Opsin is present as dimers in COS1 cells: identification of amino acids at the dimeric interface

Abstract

Rhodopsin in the disk membranes of rod outer segments serves as the dim-light photoreceptor and is a prototypic member of a G protein-coupled receptor family. Electron and atomic-force microscopy indicate that rhodopsin is present as dimers in the native membranes. Here, we have expressed the protein, opsin, in COS1 cells and have studied its molecular state by using FRET and by intermolecular cross-linking after site-directed cysteine mutagenesis. To observe FRET, the ends of the genes corresponding to the N termini of the cyan or yellow fluorescent proteins were fused to the ends of the genes corresponding to the C terminus of the opsin and the resulting fused genes were expressed in COS1 cells. The emission spectra in situ of the expressed proteins were recorded, and FRET was then calculated. The result indicated intermolecular interaction between opsin molecules in COS1 cells. To identify the amino acids involved in the interaction, those predicted by molecular modeling to be at the dimer interface were mutated one at a time to cysteine, and dimer formation was measured by the rate of disulfide bond formation in the presence of cupric orthophenanthroline. The mutants W175C and Y206C formed the dimers most rapidly, showing that the two amino acids were at the dimer interface.

Fig. 1.

Schematic representation of plasmid constructs (A) and oligomerization of opsin in vivo detected by FRET (B). Opsin fusion proteins Ops.CFP and Ops.YFP were expressed separately and together in COS1 cells for FRET experiments. Whole cells expressing these fusion proteins were excited at 425 nm, and their emission spectra were recorded. FRET data were analyzed as described in Materials and Methods. Emission spectra were recorded from cells expressing Ops.CFP and Ops.YFP separately and together. Emission due to FRET was determined by subtracting the emission spectra of cells expressing Ops.CFP alone and cells expressing Ops.YFP alone from the cells coexpressing Ops.CFP + Ops.YFP as described in Materials and Methods.

Fig. 2.

A secondary structure model of rhodopsin, showing cysteines occurring naturally (shaded circles) and cysteines that replaced amino acid residues (open circles).

Fig. 3.

Immunoblot analysis of WT opsin and cysteine mutants expressed in COS1 cells. Cells were harvested 48 h after transfection, and the extracts were prepared after lysis in 20 mM DM. Proteins were resolved by 12% SDS/PAGE in the presence or absence of DTT, and opsins were visualized by immunoblotting. The dimers are indicated by arrows. Single expression (A) or coexpression (B) of cysteine mutants in COS1 cells.

Fig. 4.

Oligomerization of WT opsin and of opsin cysteine mutants W175C and Y206C in vivo as studied by FRET. Whole cells expressing various rhodopsin fusion proteins were excited at 425 nm, and their emission spectra were recorded. FRET data were analyzed as described in Materials and Methods. (A) Emission spectra of cells expressing W175C.CFP and W175C.YFP separately and together. (B) Emission spectra of cells expressing Y206C.CFP and Y206C.YFP separately and together. The dotted line (WT FRET) is the FRET spectrum of Ops.CFP + Ops.YFP. (C) Effect of DM on FRET. Cells expressing opsin fusion proteins were suspended in PBS and 0.1 mM PMSF in the presence of varying concentrations of DM and excited at 425 nm, and their emission spectra were recorded.

Fig. 5.

Cross-linking of opsin by CuP. Cells were harvested 48 h after transfection, and membranes were prepared as described in Materials and Methods. The membranes were treated with 400 μM CuSO₄ and 1600 μM 1,10-phenanthroline for 1, 2, 5, 10, and 15 min and, then, with 20 mM NEM containing 20 mM EDTA. The proteins were resolved by SDS/PAGE and visualized by immunoblotting. (A) WT. (B) W175C. (C) Y206C.

Fig. 6.

Cross-linking of opsin by CuP. Cells were harvested 48 h after transfection, and membranes were prepared as described in Materials and Methods. The membranes were treated with CuP (0, 10/40, 25/100, 50/200, and 100/400 μM) for 10 min at room temperature and, then, with 20 mM NEM containing 20 mM EDTA. The proteins were resolved by SDS/PAGE and visualized by immunoblotting. (A) WT. (B) W175C. (C) Y206C.