Review
Activation of rhodopsin: new insights from structural and biochemical studies

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Abstract

G-protein-coupled receptors (GPCRs) are involved in a vast variety of cellular signal transduction processes from visual, taste and odor perceptions to sensing the levels of many hormones and neurotransmitters. As a result of agonist-induced conformation changes, GPCRs become activated and catalyze nucleotide exchange within the G proteins, thus detecting and amplifying the signal. GPCRs share a common heptahelical transmembrane structure as well as many conserved key residues and regions. Rhodopsins are prototypical GPCRs that detect photons in retinal photoreceptor cells and trigger a phototransduction cascade that culminates in neuronal signaling. Biophysical and biochemical studies of rhodopsin activation, and the recent crystal structure determination of bovine rhodopsin, have provided new information that enables a more complete mechanism of vertebrate rhodopsin activation to be proposed. In many aspects, rhodopsin might provide a structural and functional template for other members of the GPCR family.

Section snippets

G-protein-coupled receptors

Signal detection and transmission across biological membranes is initiated by the interaction of a chemical or physical stimulus with a specific membrane receptor which, in turn, becomes activated and initiates a chain of intracellular reactions that result in modulation of target protein activity. GPCRs are a superfamily of such membrane proteins that transmit a signal by coupling to heterotrimeric guanine nucleotide-binding proteins (known as large G proteins), which consist of three subunits

Topology of rhodopsin

Vertebrate rhodopsin is located in the membranes of discs – flat vesicles that fill the outer segment of rod cells. The extracellular (intradiscal) and intracellular regions of rhodopsin each consist of three interhelical loops (given the prefix E or I, for extracellular and intracellular, respectively) and a terminal (COOH or NH2) tail region. A fourth cytoplasmic loop (H-VIII) is formed by anchoring the C-terminal tail to the membrane via two Cys residues (Fig. 1), which carry palmitates in

Rhodopsin structure and retinal diseases

Visual pigments contain the 11-cis-retinal chromophore, bound via a protonated Schiff base linkage to a Lys side chain in the middle of H-VII. Because the retinal protonated Schiff base model compounds absorb ∼440 nm light, additional interactions between the chromophore and amino acids of the protein moiety of visual pigments cause the desirable shift of the optimal absorption (opsin shift), for instance ∼500 nm for rhodopsin and ∼400–600 nm for color pigments. Several studies on visual

Ground state

The activity of ligand-free opsin is equal to 10−6 of the activity of the all-trans-retinal-bound active metarhodopsin II state 17. However, the 11-cis-retinal-bound rhodopsin ground state exhibits an even lower level of activity against Gt, which shows that the 11-cis-retinal acts as an inverse agonist and imposes further structural constraints. The interactions can now be seen in the ground-state structure and explain why the binding of the chromophore is concomitant with a decrease in

Photochemistry and early intermediates

Absorption of a photon provides rhodopsin with the energy to form the active state. As shown in Fig. 5, three phases of the activation process can be distinguished: (1) light induced cistrans isomerization of the retinal; (2) thermal relaxation of the retinal–protein complex; and (3) the late equilibria that are affected by the interaction of rhodopsin with the G protein.

Two-thirds of the energy of 57 kcal mol−1 (238 kJ mol−1) taken up by light absorption are stored in the photoisomerized

The meta states

The intermediate MII is the signaling state capable of interacting with the G protein 6. Formation of MII from its predecessor, metarhodopsin I (MI), accompanies a large shift in the absorption maximum (first ‘bleached’ product of rhodopsin 6), the breakage of the stabilizing salt bridge between the negatively charged side chain of Glu113 and the protonated Schiff base between Lys296 and retinal 31, 32, and the motion of transmembrane helices 33, 34. All of these events occur within the

Formation of the signaling state

The positive enthalpy (ΔH, Fig. 5a) of MII formation indicates that molecular interactions built up in MI are lost upon transition to MII. To drive the conversion, the entropy, and thus the overall disorder in the protein, must increase. This observation would be consistent with the idea that formation of the active state is merely a release of constraints in the helix bundle, thus exposing cytoplasmic binding sites. Experimentally, it was indeed found that the insertion of successively

Rhodopsin as a model for other GPCRs

In the rhodopsin family of GPCRs, biogenic amine ligands are analogs of the retinal structure, with cationic ammonium groups at one end and a ring-like structure at the other 49. The inverse agonist 11-cis-retinal is covalently linked to the protein via a flexible Lys side chain. The ground state structure of rhodopsin provides clues as to how this ‘safety cord’ ensures a permanent occupancy of the ligand-binding site and stabilizing interactions, including a salt bridge-like stable apposition

Concluding remarks

Photoreceptors are designed for the extreme conditions of very low and high activities in dark and light, respectively. The strong constraints seen in the ground-state structure 4 and the early energy-rich intermediates, which can only be reached by uptake of photonic energy, reveal the necessary barriers against spontaneous activation. However, many of the structural details are similar to those in other GPCRs, and the late meta intermediates show close analogies to the low- and high-affinity

Acknowledgements

We wish to thank Thaddeus Dryja for the list of retinitis pigmentosa mutations, HartmutLücke, TorstenSchöneberg and John Spudich for discussions. Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 449), the Fonds der Chemischen Industrie, National Eye Institute (EY09339), Research to Prevent Blindness, Inc. (RPB) to the Dept of Ophthalmology at the University of Washington, Foundation Fighting Blindness, Inc., the Ruth and Milton Steinbach Fund, and the E.K. Bishop Foundation.

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