This invited paper is part of the Symposium-in-Print: Photobiology in Asia.
Mechanism of G-protein Activation by Rhodopsin†
Article first published online: 26 FEB 2007
Photochemistry and Photobiology
Volume 83, Issue 1, pages 70–75, January/February 2007
How to Cite
Shichida, Y. and Morizumi, T. (2007), Mechanism of G-protein Activation by Rhodopsin. Photochemistry and Photobiology, 83: 70–75. doi: 10.1562/2006-03-22-IR-854
- Issue published online: 26 FEB 2007
- Article first published online: 26 FEB 2007
- Received 22 March 2006; accepted 15 June 2006; published online 26 June 2006
Rhodopsin is a member of the family of G-protein-coupled receptors (GPCRs), and is an excellent molecular switch for converting light signals into electrical response of the rod photoreceptor cells. Light initiates cis–trans isomerization of the retinal chromophore of rhodopsin and leads to the formation of several thermolabile intermediates during the bleaching process. Recent investigations have identified spectrally distinguishable two intermediate states that can interact with the retinal G-protein, transducin, and have elucidated the functional sharing of these intermediates. The initial contact with GDP-bound G-protein occurs in the meta-Ib intermediate state, which has a protonated Schiff base as its chromophore. The meta-Ib intermediate in the complex with the G-protein converts to the meta-II intermediate with releasing GDP from the α-subunit of the G protein. Meta-II has a de-protonated Schiff base chromophore and induces binding of GTP to the α-subunit of the G-protein. Thus, the GDP–GTP exchange reaction, namely G-protein activation, by rhodopsin proceeds through at least two steps, with conformational changes in both rhodopsin and the G-protein.
Signal transduction cascades, which consist of receptors, G-proteins and effector enzymes, are ubiquitous in intracellular signaling. The receptors involved in these cascades are called G-protein-coupled receptors (GPCRs). So far, more than 10,000 GPCRs have been identified from various transduction systems. These receptors function to transfer the signals from the outer environment to the interior of the cell by binding with single or multiple subtypes of G-proteins. Among the various GPCRs, rhodopsin, a photoreceptive pigment present in retinal rod photoreceptor cells, is rather unique in that it has the 11-cis-retinal chromophore embedded within the protein moiety (opsin). Upon absorbing a photon of light, the 11-cis-retinal, which is an “inverse agonist”, is converted through cis–trans isomerization to the agonist all-trans-retinal. Advantages of the studies on rhodopsin in comparison with those on other GPCRs are that a large amount of rhodopsin can be isolated and purified from the highly differentiated outer segments of rod photoreceptor cells and that rhodopsin can be synchronously activated by light even at freezing temperatures. In addition, the chromophore acts as an intrinsic visible spectroscopic probe to monitor changes in the global protein structure of rhodopsin during the conversion from inactive to active states. Thus, the rhodopsin-G-protein system is one of the most suitable systems whose activation and coupling mechanisms can be elucidated at submolecular or atomic resolutions. In this article, we review recent studies on the activation mechanism of G-protein (Gt) by rhodopsin, which give new insights into the general mechanism of G-protein activation by GPCRs.
Structural motif of rhodopsin
Rhodopsin is a ∼40 kDa membrane protein which consists of a single polypeptide, opsin, and a chromophore, 11-cis-retinal (1). The three-dimensional structure of rhodopsin has been recently determined by X-ray crystallography (2,3) (Fig. 1), confirming that rhodopsin has the seven transmembrane α-helices and loop regions implicated by many previous biochemical and biophysical investigations. Additionally, there are several new and important findings from the X-ray crystallography, as follows: First, the putative 4th loop region, from the cytoplasmic border of helix VII to the positions of two cysteine residues (Cys322, Cys323) which are anchored in the plasma membrane by two palmitoyl substituents, forms a distinct α-helical structure (termed helix VIII). Second, a part of the second extracellular loop comes deeply into the center of the rhodopsin molecule, and the residues at positions 177–181 form an antiparallel β-sheet with the residues at positions 186–190. The latter sheet forms a part of the chromophore-binding pocket. The 11-cis-retinal is covalently bound to Lys296 via a protonated Schiff base, and the positive charge on the Schiff base is stabilized by the negatively charged counterion Glu113. There is a hydrogen-bonding network system, that includes Glu113, Glu181, and water molecules (3), which has been speculated to play an important role in the formation of the active state of rhodopsin (4). In addition, the 11-cis-retinal chromophore forms multiple interactions within the binding pocket to decrease the flexibility of protein. This is in agreement with the recent biochemical finding that 11-cis-retinal acts as an inverse agonist of rhodopsin (5).
From the biochemical and molecular biological analyses of rhodopsin and other GPCRs, two motifs, in particular, have been shown to be important for the G-protein activation mechanism: (1) the Glu(Asp)/Arg/Tyr triad, located at the interface between helix III and the cytoplasmic loop 3 and (2) the Asn/Pro/X/X/Tyr sequence, located at the cytoplasmic border of the helix VII. These motifs are highly conserved among GPCRs (6). According to the crystal structure of rhodopsin, the Arg residue in the former motif forms an electrostatic interaction with Glu247 and Thr251 in the cytoplasmic border of Helix VI. The Asn residue in the latter motif interacts with Asp83 (Helix II) and Asn55 (Helix III) via water molecules and keeps the structure more stable (7).
Intermediate states of rhodopsin and interaction with Gt
On absorption of light, rhodopsin is bleached (fades in color from red to pale yellow) through several thermolabile intermediates. This process is referred to as the “photobleaching process” and is composed of a photochemical reaction and subsequent thermal reactions (Fig. 2). The initial event is the cis–trans photo-isomerization of the chromophore, which starts at about 60 fs (8) and is completed within 200 fs (9,10) in the excited state of rhodopsin. As the isomerization occurs in the restricted chromophore binding site, isomerized retinal should be in a highly twisted conformation. This causes an elevation of the potential energy (11), the extent of which is about 60% of the absorbed light energy. By utilizing this potential energy, rhodopsin changes its conformation and finally dissociates into all-trans-retinal and opsin.
Among the intermediates described in Fig. 2, meta-II is the only intermediate which has a de-protonated chromophore and forms an equilibrium mixture with its precursor, meta-I (12). An important point is that meta-II has been regarded as the intermediate state which binds to and activates G-protein. This conclusion was drawn from several experimental facts. First, the addition of Gt to the equilibrium mixture caused a shift of the equilibrium in favor of meta-II, resulting in the formation of excess meta-II (referred to as “extra–meta-II”) (13,14). Second, the formation of extra–meta-II was abolished in the presence of GTP (14,15). Thus, it was thought that meta-II formed a complex with Gt in the absence of GTP, dissociating from Gt through the GDP–GTP exchange reaction (Gt activation) in the presence of GTP. However, subsequent experiments showed that the formation of extra–meta-II was also abolished in the presence of GDP (16), indicating that the Gt that forms the complex with meta-II is in a nucleotide-unbound (empty) form and that the binding of the guanine nucleotide (either GDP or GTP) to Gt causes dissociation from meta-II. Since Gt in its resting state is in a GDP-bound form, prior to forming the GDP-free complex with meta-II, it could form a complex with a state other than meta-II, where GDP is still bound to Gt.
Various techniques have been used in extensive studies to determine what kinds of conformational changes in rhodopsin are essential for the Gt activation. Important findings were obtained from the site-directed spin labeling (17,18) and photo-affinity cross-linking (19) experiments performed on the cytoplasmic region of rhodopsin. These studies revealed that the activation of rhodopsin causes a rearrangement of helices, resulting in the separation of the cytoplasmic end of helices III and VI from each other (17,18). These conformational changes originate from absorption of the light energy by the rhodopsin chromophore. Studies using a retinal analog, which has a photo-affinity substituent on its β-ionone ring, showed that the β-ionone ring of the chromophore flips over in the helical bundle by utilizing the energy stored as a highly strained chromophore structure. The position of the β-ionone ring changes from around Trp265 in helix VI to around Ala169 in helix IV (19). Thus, the light-induced positional change of the chromophore in the rhodopsin molecule induces the rearrangement of the helices.
Interaction sites between rhodopsin and Gt
Sites of interaction of photo-activated rhodopsin with Gt have been revealed by biochemical and spectroscopic studies in combination with site-directed mutagenesis and peptide inhibition (20–25) experiments. The major interaction sites were found to be located at the second, third and fourth cytoplasmic loops and at the C-terminal tail. On the other hand, the interaction sites of Gt with photo-activated rhodopsin are the C-terminus, N-terminal helix and the α4–β6–α5 region of the α-subunit of Gt (Gtα) and C-terminal region of γ-subunit of Gt (Gtγ) with its farnesyl modification. These regions should interact among each other when rhodopsin changes its conformation upon photon absorption. The crystal structure of rhodopsin in the ground state indicates that the N-terminus of loop 2, C-terminus of loop 3 and N-terminus of loop 4 form a “core” at the center of the cytoplasmic surface of the molecule (26), while the C-terminus of Gtγ is apart from the other putative interaction sites of the Gtα in the crystal structure of Gt (Fig. 3a) (27). Detailed biochemical analyses suggested that the C-terminus and N-terminus of the Gtα interact with the C-terminus of cytoplasmic loop 3 and N-terminus of cytoplasmic loop 4 (28–31). Thus, the interaction sites in the Gtα will bind to the “core” region, and the C-terminus of Gtγ would be bound to another part of the interaction surface of active rhodopsin (Fig. 3b).
Two-step interaction mechanism found in rhodopsin-Gt system
The GDP–GTP exchange reaction proceeds through at least three steps, namely, (1) the binding of photo-activated rhodopsin to GDP-bound Gt, (2) the dissociation of GDP from the rhodopsin-Gt complex, and (3) the binding of GTP to the nucleotide-unbound Gt. As already described, Gt in the meta-II-Gt complex is in the nucleotide-unbound form, indicating that the complex is the state between steps (2) and (3). Therefore, it is important to identify the rhodopsin state(s) responsible for the initial binding to the GDP-bound Gt.
As the Gt in the complex with meta-II is in the nucleotide-unbound form, it is probable that the intermediate state, which binds to the GDP-bound form of Gt, would be a precursor of meta-II. Thus, we have extensively analyzed the formation process of meta-II in the chicken rhodopsin system by means of low temperature time-resolved spectroscopy (32). With the aid of singular-value decomposition (SVD), we found an intermediate state that formed in between the formerly identified meta-I and meta-II intermediates. Because this newly identified intermediate exhibited an absorption maximum about 20 nm blue shifted from that of meta-I, but considerably different from that of meta-II, we named it meta-Ib and renamed meta-I as meta-Ia (see Fig. 4a). More importantly, meta-Ib was stabilized in the presence of Gt, and this stabilization was sustained even in the presence of GTP. Stabilization of meta-II by Gt was abolished, however. Thus, we concluded that meta-Ib can bind to Gt but cannot induce the GDP–GTP exchange reaction on Gt. The existence of meta-Ib and its properties were confirmed in the bovine rhodopsin system (33), which emphasized that the meta-Ib would be a common intermediate of rhodopsin during its activation of Gt. However, because of the limited time resolution of our spectroscopic techniques at that time, we could only identify this intermediate by using low-temperature spectroscopy and a detergent-solubilized rhodopsin–glycerol mixture. Therefore, the identification of this intermediate under physiological conditions and the elucidation of its characteristics, distinct from those of meta-II, are important for our further understanding of the molecular mechanism of G-protein activation by rhodopsin.
Fortunately, we succeeded in developing a cooled CCD spectrophotometer with a time resolution of 9.7 ms to identify meta-Ib in rod outer segment (ROS) membranes at physiological temperatures (Fig. 4a,b). The results showed that meta-Ib exists and interacts with Gt under physiological conditions in ROS membranes (34) (Fig. 4c). It was also found that meta-Ib interacts with Gt, even in the presence of excess GDP, such that extra–meta-II was no longer formed (Fig. 4c). These results suggest that meta-Ib is a physiologically relevant intermediate that interacts with Gt in a manner different from meta-II. The EC50 value of meta-Ib binding to Gt (in the presence of GDP) was estimated to be two times larger than that of meta-II binding to Gt (in the absence of GDP) (34). The Hill coefficients are approximately 2 in both cases. The higher affinity (lower EC50 value) binding of meta-II to Gt can account for the formation of extra–meta-II in the presence of Gt, even with the existence of an interaction between meta-Ib and Gt. It should be noted that the different EC50 values of these intermediates indicate the different characteristics of their binding sites. We previously reported that meta-Ib binds to the C-terminus of Gtα in a manner different from that of meta-II (35). This means that only one of the two leucine residues at the C-terminus of Gtα is needed for binding to meta-Ib; however, both of them are required for binding to meta-II. These differences demonstrate that the binding affinity of meta-II is higher than that of meta-Ib. Although the value of the Hill coefficient does not correctly reveal how many binding sites participate in the interaction, the fact that the Hill coefficients are the same may suggest that the difference in binding affinity between these states originates from the sum of the small differences in each binding site. To our knowledge, this is the first time that the binding state has been identified for the GDP-bound G-protein in either the rhodopsin system or in other GPCR systems that are activated by diffusible agonist ligand.
Predicted two-step interaction model in the activation of Gt by rhodopsin
We identified two intermediate states that interact with Gt in a manner different from each other. By using engineered cross-linkages and spin-labeling, it was demonstrated that meta-II has the protein structure that is created by the separation of the cytoplasmic ends of helix III and VI (17,18), while the preceding protonated intermediate does not have a similar arrangement (36). Based on our findings, as well as those mentioned above, it can be suggested that the massive helical rearrangement is not necessary for the binding of the rhodopsin intermediate to Gt but is essential for the GDP–GTP exchange reaction on Gt. In this sense, we speculate that the meta-Ib-like structure would be regarded as “the Gt/GDP binding state with minimum helix rearrangement,” while the extra–meta-II-like structure would be viewed as “the Gt activating state with massive helix rearrangement” (Fig. 5). The conformational change from the former state to the latter state would be necessary for the dissociation of GDP from Gt. Thus, future investigation into the structural difference between these states could elucidate the essence of the activation mechanism, with the exception of the helical movement in the meta-II state.
NMR and FTIR studies revealed that both of the C-terminal peptides of Gtα (37,38) and Gtγ (39) form the specific α-helical structures when they bind to meta-II. Combined with our finding that the interaction of the C-terminal peptide of Gtα with meta-Ib is different from that with meta-II (35), these results suggest that α-helix formation of the C-terminal peptide would be concerted with the separation of helices III and VI that occurs during the formation process of meta-II. Although we do not have strict evidence that meta-Ib is the only intermediate that can bind to the GDP-bound form of Gt, further investigation would give insight into the concerted conformational changes of receptor and G-protein during the process of G-protein activation.
We have identified meta-Ib as the intermediate of rhodopsin that binds to the GDP-bound form of Gt. However, because of the limitation of the present experiments, we cannot exclude the possibility that meta-II has multiple conformations and that one of them could bind to the GDP-bound form of Gt. As de-protonation of the Schiff base in meta-II extinguishes the connection between the Schiff base and counterion, it is reasonable to assume that the protein would be more flexible. In addition, the spectral property of the de-protonated Schiff base is less sensitive to the surrounding environments than that of the protonated Schiff base. Therefore, it is not curious that there are multiple conformations in meta-II with similar spectroscopic characteristics. In fact, the possibility that there are at least two states in meta-II is reported by several groups from evidence that meta-II uptakes a proton after its formation and that the formation rate of meta-II cannot be assigned as a single exponential process (40–42). It is possible that one of these states behaves similarly to meta-Ib in the interaction with Gt. On the other hand, it can certainly be expected that the more we investigate the molecular properties of meta-Ib, the more we understand about the dynamic activation mechanism of G-protein in rhodopsin and in GPCRs in general.
Acknowledgements— We thank Drs. Akihisa Terakita, Hiroo Imai and Takahiro Yamashita for helpful discussions.
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