 2007;Felce and Davis, 2012;Kawano et al, 2013).…”

CrossTalk opposing view: Weighing the evidence for class A GPCR dimers, the jury is still out

“…; Kawano et al . ), even when the same receptors are being studied. In addition, several reports have shown TR‐FRET efficiency that increases with receptor density (Maurel et al .…”

Selective amine labeling of cell surface proteins guided by coiled‐coil assembly

“…Contrary to untreated cells, significant FRET signals were observed in the cholesterol‐depleted cells (Figure A). The E app value linearly increased with the donor mole fraction, suggesting dimer formation (Figure B), although higher‐order oligomers should exhibit concave upward curves . The observed significant E app values (∼0.3) indicate that the interchromophore distance between the N‐termini in the dimer is comparable or shorter than R 0 of RhoG–TMR pair (53 Å).…”

“…We have developed a tag–probe labeling method based on parallel heterodimeric coiled‐coil formation between the genetic E3 tag (EIAALEK) 3 , fused with the extracellular N‐termini of membrane proteins, and the synthetic K n probe (KIAALKE) n ( n = 3 or 4), having fluorophores such as rhodamines or cyanines . The small size (5–6 kDa), cell‐surface specificity, and ease of multicolor labeling of the coiled‐coil method has advantages over conventional fusions with fluorescent proteins, such as the precise analysis of the oligomeric states of membrane proteins in living cell membranes . The affinity of the K4 probe for the E3 tag (apparent K d ≈ 6 n M ) is sufficient for the imaging of cultured cells; however, the noncovalent nature is not suitable for detecting proteins by destructive methods such as sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis (SDS‐PAGE).…”