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. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.
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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. When two Cys residues face each other in the three-dimensional 3D structure of the receptor, they have the potential to form a disulfide bridge Figure 2 , either spontaneously, or in the presence of oxidizing agents such as Cu II - 1,phenanthroline 3 referred to as 'Cu-Phen' in the following or molecular iodine. Upon cleavage with factor Xa, the disulfide bridge will keep the two cleavage products covalently linked.
General strategy used to detect the formation of disulfide bonds between vicinal Cys residues in mutant M 3 mAChRs. Following cleavage with factor Xa, the two resulting receptor fragments will remain covalently linked by the disulfide bridge.
In this case, factor Xa digestion will yield two separate cleavage products and a full-length receptor band will not appear on Western blots run under non-reducing conditions e , right lane. During the past few years, we have used the approach summarized in Figure 2 to analyze more than different double Cys mutant M 3 mAChRs [ 18 — 24 ], using membrane preparations from transiently transfected COS-7 cells.
The positions that were targeted by Cys substitution mutagenesis are highlighted in Figure 1. To ensure that the double Cys mutant receptors were properly folded, all receptors were characterized in radioligand binding and second messenger studies for their ability to bind muscarinic ligands and to mediate agonist-dependent G protein activation, respectively [ 18 — 24 ].
Agonist binding to GPCRs involves residues located on the extracellular surface of the receptor proteins [ 30 — 32 ]. ACh binding to the M 3 mAChR is predicted to trigger conformational changes within the TM receptor core, which are then propagated to the cytoplasmic surface of the receptor which is involved in G protein recognition and activation. The general view is that agonist binding leads to the disruption of existing interhelical intramolecular interactions, thereby promoting a set of interactions that leads to a new, energetically favorable conformational state of the receptor [ 17 , 34 ].
Interestingly, a recent study [ 21 ] demonstrated that muscarinic agonists promoted disulfide cross-linking between a Cys residue introduced into TM III at position S position 3. A 3D model of the inactive form of the M 3 receptor indicates that SC 3.
The observed cross-linking pattern is therefore consistent with a model in which agonist binding to the M 3 receptor increases the proximity of the exofacial segments of TM III and VII, thus allowing the formation of a disulfide bridge between SC 3. A 3D model of the inactive state of the rat M 3 mAChR was built via homology modeling using the high-resolution X-ray structure of bovine rhodopsin as a template [ 2 , 20 ].
As discussed in the text, muscarinic agonists promote the formation of a disulfide bond between C 7. K 6. Disulfide cross-linking data support the view that muscarinic agonists trigger a conformational change that includes a clockwise rotational movement of the cytoplasmic segment of TM VI [ 22 ]. Among the five highlighted TM V residues, only Y 5. Disulfide scanning mutagenesis studies strongly suggest that the cytoplasmic end of TM VI, but not the corresponding region of TM V, undergoes a major activity-dependent conformational change see text for more details; [ 24 ].
Cys substitution of the residues highlighted in yellow resulted in double Cys mutant receptors that showed agonist-dependent disulfide cross-linking 88 1. Whereas V88 1.
Disulfide cross-linking data suggest that muscarinic agonists increase the distance between Cys residues introduced at a positions 91 1. In contrast, inverse muscarinic agonists are predicted to reduce the distance between these residues [ 23 ]. The individual figure panels were taken from Refs [ 21 ]-[ 24 ]. Figure 1 indicates that S 3. The positively charged ammonium head group of ACh is known to form a salt bridge with the negatively charged side chain of D 3. Replacement of the three N-methyl groups of ACh with ethyl groups resulted in a muscarinic antagonist which, like the classical muscarinic antagonist atropine, was unable to promote disulfide bond formation between SC 3.
These data support the view that muscarinic antagonists are unable to mimic the proposed ACh-induced 'tightening' of the aromatic cage around the ACh quaternary ammonium group, most likely due to steric hindrance caused by the increased size of their ammonium head groups [ 21 , 34 ]. Distance measurements derived from molecular modeling studies suggested that metal ion-mediated receptor activation involves a movement of the extracellular segments of TM VII and VI inward towards TM III [ 37 ], in agreement with the disulfide cross-linking data reported in Ref.
At present, little is known about the series of conformational events that must occur within the TM receptor core in order to propagate these structural changes to the intracellular receptor surface where G protein coupling is known to occur. As has been discussed in detail elsewhere for a recent review, see Ref.
To study the ability of muscarinic ligands to trigger conformational changes on the cytoplasmic side of the M 3 receptor protein, we carried out disulfide cross-linking studies using a large number of mutant receptors containing pairs of Cys residues on the intracellular receptor surface [ 18 , 21 — 24 ]. For example, a recent study [ 22 ] demonstrated that the muscarinic agonist, carbachol, promoted disulfide cross-linking in two mutant receptors that contained one Cys residue at the bottom of TM III IC 3.
A 3D model of the rat M 3 mAChR, which was established via homology modeling using the dark state of bovine rhodopsin as a template [ 2 , 20 ], predicts that K 6. The observed cross-linking pattern therefore supports a model in which M 3 receptor activation is associated with a clockwise rotation of the cytoplasmic end of TM VI cytoplasmic view , which would allow the formation of disulfide cross-links between IC 3.
This conclusion is in agreement with the results of a site-directed spin labeling SDSL study carried out with Cys-substituted mutant versions of bovine rhodopsin [ 8 ]. This analysis demonstrated that agonist treatment promoted the formation of disulfide cross-links in four of the ten analyzed mutant receptors YC 5.
Since the A 6. The cross-linking data reported in ref. Summary of activity-dependent structural changes predicted to occur at the cytoplasmic surface of the M 3 mAChR. Costanzi and J. Karpiak; unpublished results.
The intracellular surface of the M 3 mAChR is viewed from the cytoplasm. As discussed in detail in the text, disulfide cross-linking data suggest that agonist binding to the M 3 mAChR causes the following structural changes: 1 The cytoplasmic end of TM VI is predicted to undergo a rotational movement and perhaps a partial unfolding and to move closer to the corresponding segment of TM V [ 18 , 22 , 24 ].
The agonist-induced structural changes are thought to expose previously inaccessible receptor residues or surfaces e. To obtain additional structural information regarding activity-dependent changes in the relative orientation of TM V and VI, a recent disulfide cross-linking study [ 24 ] examined a series of double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V L 5.
This analysis showed that muscarinic agonists promoted the formation of disulfide bonds in six of the twenty analyzed double Cys mutant M 3 receptors YC 5. Whereas A 6. Thus, the ability of muscarinic agonists to promote the formation of disulfide bonds in constructs containing the QC 6. Interestingly, in the same study [ 24 ], muscarinic agonists failed to induce the formation of disulfide bonds in all double Cys mutant M 3 receptors containing the LC 5. In contrast to Y 5. It is therefore likely that the cytoplasmic end of TM V does not undergo major activity-dependent conformational changes in the M 3 mAChR.
To test the hypothesis that this receptor region undergoes conformational changes during M 3 receptor activation, a recent cross-linking study [ 20 ] analyzed a series of double Cys mutant M 3 receptors containing one Cys substitution within the C-terminal portion of TM VII V 7. These two receptor regions are predicted to lie adjacent to each other in the 3D structure of class I GPCRs [ 2 — 6 ].
In the inactive state of the M 3 receptor in the absence of ligands , only the V88C 1. Interestingly, agonist carbachol treatment further enhanced the intensity of the disulfide cross-linking signal displayed by this mutant receptor, suggesting that V88 1.
Agonist binding also led to the formation of disulfide bonds in two additional double Cys mutant receptors, A91C 1. In the inactive state of the M 3 receptor, L 7. The observed cross-linking pattern therefore suggests that M 3 receptor activation involves a major conformational change at the cytoplasmic end of TM VII. Consistent with these findings, biochemical and biophysical studies with bovine rhodopsin indicate that the C-terminal segment of TM VII undergoes a significant light-induced conformational change [ 39 — 41 ].
Y 7. As discussed elsewhere [ 21 , 36 ], it is possible that agonist binding triggers a reorientation of the conserved TM VII proline bend, which in turn leads to a conformational rearrangement of the C-terminal segment of TM VII. A recent study [ 23 ] therefore tested the hypothesis that M 3 receptor activation involves a reorientation of helix 8.
Given the observation that several helix 8 residues are located close to the cytoplasmic end of TM I in bovine rhodopsin [ 2 , 12 ], this disulfide cross-linking study [ 23 ] examined a series of double Cys mutant M 3 receptors, all of which contained one Cys substitution within the cytoplasmic end of TM I A91 1. This analysis showed that muscarinic agonists inhibited constitutive disulfide cross-linking displayed by the A91C 1.
Figure 3e indicates that residues 91 1. One likely scenario is that the reorientation of helix 8 is triggered by the agonist-induced conformational rearrangement of the cytoplasmic end of TM VII see discussion above.
Interestingly, the same study [ 23 ] also reported that atropine and Nmethylscopolamine, two classical muscarinic antagonists that have recently been reclassified as inverse agonists [ 47 , 48 ], enhanced disulfide bond formation in the A91C 1.
This finding suggests that inverse muscarinic agonists, in contrast to muscarinic agonists, decrease the distance between the cytoplasmic end of TM I and the N-terminal portion of helix 8. A related study examining the formation of disulfide cross-links between Cys residues introduced at the cytoplasmic ends of TM V and VI also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors [ 24 ].
These studies [ 23 , 24 ] therefore provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists, highlighting the usefulness of disulfide cross-linking approaches to study changes in GPCR structure caused by different classes of agonists also see Refs.
Overall, agonist binding to the M 3 mAChR is predicted to cause the following structural changes at the intracellular receptor surface Figure 4 ; for a summary of disulfide crosslinking data, also see Table 1.
As discussed in more detail below 'Caveats inherent in the use of disulfide cross-linking strategies' , the possibility exists that at least some of the observed changes in agonist-dependent disulfide cross-linking patterns are also affected by the overall increase in dynamic flexibility of the active receptor confirmation.
Summary of the effects of muscarinic agonists on the formation of disulfide cross-links in double Cys mutant M 3 mAChRs. It remains unclear at present whether these predicted conformational changes occur in a concerted fashion or whether a primary structural change e.
In any case, the disulfide cross-linking data are consistent with the concept that agonist activation of the M 3 mAChR opens a cleft on the intracellular receptor surface that increases the accessibility of various residues located at the cytoplasmic ends of different TM helices including TM III, VI, and VII Figure 4.
As mentioned in the introduction, biochemical and biophysical studies with bovine rhodopsin, including the use of sophisticated SDSL techniques, have led to a rather detailed view of the activity-dependent structural changes occurring at the cytoplasmic surface of this photoreceptor protein [ 8 — 13 ].
Several of the dynamic changes observed in these studies, including conformational changes at the cytoplasmic ends of TM VI, VII, and helix 8 summarized in Ref. However, while the cytoplasmic end of TM VII is thought to move away from the TM helical bundle during rhodopsin activation [ 12 ], studies with the M 3 mAChR suggest that this region moves closer to the intracellular end of TM I following agonist stimulation.
Moreover, rhodopsin activation is predicted to be accompanied by small but significant outward movements of the cytoplasmic ends of TM I-III [ 12 ], movements that were not observed or not systematically studied in the M 3 mAChR.
It is unclear at present to which extent these observations reflect true differences between the pattern of structural changes that accompany the activation of these two receptor proteins.
However, it is likely that at least some of these phenomena are caused by differences in experimental techniques and conditions used. Whereas the vast majority of the rhodopsin studies were carried out with mutant versions of rhodopsin in the solution state, all disulfide cross-linking studies were carried out with Cys-substituted mutant M 3 receptors present in cellular membranes.
Rhodopsin has been shown to have increased flexibility in the solution state as compared to native membranes [ 12 , 51 ], providing a possible explanation for at least some of the differences observed between the two receptor systems. Moreover, while biophysical approaches, including SDSL and fluorescence-based techniques, usually provide information only about average receptor conformations, disulfide cross-linking can occur by trapping rather transient receptor conformations for example, conformations that are intermediate between the ground state and the fully active state of the receptor in which the two Cys residues are temporarily close to each other in the 3D structure of the receptor.
The two different techniques therefore yield results that are complementary in nature. Interestingly, a high-resolution crystal structure of ligand-free native opsin from bovine retinal rod cells has been published very recently [ 52 ]. When compared to rhodopsin, opsin displays several striking structural changes, resembling in many aspects the structure of the active state of rhodopsin predicted by biophysical and biochemical studies [ 12 ].
Many, but not all, of the activity-dependent conformational changes proposed for the M 3 mAChR on the basis of disulfide cross-linking studies were also observed in the opsin structure. For example, when compared rhodopsin, the cytoplasmic end of TM VI displays an outward, rotational movement in opsin, the cytoplasmic ends of TM V and VI move closer towards each other, and the C-terminal portion of TM VII undergoes a rotational movement clockwise as viewed from the cytoplasm; ref.
One caveat associated with the use of disulfide cross-linking approaches is that negative results are difficult to interpret. Previous studies have shown that the efficiency of disulfide bond formation is determined not only by the distance between the two Cys residues under investigation but also by their relative orientation and the environment surrounding the Cys residues which can strongly affect the pK a values of the SH groups [ 28 , 53 ]. Such cross-links can form when the Cys residues are contained in receptor regions endowed with a high degree of conformational flexibility.
In fact, structural and biophysical evidence suggests that several cytoplasmic GPCR domains, including the C-terminal tail beyond helix 8 and the central portion of the i3 loop, are characterized by a high degree of structural flexibility [ 2 — 5 , 16 , 17 , 55 ]. In view of the caveats raised above, it is difficult or impossible to draw meaningful conclusions from disulfide cross-linking data obtained with only a small number of double Cys mutant receptors.
It is therefore recommended to perform systematic scans in which consecutive residues in two receptor regions of interest are replaced, one by one, with Cys. Disulfide cross-linking data obtained with such a larger collection of double Cys mutant receptors are more likely to yield useful structural information. Cu-Phen is a commonly used oxidizing agent to induce the formation of disulfide bonds in receptor-containing membrane preparations. One caveat associated with the use of Cu-Phen is that this agent may induce the formation of disulfide cross-links of proteins in non-native conformations when used at high concentrations [ 9 ; J.
Wess, unpublished observations. To reduce the likelihood of such events, we recommend to use Cu-Phen at relatively low concentrations 2. Previous work suggests that Cu-Phen-catalyzed disulfide bond formation does not require the generation of hydrogen peroxide as an oxidizing intermediate [ 56 ], suggesting that the Cu-Phen complex must be present in the direct vicinity of the SH groups to be cross-linked.
This concept is supported by the results of a previous disulfide cross-linking study suggesting that muscarinic ligands compete with the relatively bulky Cu-Phen moiety for access to the hydrophilic binding crevice [ 19 ]. In such cases, the use of oxidizing agents that are smaller in size, such as molecular iodine, may yield more meaningful cross-linking data [ 19 ].
Mercury II salts HgCl 2 which can bridge vicinal pairs of Cys residues have also been used successfully to achieve cross-linking between Cys residues buried in the TM receptor core [ 57 , 58 ]. In conclusion, disulfide cross-linking studies with the M 3 mAChR have shed new light on the conformational changes associated with M 3 mAChR activation.
Overall, the activity-dependent conformational changes postulated for the M 3 mAChR are similar but not identical to those observed with the photoreceptor rhodopsin, highlighting the usefulness of disulfide cross-linking strategies to examine dynamic changes in GPCR structure.
Future cross-linking studies are likely to provide even more detailed insights into the molecular mechanisms involved in GPCR activation. Since GPCRs represent ideal drug targets, the novel structural information obtained from these studies might facilitate the development of novel classes of therapeutic agents.
All disulfide cross-linking studies carried out with the M 3 mAChR have so far relied on the use of a receptor construct that contained a protease cleavage site within the i3 loop Figure 1. As a result, this construct can be used only to detect the formation of disulfide bonds in double Cys mutant receptors where one Cys residue is located N-terminal and the other one C-terminal of the protease cleavage site.
Studies with several GPCRs suggest that structurally diverse classes of agonists stabilize different receptor conformations which may differ in their ability to interact with G proteins and other receptor-associated proteins such as arrestins [ 7 , 16 , 17 ]. How these various conformations differ at the molecular level remains unclear at present. The disulfide cross-linking strategy described in this review may therefore prove useful to define the structural differences between these various active receptor states.
Little is known about the sequence of conformational events that link the activity-dependent structural changes in the immediate vicinity of the agonist binding site to the conformational rearrangement of the cytoplasmic receptor surface.
It should therefore be highly informative to systematically apply the disulfide cross-linking approach described in this review to the entire TM receptor core. Moreover, recent data suggest that GPCR activation may lead to changes in receptor quaternary structure. For example, disulfide cross-linking studies with the D 2 dopamine [ 62 ] and the 5HT 2c serotonin receptors [ 63 ] have shown that receptor activation is associated with dynamic changes at the dimer interface.
These studies indicate that a full understanding of the molecular mechanisms that underlie GPCR activation also requires more detailed information about changes in GPCR quaternary structure. We thank Dr. Stefano Costanzi and Mr. Joel D. Read article at publisher's site DOI : Elife , 8, 12 Nov Nat Struct Mol Biol , 25 6 , 28 May Cited by: 14 articles PMID: Front Synaptic Neurosci , , 21 Feb Insect Biochem Mol Biol , , 08 Dec Cited by: 13 articles PMID: 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 beta 2 -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 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.
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