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.2004 Nov-Dec;21(6):435-46.
doi: 10.1080/09687860400020291.

The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

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The supramolecular structure of the GPCR rhodopsin in solution and native disc membranes

Kitaru Suda et al. Mol Membr Biol.2004 Nov-Dec.

Abstract

Rhodopsin, the prototypical G-protein-coupled receptor, which is densely packed in the disc membranes of rod outer segments, was proposed to function as a monomer. However, a growing body of evidence indicates dimerization and oligomerization of numerous G-protein-coupled receptors, and atomic force microscopy images revealed rows of rhodopsin dimers in murine disc membranes. In this work we demonstrate by electron microscopy of negatively stained samples, blue native- and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, chemical crosslinking, and by proteolysis that native bovine rhodopsin exists mainly as dimers and higher oligomers. These results corroborate the recent findings from atomic force microscopy and molecular modeling on the supramolecular structure and packing arrangement of murine rhodopsin dimers.

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Figures

Figure 1
Figure 1
Transmission electron microscopy of negatively stained native disc membranes adsorbed on carbon film. (a) Morphology of a native disc membrane from bovine. (b) Average of five power spectra calculated from circular regions as marked on the displayed disc membrane by the broken circle (1). A diffuse powder diffraction signal is evident, indicating paracrystallinity of rhodopsin. (c) Average of five power spectra calculated from circular regions on the carbon film as marked by the broken circle (2). No powder diffraction is evident. Scale bars: 2000 Å (a) and (40 Å )−1 (b andc).
Figure 2
Figure 2
BN-PAGE of unbleached and bleached disc membranes. (a) Unbleached disc membranes solubilized in 0.3% (lanes 1–3) and 0.6% DM (lanes 4–6) at protein concentrations of ≈0.9 mg/ml (lane 1 and 4), ≈0.6mg/ml (lane 2 and 5) and ≈0.3 mg/ml (lane 3 and 6). (b) Comparison between unbleached (lane R) and bleached (lane O) disc membranes. Both were solubilized in 0.6% DM at a protein concentration of ≈1.1 mg/ml. The BN-gels displayed in panels (a) and (b) are 5–12% linear gradient gels. Lanes labeled M: soluble markers thyroglobulin (669 kDa), ferritin (440 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (66 kDa). Protein per lane: ≈18 μg (lanes 1, 4, R, and O), ≈12 μg (lane 2 and 5), and ≈6 μg (lane 3 and 6). This figure is reproduced in colour inMolecular Membrane Biology online.
Figure 3
Figure 3
Transmission electron microscopy of negatively stained DM-solubilized disc membranes. Rhodopsin dimers are clearly discerned on the carbon film. The selected particles which are marked by broken circles were magnified and are displayed in the gallery. The scale bar represents 500 Å. The frame size of the magnified particles in the gallery is 104 Å.
Figure 4
Figure 4
SDS-PAGE of bovine disc membranes crosslinked in the presence of DSP. (a) Crosslinking of isolated disc membranes at different concentrations of DSP. Incubation for 15 h at 4ºC. No 2-ME in the SB. (b) Same as (a) but with 2-ME in the SB. (c) Time course of crosslinking at 2.5 mM DSP. No 2-ME in the SB. (d) Same as (c ) but with 2-ME in the SB. Protein concentration during the crosslinking reactions in (a–d): 100 μg/ml. (e) Crosslinking at 2.5 mM DSP and different disc membrane protein concentrations (50, 100 and 200 μg/ml). No 2-ME in the SB. (f ) Same as (e) but with 2-ME in the SB. (g) Disc membranes crosslinked in the presence of 2.5 mM DSP and subsequently digested with thermolysin. Incubation for 15 h at 4ºC for both, crosslinking and protease treatment, respectively. No 2-ME in the SB. (h) Same as (g) but with 2-ME in the SB. (i ) Crosslinked and thermolysin-digested rhodopsin monomers (m; left lane) and dimers (d; right lane) after extraction from gels such as that displayed in panel (g). No 2-ME in the SB. (j ) Same as (i ) but with 2-ME in the SB. Band labels: m, rhodopsin monomer; d, rhodopsin dimer; F1 and F2, proteolytic fragments of rhodopsin after thermolysin digestion. The 13.5% SDS-polyacrylamide gels (a–j ) are silver-stained. ‘+’ means sample was mixed with SB containing 2-ME (final concentration: 3% 2-ME) and ‘−’ means sample was mixed with SB without 2-ME.
Figure 5
Figure 5
SDS-PAGE of bovine disc membranes crosslinked in the presence of LC-SPDP. (a) Crosslinking of isolated disc membranes at different concentrations of LC-SPDP. Incubation for 15 h at 4ºC. No 2-ME in SB. (b) Same as (a) but with 2-ME in the SB. (c ) Time course of crosslinking at 250 μM LC-SPDP. No 2-ME in the SB. (d) Same as (c ) but with 2-ME in the SB. Protein concentration during the crosslinking reactions in (a–d): 100 μg/ml. (e) Disc membranes crosslinked in the presence of 250 μM LC-SPDP and subsequently digested with thermolysin. Incubation for 15 h at 4ºC for crosslinking and protease treatment, respectively. No 2-ME in the SB. (f ) Same as (e) but with 2-ME in the SB. Band labels: m, rhodopsin monomer; F1 and F2, proteolytic fragments of rhodopsin after thermolysin digestion. The 13.5% SDS-polyacrylamide gels (a–f ) are silver-stained. ‘+’ means sample was mixed with SB containing 2-ME (final concentration: 3% 2-ME) and ‘−’ means sample was mixed with SB without 2-ME.
Figure 6
Figure 6
Location of selected Lys and Cys residues on the cytoplamic surface of the molecular model of the rhodopsin oligomer (model IV–V). Red balls indicate the reactive Lys ɛ-amino groups while the green balls indicate the reactive β-thiol groups of the Cys residues. Distances between selected groups are summarized in Table 2. The intra- (contact 1) and interdimeric contacts (contact 2) as well as the row–row contacts (contact 3) forming the higher-order structure of rhodopsin are indicated. For better clarity, the C-termini (labeled c) as well as parts of the C-III loops (labeled C-III) are displayed as wires and the one letter instead of the three letter code was used for the highlighted amino acid residues. Unlabelled balls can be assigned by symmetry. The coordinates of model IV–V are deposited at the Protein Data Bank (accession code 1N3M) [–44]. The scale bar represents 10 Å.
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