In dark rhodopsin, a salt bridge, termed the ionic lock, is made between R 3. Collectively, these observations suggest that the salt bridge stabilizes the inactive state of family A GPCRs but forms transiently in receptors that display basal activity. Consistent with this notion, the mutations D 3. As described below, R 3. The importance of the salt bridge in stabilizing the inactive state is further illustrated by the virally encoded US28 chemokine receptor, a constitutively active GPCR, in which E 3.
The packing of R 3. Bending near the cytoplasmic end of TM6 results in an electrostatic interaction between R 3. Polar interactions are shown with dashed lines. The interactions of Y 5. The conserved D 3. D 3. The interaction of D E 3. Y 3. The changes in the DRY motif also indirectly enable other contacts with the bound G protein. For example, L 6. A second conserved motif, NP 7.
This motif does not interact directly with the bound G protein but is essential for forming the active conformation. Owing to the break in hydrogen bonding, TM7 can rotate at P 7. This moves Y 7. Also, the last turn of the helix in TM7 in the inactive structure unravels as part of this transition.
The NP 7. Water-mediated networks that connect side chains of conserved polar residues as well as the backbone, most prominently on the cytoplasmic halves of TM2, TM3, TM6, and TM7, have been observed in a number of GPCR crystal structures 34 Figure 5. For example, in the inactive state, water molecules link the conserved N 7. In the active conformation, N 7.
Closer to the orthosteric site and connector that links the orthosteric and G protein sites see below , N 3. Although the resolution of many GPCR crystal structures does not allow visualization of water molecules, the conservation of polar residues in the interior of the protein strongly suggests that these water-mediated networks are common to family A GPCRs and must rearrange as part of forming the active state.
The barrier to rearranging hydrogen-bonded water networks is likely lower than that involving extensive repacking of hydrophobic groups. Polar side chains conserved in the family A receptors are shown in stick representation and are labeled with their Ballesteros—Weinstein numbers.
These two receptors are highly homologous, and these structures are at sufficiently high resolution 1. In accord with the diverse ligands with which they interact, the detailed geometry and chemical nature of the ligand-binding sites vary among different GPCRs, and the changes in the sites upon activation also vary.
A general feature of agonist binding relative to inverse agonists is a small compaction of the orthosteric ligand—binding site arising from inward movements of one or more surrounding helices as well as changes in side chain rotamers. In agonists, the ethanolamine moiety is linked to a catechol ring or structures of similar size bearing hydrogen-bonding groups with equivalent spacings; inverse agonists typically contain a larger ring such as the carbazole ring system of carazolol and lack one of these polar groups.
Structures bound to the high-affinity BI agonist as well as to the natural agonist adrenaline epinephrine reveal that agonists form a hydrogen bond with S 5.
This interaction arises from an inward bulge of the TM5 helix enabled by the break in hydrogen bonding at the conserved Pro 5. Antagonists lack this hydrogen-bonding group and are typically bulkier in this region; therefore, they require a more expanded binding site. Binding of the agonist also slightly changes the position of W 6.
Carazolol is shown in blue sticks, and adrenaline in yellow sticks. In panel b , key hydrogen bonds formed with adrenaline are shown with dashed lines. The conserved nonpolar residues P 5. The inward bulge of TM5 near P 5. This in turn requires a shift in the position of F 6. In addition, W 6. The shifts needed to repack W 6.
These changes swing the lower half of TM6 outward, as described above. Similarly, the changes at N 7. Thus, the small changes in the ligand-binding site that form the agonist-bound conformation favor repacking of neighboring residues, which is coupled to the rotations in TM6 and TM7 that produce the G protein—binding conformation.
The rigidity of the helices surrounding the kinks provides a lever arm that amplifies these small changes into the large movements that create the G protein—binding pocket. Data from crystallography, spectroscopy, and MD simulations have provided evidence for stable intermediate states that may link the fully inactive and fully active conformations.
Structural data from several GPCRs strongly suggest that ligand binding to the orthosteric site and the large changes observed in the cytoplasmic portion of the receptor are not strictly coupled. Moreover, no other changes relative to the inactive structure occurred, such as the repacking of the P 5. In contrast, agonist-bound structures of the A 2a R in the absence of a G protein revealed the inward movement and contraction of the orthosteric site and also the altered packing of P 5.
These structural data imply that the relative energies of the different intermediates differ in different receptors. Likely activation intermediate observed in the A 2a R. An intermediate that resembles the agonist-bound intermediate of the A 2a R is seen in these simulations Moreover, changes in the orthosteric site bulging of TM5 at S 5.
The simulations also suggested that the G protein—binding site can leave its inactive conformation before the connector assumes the active conformation Moreover, the G protein or nanobody slows dissociation of the agonist from the orthosteric site Mutation of Y 7.
These observations demonstrate that the G protein can stabilize the high-affinity, agonist-binding conformation of the receptor. Analysis of correlated movements defines allosteric pipelines that couple different regions of the receptor 47 and confirms that the intermediate and active states have fewer correlated movements than the inactive states and are more conformationally heterogeneous.
Overall, the biochemical, structural, and simulation data indicate the formation of stable intermediates in the presence of agonists, an idea that is consistent with the notion that the G protein lowers the free energy of the agonist-bound state and thereby shifts the conformational equilibrium such that the active conformation is significantly populated.
Energy landscapes describe the relative energies of conformational states and the barriers to their interconversion 48 Figure 8. Ligand binding changes the free energy of states and thereby changes their relative populations at equilibrium, so defining the energy landscape that governs the equilibrium among functional GPCR states is essential for understanding GPCR pharmacology.
When combined with structural data, spectroscopically distinct states can be related to particular conformations and thereby deepen our understanding of how ligands activate GPCRs. The gray lines solid and dashed indicate the energy landscape of the ligand-free basal state. The solid black lines indicate the effect of the indicated ligand and G protein on the energy landscape.
The two inactive states detected by spectroscopy are denoted S1 intact ionic lock and S2 broken ionic lock. S3 is the intermediate detected in the presence of an agonist without G protein, and S4 states are active states in the presence of an agonist and a G protein. Single-molecule FRET analysis provides evidence for distinct active states in the presence of an agonist that are dependent on the nucleotide state of the G protein.
NMR spectroscopy using labels at different positions has been extremely powerful for constructing GPCR energy landscapes. For an NMR label at a given site, the observation of peaks at more than one chemical shift value provides evidence for multiple structural states, and measurements of exchange rates between these states provide information about the energy barrier between them.
Spectra measured in the presence of ligands of different efficacies allow assignment of these peaks to a functional conformation.
Differences in the relative peak intensities in the presence of different ligands without changes in chemical shift values provide strong evidence for ligands being able to shift populations among different preexisting conformations.
However, the appearance of peaks at distinct chemical shifts that depend on the ligand is more challenging to interpret. This can arise either from populating conformations that are rarely sampled in the absence of the ligand or from changes in exchange rate between preexisting populations. Double electron-electron resonance DEER spectroscopy, which measures the interaction between spin probe labels, provides information complementary to NMR. DEER provides distance distributions i. Changes in the distance distributions in the presence of different ligands give a readout of the effect of a ligand on the relative populations of the conformations.
Fluorescence resonance energy transfer FRET , which is sensitive to changes in distances between probes, can also be used to monitor conformational changes. DEER probes are also smaller than typical fluorophores, making it less likely that they will perturb the conformational equilibrium Nonetheless, single-molecule FRET has provided important insights into the dynamics of the equilibrium see below. The outward movement of TM6 associated with forming the G protein—binding site is the largest change upon receptor activation and has proven experimentally accessible with different probes.
Early studies employing fluorescent labels at a native cysteine near the cytoplasmic region of TM6 C 6. These experiments also confirmed that receptors sample multiple conformational states in the ligand-free or neutral antagonist—bound basal state as well as when bound to ligands of different efficacies. Two peaks were observed, even in the presence of the strong inverse agonist carazolol. Crucially, the positions and line widths of the NMR peaks were essentially unchanged in the presence of different ligands; only their intensities differed.
These observations demonstrated that ligands promote population shifts between preexisting conformations rather than stabilizing unique conformations. Conformational se-lection has also been observed directly in NMR studies of the A 2a R labeled at an equivalent site As noted above, the ionic lock between R 3.
These spectroscopically defined states, designated S1 and S2 Figure 8 , were equally populated i. In the ligand-free receptor, the populations were also equal but the lifetimes increased and the exchange rate dropped approximately two-fold, indicating that the energy of these states is the same but that the inverse agonist lowers the barrier between them Figure 8.
When a nanobody that mimics the G protein was added to the agonist-bound receptor, a fourth state S4 with a distinct chemical shift was observed. These observations suggest that S3 is an activation intermediate favored by agonist binding but is not the fully active conformation, and they also suggest that the nanobody shifts the equilibrium strongly to the activated conformation Figure 8. Isoproterenol also increases the fraction of receptors in the broken ionic lock state S2 relative to the intact ionic lock state S1 Figure 8.
In the presence of a high-affinity agonist, the inactive and active intermediate conformations exchange slowly, in the millisecond—second regime, indicating a high energy barrier between these functional states Figure 8. In the presence of inverse agonists or neutral antagonists, the receptor was in a high FRET state corresponding to the distances predicted from the inactive structure.
In the presence of partial or full agonists, lower FRET states were observed, indicating relative separation of the fluorophores resulting from an outward displacement of TM6.
The extent to which agonists and partial agonists reduced FRET correlated with ligand efficacy. The single-molecule FRET study also provided evidence for distinct receptor conformational states associated with the nucleotide state of bound G s Nucleotide-free G s dissociated very slowly lifetimes of minutes from receptors bound to partial or full agonists. Thus, in the presence of guanine nucleotides, G s remains associated with the receptor for multiple seconds. These data imply that the receptor accesses conformations that can bind GDP-loaded G s prior to nucleotide exchange, and the post-exchange GTP-bound G s may associate with a distinct receptor conformation.
The structural differences among these states is not known, however. Different GPCRs display differences in their energy landscapes.
In contrast, the A 2a R appears to more readily form an active state, as NMR analysis of the A 2a R labeled with 19 F-BTFA at a position near the cytoplasmic end of TM6 revealed two activated states, as well as two inactive states, in the absence of a ligand Addition of a partial or full agonist shifted the populations to favor these active states, supporting the conformational selection mechanism. Notably, the inactive and active states exchange slowly, in the 1—3 s time frame, and one of the active states is long lived.
Importantly, one active state was stabilized by a partial agonist, whereas the other was stabilized by a full agonist, suggesting that these agonists form distinct conformations even in the presence of a G protein.
The state stabilized by the full agonist was shifted upfield relative to that associated with the partial agonist, indicating that the label at the end of TM6 in the full agonist form is solvent exposed. These data support a model in which partial agonists form a unique, less efficacious state than full agonists, rather than simply not being able to shift the equilibrium as far toward a single active structure. The spectroscopic studies summarized above focused on the large conformational changes associated with formation of the G protein—binding site.
These regions are unlikely to be accessible to labeling by large probes, which also would likely perturb the local structure. The native M82 2. This timescale is too slow to be attributed to side chain rotamer changes and suggests that the two peaks reflect larger conformational differences. In the inactive structure, Met82 2. Thus, it appears that in the inactive state the structure in this region samples at least two conformations, one of which may be an intermediate to the formation of the active state.
Upon addition of the agonist BI and a nanobody mimic of the G protein, most of the methionine peaks shift, indicating a distinct conformation With BI alone, M82 2. Again, this is consistent with loose coupling of agonist and G protein—binding described from MD simulations. These data also show that with an agonist alone, the receptor does not simply populate a mixture of the inactive and active crystallographic conformations but adopts new, intermediate conformations, particularly in the vicinity of M 5.
MD simulations suggest that in this state, TM7 is in an inactive conformation but TM5 and TM6 differ, with their intracellular ends having substantial mobility It is important to note that the intermediates detected spectroscopically likely depend on the probe, so it cannot be rigorously established that different probes are detecting the same intermediate even in the presence of a particular ligand.
The receptor environment has a critical role in determining the dynamics of the receptor. Likewise, Kofuku et al. Transitions between the two inactive conformations and between them and the active conformation observed at Met82 2. Moreover, in the presence of a partial agonist, the population of the active conformation was higher than in detergents. Specific lipids also influence the energy landscape. Isogai et al. Individual valine peaks were assigned by mutational analysis and examined in the presence of an inverse agonist, no ligand, or an agonist.
Changes in the orthosteric site correlated well with contacts made by different classes of ligands. Nonetheless, changes in the back-bone at several key positions in TM5, TM6, and TM7 observed between inactive and activated GPCR crystal structures were detected as chemical shifts, suggesting that agonists can promote an intermediate conformation on the pathway to full activation. The stabilized protein included the mutations Y 5. These positions are critical for forming the active state Figure 4 , and when the native tyrosines were restored, the protein could now activate G protein.
In the presence of a nanobody mimic of a G protein, the protein displayed the expected conformational changes. In addition to highlighting the importance of the conserved tyrosines, these results support the notions that the active state is less stable and that its relative instability is essential for agonist-mediated activation. The loose coupling between agonist and G protein binding sites is not unique to adrenergic receptors. In contrast, lysine residues in ICL1 and helix 8 changed in the presence of an agonist alone, leading to the suggestion that these regions may first engage the G protein before formation of the stable ternary complex.
The mechanism of partial agonism, that is, how certain ligands at saturation activate activity to a level below that of full agonists, is not established. This could mean that, in the presence of a partial agonist, a G protein cannot shift the population as far toward the fully active conformation as it does when the receptor is bound to a full agonist. Conversely, the A 2a R study employing 19 F label at the base of TM6 suggested distinct conformations for the partial and fully active states This difference could be due to the different positions probed in these experiments.
Alternatively, although the partial agonist-bound state observed by NMR might be an intermediate similar to that on the pathway to full activation, it is possible that in the presence of a G protein the partial agonist-bound receptor adopts a unique conformation that is less effective in promoting nucleotide exchange.
Gregorio et al. Overall, it appears that the receptor can adopt even more conformations than those observed using conformational probes at just a few sites and that these conformations probably relate to the ultimate level of efficacy of particular ligands. We have focused on activation of GPCR guanine nucleotide exchange activity. Much less is known about coupling to the arrestin pathway Figure 1b. Recently, the crystal structure of rhodopsin bound to visual arrestin was determined 70 , The receptor displays similar rearrangements on the cytoplasmic side of the TM region that occur as part of G protein binding.
However, the principal contacts made by arrestin are with TM7; in contrast, the G protein does not interact directly with this part of the receptor. In the Liu et al. Interestingly, C 7. Thus, while the pharmacology implies distinct coupling mechanisms for agonists that target arrestin versus those that activate G proteins, these agonists likely share allosteric transmission mechanisms.
Low barriers between different activation intermediate states may enable signaling through different pathways 47 by a given ligand. Higher resolution structures of arrestin complexes with GPCRs that bind to diffusible ligands, as well as spectroscopic characterization, are needed to address these mechanistic problems. This work was supported by a grant from the Mathers Foundation W. National Center for Biotechnology Information , U. Annu Rev Biochem. Author manuscript; available in PMC May William I.
Weis 1, 2 and Brian K. Kobilka 2. Brian K. Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Annu Rev Biochem. See other articles in PMC that cite the published article. Abstract G protein—coupled receptors GPCRs mediate the majority of cellular responses to external stimuli.
Open in a separate window. Figure 1. Figure 2. Active State Crystal Structures For most GPCRs, the active state is not sufficiently stable to crystallize even in the presence of a high-affinity or covalently bound agonist 23 ; the G protein is needed to stabilize a state of lower free energy—in other words, to shift the equilibrium to make the active conformation the dominant one in solution.
Figure 3. Figure 4. Figure 5. The Orthosteric Site In accord with the diverse ligands with which they interact, the detailed geometry and chemical nature of the ligand-binding sites vary among different GPCRs, and the changes in the sites upon activation also vary. Figure 6. Figure 7. In inactive rhodopsin, there is virtually no activity towards the G protein transducin. Absorption of a photon of light converts covalently bound Cisretinal an inverse agonist to all trans retinal a full agonist within femptoseconds.
The structural changes associated with the formation of metarhodopsin II, the active form of rhodopsin, are observed within microseconds of photoactivation [ 79 ]. Photoisomerization of bound retinal is extremely efficient in using the energy of the captured photon to induce protein structure changes. For the vast majority of other GPCRs, activation occurs when an agonist diffuses into an unliganded receptor. In many cases the unliganded receptor has some basal constitutive activity towards a G protein.
Agonists are defined as ligands that fully activate the receptor. Partial agonists induce submaximal activation of the G protein even at saturating concentrations. Inverse agonists inhibit basal activity. Antagonists have no effect on basal activity, but competitively block access of other ligands.
The efficacy of a given drug may vary depending on the signaling pathway being examined [ 83 ]. A number of kinetic models have been developed to explain GPCR activation using information derived from indirect measures of receptor conformation, such as ligand binding affinity and the activation of G proteins or effector enzymes [ 84 — 87 ].
The efficacy of ligands reflects their ability to alter the equilibrium between these two states. The two-state model can describe much of the functional behavior of GPCRs and explain the spectrum of responses to ligands of different efficacy in simple experimental systems consisting of one receptor and one G protein.
However, there is a growing body of experimental evidence for the existence of multiple conformational states [summarized in [ 83 ]]. Within this framework, each ligand may induce or stabilize a unique conformational state that can be distinguished by the activity of that state towards different signaling molecules G proteins, kinases, arrestins. Moreover, there is a rich source of structurally similar ligands having a spectrum of efficacies ranging from inverse agonists to full agonists Fig 3B.
The amine nitrogen interacts with Asp in TM3 [ ], the catechol hydroxyls interact with serines in TM5 [ — ]. Catechol is a very weak partial agonist. Dopamine and salbutamol are partial agonists. Norepinephrine, epinephrine and isoproterenol are full agonists. ICI, is an inverse agonist. Based on these studies we have proposed a working model Figure 4 where agonist binding and conformational changes occur through a sequence of conformational intermediates.
Biophysical support for this model will be briefly summarized below. Sequential Binding Model. Cartoon representing structural components of norepinephrine. In the absence of ligand, the receptor R is conformationally flexible.
Conformational state R 1 is stabilized by interactions between TMs 5 and 6 and the catechol ring. The transition to state R 2 occurs when Asp in TM3 binds the amine nitrogen. The transitions from R to R 2 are rapid. Adapted from Swaminath et al. The resulting minimal cysteine receptor exhibits normal ligand binding and G protein coupling behavior. A new reactive cysteine is introduced into a specific structural domain by site-directed mutagenesis.
Therefore it is possible to monitor ligand-induced conformational changes in different receptor domains, and to correlate ligand structure with specific structural changes in the receptor. An environmentally sensitive fluorophore covalently bound to Cys is well positioned to detect agonist-induced conformational changes relevant to G protein activation. Based on homology with rhodopsin, Cys is located in the third intracellular loop IC3 at the cytoplasmic end of the TM6.
Mutagenesis studies have shown this region of IC3 to be important for G protein coupling [ 90 , 91 ]. Fluorescence lifetime analysis can detect discrete conformational states in a population of molecules, while fluorescence intensity measurements reflect the weighted average of one or more discrete states.
Therefore, lifetime analysis of fluorescein bound to Cys is well suited to capture even short-lived, agonist-induced conformational states. We observed that the full agonist isoproterenol induced a conformation that was distinct from the conformations induced by the partial agonists salbutamol and dobutamine [ 42 ]. These studies also revealed the existence of an intermediate state in equilibrium with the active state for both full agonists and partial agonists. In contrast, the neutral antagonist appeared to stabilize a state that was indistinguishable from the unliganded receptor.
The increase in fluorescence intensity as a function of time following activation by the agonist norepinephrine is best fit with a two component exponential function [ 88 ] Fig 5 A. In contrast, the response to dopamine a partial agonist is adequately fit by a one component exponential function. Of interest, the rapid component of norepinephrine is very similar to the response to dopamine Fig. A rapid component of fluorescence change is observed with all ligands containing a catechol ring and can be observed with catechol alone Fig.
In fact, catechol is a weak partial agonist Fig 5C. Based on this observation we proposed that the catechol-induced conformation might represent an intermediate in the conformational response to dopamine and norepinephrine Fig.
The response to norepinephrine is best fit by a two-site exponential function. The rapid and slow components of the response are illustrated by the dotted lines.
The data presented here are adapted from Swaminath et al. The slow component of the response to norepinephrine Fig. There are several possible explanations for these observed differences in kinetics. The use of a large fluorescence reporter such as CFP does not allow one to determine the nature of the structural change responsible for the change in FRET; therefore, these FRET experiments may be detecting a different conformational switch, possibly similar to the rapid response observed with dopamine and catechol.
It is known that receptors form complexes with G proteins in the plasma membrane precoupling , and that these complexes have a higher affinity for agonists than do receptors alone. Other factors that might contribute to differences between experiments using purified receptor and those on receptors in cells could be the influence of the pH and salt gradients across the plasma membrane in living cells, as well as the asymmetry of plasma membrane lipids.
Nevertheless, while the slow component of the conformational response to agonists may be attributable to the use of purified receptor protein, we believe the structural changes are physiologically relevant because they can be linked to specific interactions between the ligand and the receptor e.
Based on what is known about the binding site for the catechol ring of catecholamines Fig. This conformational change, known as a rotamer toggle switch, has been proposed to be involved in the activation of amine and opsin receptor families [ 97 ]. Upon binding, the aromatic catechol ring of catecholamines would interact directly with the aromatic residues of the rotamer toggle switch, Trp 6. Molecular dynamics simulations suggest that rotamer configurations of Cys 6.
This movement could be detected by tetramethylrhodamine bound to Cys at the cytoplasmic end of TM6. This suggests that the aromatic ring of salbutamol does not occupy the same binding space as catechol and does not activate the rotamer toggle switch [ 89 ].
Thus, the active state induced by salbutamol would be different from that induced by catecholamine agonists [ 89 ]. This is in agreement with fluorescent lifetime experiments discussed above [ 42 ].
This is shown in Fig. This suggests that ICI, does not occupy the catechol binding pocket and does not prevent activation of the rotamer toggle switch by catechol. We investigated another proposed molecular switch, the ionic lock between the Asp 3. Disruption of the ionic lock would allow Trp to contact and quench bimane fluorescence. Our results demonstrated that the disruption of the ionic lock is an obligatory step for maximal receptor activation and is triggered by nearly all agonists, independent of efficacy Fig.
However, we found that disruption of the ionic lock is not directly coupled to the rotamer toggle switch in TM6 since catechol, which is capable of activating the rotamer toggle switch, was not able to activate the ionic lock [ 71 ]. Moreover, salbutamol which does not activate the rotamer toggle switch [ 89 ] is able to fully activate the ionic lock [ 71 ] Fig.
Close up view of the ionic lock and the modifications made to monitor conformational changes in this region. Alanine was mutated to cysteine C and isoleucine was mutated to tryptophan W Upon activation, W moves closer to bimane on C and quenches fluorescence. Emission spectrum of bimane on C before and after activation by the agonist isoproterenol. Effect of different ligands on disruption of the ionic lock as determined by bimane fluorescence. The partial agonists dopamine and salbutamol are as effective at disrupting the ionic lock as the full agonists norepinephrine and isoproterenol.
Only catechol has no effect on the ionic lock. These data are adapted from Yao et al. This is surprising considering that the interaction between the primary amine of dopamine and Asp makes the strongest contribution to the binding energy. Since dopamine and catechol bind with the same affinity, but only dopamine disrupts the ionic lock, part of the binding energy associated with the interaction between dopamine and Asp may be used to offset by the energetic cost of breaking the ionic lock.
Based on these fluorescence studies we proposed a model where agonists stabilize partially or fully active states by using different chemical groups to activate different combinations of molecular switches, which are not necessarily interdependent. In the unliganded inactive state of a GPCR, the arrangement of TM segments is stabilized by non-covalent interactions between side chains.
Structurally distinct ligands are able to break different combinations of the basal state stabilizing interactions either directly by binding to amino acids that are involved in these intramolecular interactions, or indirectly by stabilizing new intramolecular interactions. These ligand-specific conformational changes may be responsible for differential activation of the signaling cascades of the receptor.
The affinity of a particular ligand will then be dependent on the energy costs and gains associated with each disrupted and created interaction, while its efficacy will be dependent on the ability to trigger the switches associated with activation. These molecular switches are normally activated by agonist binding, but will also be revealed in constitutively active mutants, where single point mutations in virtually any structural domain can lead to elevated basal activity[ 99 ].
A better understanding of the process by which ligands bind and modify GPCR structure may ultimately help in the design of more selective drugs with the appropriate efficacy for the desired physiologic function. YY, where X refers to the TM segment and YY to the position relative to the most highly conserved amino acid in the TM segment, which is assigned an arbitrary position of Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication.
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Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. National Center for Biotechnology Information , U. Biochim Biophys Acta. Author manuscript; available in PMC May Brian K.
Author information Copyright and License information Disclaimer. Copyright notice. The publisher's final edited version of this article is available at Biochim Biophys Acta.
See other articles in PMC that cite the published article. Open in a separate window. Figure 1. Three-dimensional crystals More recently, three dimensional crystals structures of rhodopsin have been obtained by several groups [ 21 — 26 ]. Comparison of P4 1 and P3 1 rhodopsin structures The structures obtained from P4 1 and P3 1 crystals are very similar overall, particularly in the transmembrane, and extracellular domains Fig.
Figure 2. Figure 3. Figure 4. Figure 5. Catechol activates of the rotamer toggle switch Based on what is known about the binding site for the catechol ring of catecholamines Fig. Activation of the ionic lock We investigated another proposed molecular switch, the ionic lock between the Asp 3. Figure 6. Conclusions Based on these fluorescence studies we proposed a model where agonists stabilize partially or fully active states by using different chemical groups to activate different combinations of molecular switches, which are not necessarily interdependent.
Ballesteros JA, Weinstein H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein coupled receptors. Meth Neurosci. Transduction of receptor signals by beta-arrestins. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci. Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.
The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol. Orphan G-protein-coupled receptors and natural ligand discovery. Trends Pharmacol Sci. G protein-coupled receptors. Diversity of receptor-ligand interactions. J Biol Chem. Pharmacol Ther. Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site.
BAY a potent non-competitive mGlu1 receptor antagonist with inverse agonist activity. The non-competitive antagonists 2-methyl phenylethynyl pyridine and 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester interact with overlapping binding pockets in the transmembrane region of group I metabotropic glutamate receptors. CPCCOEt, a noncompetitive metabotropic glutamate receptor 1 antagonist, inhibits receptor signaling without affecting glutamate binding.
Ray K, Northup J. The three-dimensional structure of bovine rhodopsin determined by electron cryomicroscopy. Characterisation of an improved two-dimensional p crystal from bovine rhodopsin. J Mol Biol. Schertler GF. Structure of rhodopsin. Arrangement of rhodopsin transmembrane alpha-helices.
Projection structure of rhodopsin. An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors. X-Ray diffraction analysis of three-dimensional crystals of bovine rhodopsin obtained from mixed micelles. J Struct Biol. Crystal structure of rhodopsin: A G protein-coupled receptor [see comments] Science.
Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography. Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors GPCRs Biochemistry. The retinal conformation and its environment in rhodopsin in light of a new 2. Structure of bovine rhodopsin in a trigonal crystal form. Highly selective separation of rhodopsin from bovine rod outer segment membranes using combination of divalent cation and alkyl thio glucoside.
Photochem Photobiol. Crystals of native and modified bovine rhodopsins and their heavy atom derivatives. Structure of rhodopsin and the metarhodopsin I photointermediate. Curr Opixn Struct Biol. Structure of human follicle-stimulating hormone in complex with its receptor.
Shi L, Javitch JA. The binding site of aminergic G protein-coupled receptors: the transmembrane segments and second extracellular loop. Annu Rev Pharmacol Toxicol. Metal-ion sites as structural and functional probes of helix-helix interactions in 7TM receptors. Ann N Y Acad Sci. Structural mimicry in G protein-coupled receptors: implications of the high-resolution structure of rhodopsin for structure-function analysis of rhodopsin-like receptors.
Rhodopsin crystal: new template yielding realistic models of G-protein-coupled receptors? Tate CG, Grisshammer R. Heterologous expression of G-protein-coupled receptors. Trends Biotechnol. Massotte D. G protein-coupled receptor overexpression with the baculovirus-insect cell system: a tool for structural and functional studies. Heterologous expression of G-protein-coupled receptors: comparison of expression systems fron the standpoint of large-scale production and purification.
Cell Mol Life Sci. Improved purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem Soc Trans. Weiss HM, Grisshammer R. Purification and characterization of the human adenosine A 2a receptor functionally expressed in Escherichia coli.
Eur J Biochem. Ostermeier C, Michel H. Crystallization of membrane proteins. Curr Opin Struct Biol. Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor. Dimerization: an emerging concept for G protein-coupled receptor ontogeny and function. Devi LA. Heterodimerization of G-protein-coupled receptors: pharmacology, signaling and trafficking.
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