The G protein-coupled receptor rhodopsin in the native membrane

Abstract

The higher-order structure of G protein-coupled receptors (GPCRs) in membranes may involve dimerization and formation of even larger oligomeric complexes. Here, we have investigated the organization of the prototypical GPCR rhodopsin in its native membrane by electron and atomic force microscopy (AFM). Disc membranes from mice were isolated and observed by AFM at room temperature. In all experimental conditions, rhodopsin forms structural dimers organized in paracrystalline arrays. A semi-empirical molecular model for the rhodopsin paracrystal is presented validating our previously reported results. Finally, we compare our model with other currently available models describing the supramolecular structure of GPCRs in the membrane.

Fig. 1

Morphology of isolated ultrathin sectioned ROS. AFM height (A) and amplitude image (B) recorded by tapping mode in air, and TEM micrograph (C). Disc membranes in a rod are stacked and surrounded by plasma membrane (arrow). The calculated power spectrum of the corresponding image is shown as inset. Vertical brightness ranges: 10 nm (A) and 1 nm (B).

Fig. 2

EM of negatively stained native disc membranes adsorbed on carbon film. A: Morphology of a native disc membrane from mouse isolated at room temperature and never exposed to low temperatures. B: Average of six power spectra calculated from regions on the displayed disc membrane, e.g. area marked by the broken circle (1). Powder diffraction is evident indicating paracrystallinity of the disc. C: Average of six power spectra calculated from regions on the carbon film, e.g. area marked by the broken circle (2). No powder diffraction is evident.

Fig. 3

AFM of a native disc membrane adsorbed on mica and imaged in buffer solution. A: Overview height image of an open, spread-flattened single-layered disc membrane isolated at room temperature and never exposed to low temperatures. Four different surface types are discerned: the rhodopsin packed areas (types 1 and 2), lipid (type 3) and mica (type 4). B: Height profile along the broken line in A. C: Deflection image of A at higher magnification: rhodopsin paracrystals are evident (broken ellipse). Inset in C: Deflection image of a rhodopsin paracrystal at higher resolution. One row (arrow) of rhodopsin dimers (arrowheads) forming the paracrystal is indicated. Frame size of the inset in C: 56 nm. Vertical brightness ranges: 19 nm (A), 0.5 nm (C) and 0.3 nm (C, inset).

Fig. 4

Model for the packing arrangement of rhodopsin molecules within the paracrystalline arrays of native disc membranes (based on [45,46]). A: Side view of a rhodopsin dimer, the building block of the paracrystal. Contacts between monomers are formed by the transmembrane helices IV (yellow-green) and V (yellow). Most of the interacting residues are located on the cytoplasmic loop between helices H-III and H-IV, and on the C-terminal region. Other interaction sites are located within the membrane. Both areas contain hydrogen bonds as well as interactions of hydrophobic nature. B: Cytoplasmic side (top) and extracellular side (bottom) of rhodopsin oligomers. Positions of helix ends are marked by colored discs and the corresponding helix numbers. Extracellular and cytoplasmic loops are drawn schematically at the corresponding locations. Contacts between dimers are created entirely by the intracellular loop between H-V and H-VI from one monomer with the loop between H-I and H-II and the C-terminal residues from the partner monomer. Only half of a second row of rhodopsins is shown. The contact between double rows is created mainly by hydrophobic residues from H-I close to the extracellular side. The lipid molecules initially included in the model for molecular dynamics are not displayed.

Fig. 5

Alternative H-IV arrangement of rhodopsin in the membranes (based on [,,—54]). Two views are displayed: cytoplasmic (left) and extracellular side (right). Intradimeric contacts are formed mainly by H-IV. The distance between two monomers in the dimer is 35 Å (horizontal, solid line), between dimers along the rows (oblique, solid line) 45 Å, and between double rows (horizontally, e.g. yellow to yellow monomer distance) maximally 77 Å. Contact between dimers is only possible at the cytoplasmic side between H-8 and the ends of helices III and V. Contacts between double rows are formed by IC-3, the cytoplasmic loop between H-V and H-VI. The lipid molecules initially included in the model for molecular dynamics are not displayed