G-protein-coupled receptor rhodopsin regulates the phosphorylation of retinal insulin receptor

We have shown previously that phosphoinositide 3-kinase in the retina is activated in vivo through light-induced tyrosine phosphorylation of the insulin receptor (IR). The light effect is localized to photoreceptor neurons and is independent of insulin secretion (Rajala, R. V., McClellan, M. E., Ash, J. D., and Anderson, R. E. (2002) J. Biol. Chem. 277, 43319-43326). These results suggest that there exists a cross-talk between phototransduction and other signal transduction pathways. In this study, we examined the stage of phototransduction that is coupled to the activation of the IR. We studied IR phosphorylation in mice lacking the rod-specific alpha-subunit of transducin to determine if phototransduction events are required for IR activation. To confirm that light-induced tyrosine phosphorylation of the IR is signaled through bleachable rhodopsin, we examined IR activation in retinas from RPE65(-/-) mice that are deficient in opsin chromophore. We observed that IR phosphorylation requires the photobleaching of rhodopsin but not transducin signaling. To determine whether the light-dependent activation of IR is mediated through the rod or cone transduction pathway, we studied the IR activation in mice lacking opsin, a mouse model of pure cone function. No light-dependent activation of the IR was found in the retinas of these mice. We provide evidence for the existence of a light-mediated IR pathway in the retina that is different from the known insulin-mediated pathway in nonneuronal tissues. These results suggest that IR phosphorylation in rod photoreceptors is signaled through the G-protein-coupled receptor rhodopsin. This is the first study demonstrating that rhodopsin can initiate signaling pathway(s) in addition to its classical phototransduction.     

Internal hydration increases during activation of the G-protein-coupled receptor rhodopsin

Rhodopsin, the membrane protein responsible for dim-light vision, until recently was the only G-protein-coupled receptor (GPCR) with a known crystal structure. As a result, there is enormous interest in studying its structure, dynamics, and function. Here we report the results of three all-atom molecular dynamics simulations, each at least 1.5 μs, which predict that substantial changes in internal hydration play a functional role in rhodopsin activation. We confirm with ¹H magic angle spinning NMR that the increased hydration is specific to the metarhodopsin-I intermediate. The internal water molecules interact with several conserved residues, suggesting that changes in internal hydration may be important during the activation of other GPCRs. The results serve to illustrate the synergism of long-time-scale molecular dynamics simulations and NMR in enhancing our understanding of GPCR function.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

Three-dimensional model for meta-II rhodopsin,an activated G-protein-coupled receptor

A novel approach that iteratively combined the results of energy calculations and experimental data was used to generate a three-dimensional (3D) model of the photoactivated state (R*) of bovine rhodopsin (Rh). The approach started with simplified energy calculations in an effort to find a set of sterically and energetically reasonable options for transmembrane (TM) helix arrangements with all-trans-retinal. Various 3D models of TM helix packing found by computations were then compared to limited site-directed spin-label experimental data regarding the transition of the TM helices of Rh in the inactive state (R) to those in the R* state to identify the most plausible model of the TM helical bundle. At the next step, all non-TM structural elements, such as the non-TM helix 8, the N- and C-terminal fragments, and the loops connecting TM helices, were reconstructed, and after the entire R* structure had been relaxed, all other currently available additional experimental data, both mutational and spectroscopic, on the structure of the meta-II state of rhodopsin were used to validate the resulting 3D model.     

Solid state 15N NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin.

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [alpha,epsilon-15N2]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [alpha, epsilon-15N2]Lys-rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [alpha, epsilon-15N2]Lys-rhodopsin resonates with an isotropic shift sigmaI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, numax, associated with the pi-pi transition of the polyene chromophore. Experimental modeling of the relation between the numax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center-center distance (deff) between the counterion and the pSB of 0.43 +/- 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.     

Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin

Low resolution electron density maps have revealed the general orientation of the transmembrane helices of rhodopsin. However, high resolution structural information for the transmembrane domain of the G-protein-coupled receptor, rhodopsin, is as yet unavailable. In this study, a high resolution solution structure is reported for a 15 residue portion of the sixth transmembrane helix of rhodopsin (rhovih) as a free peptide. Helix 6 is one of the transmembrane helices of rhodopsin that contains a proline (amino acid residue 267) and the influence of this proline on the structure of this transmembrane domain was unknown. The structure obtained shows an alpha-helix through most of the sequence. The proline apparently induces only a modest distortion in the helix. Previously, the structure of the intradiskal loop connected to helix 6 was solved. The sequence of this loop contained five residues in common (residues 268-272) with the peptide reported here from the rhovih. The five residues in common between these two structures were superimposed to connect these two structures. The superposition showed a root mean square deviation of 0.2 A. Thus, this five residue sequence formed the same structure in both peptides, indicating that the structure of this region is governed primarily by short range interactions.     

Membrane model for the G-protein-coupled receptor rhodopsin: hydrophobic interface and dynamical structure

Rhodopsin is the only member of the pharmacologically important superfamily of G-protein-coupled receptors with a known structure at atomic resolution. A molecular dynamics model of rhodopsin in a POPC phospholipid bilayer was simulated for 15 ns, revealing a conformation significantly different from the recent crystal structures. The structure of the bilayer compared with a protein-free POPC control indicated hydrophobic matching with the nonpolar interface of the receptor, in agreement with deuterium NMR experiments. A new generalized molecular surface method, based on a three-dimensional Voronoi cell construction for atoms with different radii, was developed to quantify cross-sectional area profiles for the protein, lipid acyl chains and headgroups, and water. Thus, it was possible to investigate the bilayer deformation due to curvature of the individual lipid monolayers. Moreover, the generalized molecular surface derived hydrophobic interface allowed benchmarking of the hydropathy sequence analysis, an important structural genomics tool. Five water molecules diffused into internal hydration sites during the simulation, yielding a total of 12 internal waters. The cytoplasmic loops and the C-terminal tail, containing the G-protein recognition and protein sorting sequences, exhibited a high mobility, in marked contrast to the extracellular and transmembrane domains. The proposed functional coupling of the highly conserved ERY motif to the lipid-water interface via the cytoplasmic loops provides insight into lipid effects on G-protein-coupled receptor activation in terms of a flexible surface model, involving the spontaneous monolayer curvature.     

Insights into the activation mechanism of the visual receptor rhodopsin

Recent advances in the structural biology of GPCRs (G-protein-coupled receptors) have provided insights into their structure and function. Comparisons of the visual and ligand-activated receptors highlight the unique elements of rhodopsin that allow it to function as a highly sensitive dim-light photoreceptor in vertebrates, as well as the common elements that it shares with the large class A GPCR family. However, despite progress, a number of questions remain unanswered about how these receptors are activated.     

Modeling of the complex between transducin and photoactivated rhodopsin, a prototypical G-protein-coupled receptor

Obtaining a reliable 3D model for the complex formed by photoactivated rhodopsin (R*) and its G-protein, transducin (Gtalphabetagamma), would significantly benefit the entire field of structural biology of G-protein-coupled receptors (GPCRs). In this study, we have performed extensive configurational sampling for the isolated C-terminal fragment of the alpha-subunit of transducin, Gtalpha 340-350, within cavities of photoactivated rhodopsin formed by different energetically feasible conformations of the intracellular loops. Our results suggested a new 3D model of the rhodopsin-transducin complex that fully satisfied all available experimental data on site-directed mutagenesis of rhodopsin and Gtalphabetagamma as well as data from disulfide-linking experiments. Importantly, the experimental data were not used as a priori constraints in model building. We performed a thorough comparison of existing computational models of the rhodopsin-transducin complex with each other and with current experimental data. It was found that different models suggest interactions with different molecules in the rhodopsin oligomer, that providing valuable guidance in design of specific novel experimental studies of the R*-Gtalphabetagamma complex. Finally, we demonstrated that the isolated Gtalpha 340-350 fragment does not necessarily bind rhodopsin in the same binding mode as the same segment in intact Gtalpha.     

Modeling flexible loops in the dark-adapted and activated states of rhodopsin, a prototypical G-protein-coupled receptor

Conformational possibilities of flexible loops in rhodopsin, a prototypical G-protein-coupled receptor, were studied by modeling both in the dark-adapted (R) and activated (R*) states. Loop structures were built onto templates representing the R and R* states of the TM region of rhodopsin developed previously (G. V. Nikiforovich and G. R. Marshall. 2003. Biochemistry. 42:9110). Geometrical sampling and energy calculations were performed for each individual loop, as well as for the interacting intracellular loops IC1, IC2, and IC3 and the extracellular loops EC1, EC2, and EC3 mounted on the R and R* templates. Calculations revealed that the intra- and extracellular loops of rhodopsin possess low-energy structures corresponding to large conformational movements both in the R and R* states. Results of these calculations are in good agreement with the x-ray data available for the dark-adapted rhodopsin as well as with the available experimental biophysical data on the disulfide-linked mutants of rhodopsin. The calculated results are used to exemplify how the combined application of the results of independent calculations with emerging experimental data can be used to select plausible three-dimensional structures of the loops in rhodopsin.     

Studies on the structure of the G-protein-coupled receptor rhodopsin including the putative G-protein binding site in unactivated and activated forms

Activation of G-protein coupled receptors (GPCR) is not yet understood. A recent structure showed most of rhodopsin in the ground (not activated) state of the GPCR, but the cytoplasmic face, which couples to the G protein in signal transduction, was not well-defined. We have determined an experimental three-dimensional structure for rhodopsin in the unactivated state, which shows good agreement with the crystal structure in the transmembrane domain. This new structure defines the cytoplasmic face of rhodopsin. The G-protein binding site can be mapped. The same experimental approach yields a preliminary structure of the cytoplasmic face in the activated (metarhodopsin II) receptor. Differences between the two structures suggest how the receptor is activated to couple with transducin.     

Cooperative conformational changes in a G-protein-coupled receptor dimer, the leukotriene B(4) receptor BLT1

We have used an isolated receptor, the leukotriene B(4) receptor BLT1, to analyze the mechanism of receptor activation in a G-protein-coupled receptor dimer. The isolated receptor is essentially a dimer whether the agonist is present or not, provided the detergent used stabilizes the inactive dimeric assembly. We have produced a receptor mutant where Cys(97) in the third transmembrane domain has been replaced by a serine. This mutation leads to an approximately 100-fold decrease in the affinity for the agonist. 5-Hydroxytryptophan has then been introduced at position 234 in the C97A mutant sixth transmembrane domain. Agonist binding to the labeled receptor is associated with variations in the fluorescence properties of 5-hydroxytryptophan due to specific agonist-induced conformational changes. The C97A mutant labeled with 5-hydroxytryptophan has then been associated with a wild-type receptor in a dimeric complex that has been subsequently purified. The purified complex activates its G-protein partner in a similar manner as the wild-type homodimer. Due to the difference in the affinity for the agonist between the wild-type and mutant protomers in this dimer, we have been able to reach a state where one of the protomers, the mutant, is in its unliganded state, whereas the other, the wild type, is loaded with the agonist. We show that agonist binding to the wild-type receptor induces specific changes in the conformation of the unliganded protomer, as evidenced by the variations in the emission of the 5-hydroxytryptophan residue in the mutant receptor. These data provide a direct demonstration for agonist-induced cooperative conformational changes in a GPCR dimer.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors

G-protein-coupled receptors (GPCRs) participate in virtually all physiological processes. They constitute the largest and most structurally conserved family of signaling molecules. Several class C GPCRs have been shown to exist as dimers in their active form and growing evidence indicates that many, if not all, class A receptors also form dimers and/or higher-order oligomers. High-resolution crystal structures are available only for the detergent-solubilized light receptor rhodopsin (Rho), the archetypal class A GPCR. In addition, Rho is the only GPCR for which the presumed higher-order oligomeric state has been demonstrated, by imaging native disk membranes using atomic force microscopy (AFM). Based on these data and the X-ray structure, an atomic model of Rho dimers has been proposed, a model that is currently scrutinized in various ways. AFM has also been used to measure the forces required to unfold single Rho molecules, thereby revealing which residues are responsible for Rho's stability. Recent functional analyses of fractions from solubilized disk membranes revealed that higher-order Rho oligomers are the most active species. These and other results have enhanced our understanding of GPCR structure and function.     

Roles of G-protein-coupled receptor dimerization

The classical idea that G-protein-coupled receptors (GPCRs) function as monomeric entities has been unsettled by the emerging concept of GPCR dimerization. Recent findings have indicated not only that many GPCRs exist as homodimers and heterodimers, but also that their oligomeric assembly could have important functional roles. Several studies have shown that dimerization occurs early after biosynthesis, suggesting that it has a primary role in receptor maturation. G-protein coupling, downstream signalling and regulatory processes such as internalization have also been shown to be influenced by the dimeric nature of the receptors. In addition to raising fundamental questions about GPCR function, the concept of dimerization could be important in the development and screening of drugs that act through this receptor class. In particular, the changes in ligand-binding and signalling properties that accompany heterodimerization could give rise to an unexpected pharmacological diversity that would need to be considered.     

Localization of light-induced conformational changes in bovine rhodopsin

Conformational changes in the extradiscal regions of rhodopsin induced by illumination were investigated by modifying the visual pigment by mild treatment with cyanogen bromide prior to and after light exposure. Light induced an increased yield of cleavage of the Met bond 253-254 and a new cleavage at the Met bond 155-156 of the rhodopsin polypeptide chain. These residues, located at the beginnings of the membrane-buried helices 6 and 4, respectively, were concluded to become extradiscally exposed upon illumination.     

Molecular physiology of visual pigment rhodopsin

The key physiological functions of the rhodopsin molecule are reviewed. Molecular mechanisms of visual pigments spectral tuning, photoisomerization of the 11-cis-retinal chromophore that triggers the phototransduction process, formation of physiologically active state of rhodopsin as a G-protein-coupled receptor, rhodopsin visual cycle, and consequences of its impairment are evaluated. Visual pigment rhodopsin performs several functions, providing spectral sensitivity of photoreceptor cells, phototransduction processes and light and dark adaptation. Genetically determined defects of visual pigment molecule and proteins involved into mechanisms of phototransduction and adaptation or into mechanism of visual cycle are directly linked to pathogenesis of different forms of degenerative retina diseases. Understanding the molecular mechanisms of these physiological processes uncovers the way to direct investigation of pathogenesis of these severe eye diseases.     

Prediction of G-protein-coupled receptor classes

Being the largest family of cell surface receptors, G-protein-coupled receptors (GPCRs) are among the most frequent targets of therapeutic drugs. The functions of many of GPCRs are unknown, and it is both time-consuming and expensive to determine their ligands and signaling pathways. This forces us to face a critical challenge: how to develop an automated method for classifying the family of GPCRs so as to help us in classifying drugs and expedite the process of drug discovery. Owing to their highly divergent nature, it is difficult to predict the classification of GPCRs by means of conventional sequence alignment approaches. To cope with such a situation, the CD (Covariant Discriminant) predictor was introduced to predict the families of GPCRs. The overall success rate thus obtained by jack-knife test for 1238 GPCRs classified into three main families, i.e., class A-"rhodopsin like", class B-"secretin like", and class C-"metabotrophic/glutamate/pheromone", was over 97%. The high success rate suggests that the CD predictor holds very high potential to become a useful tool for understanding the actions of drugs that target GPCRs and designing new medications with fewer side effects and greater efficacy.