Quaternary structures of opsin in live cells revealed by FRET spectrometry.
Rhodopsin is a prototypical G-protein-coupled receptor (GPCR) that initiates phototransduction in the retina. The receptor consists of the apoprotein opsin covalently linked to the inverse agonist 11-cis retinal. Rhodopsin and opsin have been shown to form oligomers within the outer segment disc membranes of rod photoreceptor cells. However, the physiological relevance of the observed oligomers has been questioned since observations were made on samples prepared from the retina at low temperatures. To investigate the oligomeric status of opsin in live cells at body temperatures, we utilized a novel approach called Förster resonance energy transfer spectrometry, which previously has allowed the determination of the stoichiometry and geometry (i.e. quaternary structure) of various GPCRs. In the current study, we have extended the method to additionally determine whether or not a mixture of oligomeric forms of opsin exists and in what proportion. The application of this improved method revealed that opsin expressed in live Chinese hamster ovary (CHO) cells at 37°C exists as oligomers of various sizes. At lower concentrations, opsin existed in an equilibrium of dimers and tetramers. The tetramers were in the shape of a near-rhombus. At higher concentrations of the receptor, higher-order oligomers began to form. Thus, a mixture of different oligomeric forms of opsin is present in the membrane of live CHO cells and oligomerization occurs in a concentration-dependent manner. The general principles underlying the concentration-dependent oligomerization of opsin may be universal and apply to other GPCRs as well.
Department of Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA.
Ashish K
AK
Mishra
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, USA.
Megan
M
Gragg
Department of Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA.
Michael R
MR
Stoneman
Department of Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA.
Gabriel
G
Biener
Department of Biological Sciences, University of Wisconsin Milwaukee, Milwaukee, WI, USA.
Julie A
JA
Oliver
Laboratory of Biomodeling, Faculty of Chemistry and Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland.
Przemyslaw
P
Miszta
Laboratory of Biomodeling, Faculty of Chemistry and Biological and Chemical Research Centre, University of Warsaw, Warsaw, Poland.
Slawomir
S
Filipek
Department of Physics, University of Wisconsin Milwaukee, Milwaukee, WI, USA Department of Biological Sciences, University of Wisconsin Milwaukee, Milwaukee, WI, USA.
Valerică
V
Raicu
Department of Ophthalmology and Visual Sciences, Case Western Reserve University, Cleveland, OH, USA.
Paul S-H
PS
Park
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D055355:Opsins / Q000737:chemistry* / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism
D055355:Opsins; D043562:Receptors, G-Protein-Coupled
G-protein-coupled receptors; fluorescence resonance energy transfer; membrane proteins; oligomerization; quaternary structure