Conformational complexity of G-protein-coupled receptors
2007; Elsevier BV; Volume: 28; Issue: 8 Linguagem: Inglês
10.1016/j.tips.2007.06.003
ISSN1873-3735
AutoresBrian K. Kobilka, Xavier Deupí,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoG-protein-coupled receptors (GPCRs) are remarkably versatile signaling molecules. Members of this large family of membrane proteins respond to structurally diverse ligands and mediate most transmembrane signal transduction in response to hormones and neurotransmitters, and in response to the senses of sight, smell and taste. Individual GPCRs can signal through several G-protein subtypes and through G-protein-independent pathways, often in a ligand-specific manner. This functional plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or to stabilize ligand-specific conformations. Here, we review what has been learned about the dynamic nature of the structure and mechanism of GPCR activation, primarily focusing on spectroscopic studies of purified human β2 adrenergic receptor. G-protein-coupled receptors (GPCRs) are remarkably versatile signaling molecules. Members of this large family of membrane proteins respond to structurally diverse ligands and mediate most transmembrane signal transduction in response to hormones and neurotransmitters, and in response to the senses of sight, smell and taste. Individual GPCRs can signal through several G-protein subtypes and through G-protein-independent pathways, often in a ligand-specific manner. This functional plasticity can be attributed to structural flexibility of GPCRs and the ability of ligands to induce or to stabilize ligand-specific conformations. Here, we review what has been learned about the dynamic nature of the structure and mechanism of GPCR activation, primarily focusing on spectroscopic studies of purified human β2 adrenergic receptor. IntroductionG-protein-coupled receptors (GPCRs) for hormones and neurotransmitters are often depicted as bimodal switches with inactive and active states. This depiction might be close to the truth for rhodopsin, where basal signaling is almost non-existent and absorption of a single photon of light is sufficient for maximal activation. Much evidence indicates, however, that GPCR signaling is much more complex than was originally envisaged. GPCRs can activate more than one G protein isoform, and recent evidence suggests that they can also signal through G-protein-independent pathways [1Lefkowitz R.J. Shenoy S.K. Transduction of receptor signals by β-arrestins.Science. 2005; 308: 512-517Crossref PubMed Scopus (1408) Google Scholar, 2Luttrell L.M. Lefkowitz R.J. The role of β-arrestins in the termination and transduction of G-protein-coupled receptor signals.J. Cell Sci. 2002; 115: 455-465Crossref PubMed Google Scholar, 3Azzi M. et al.β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (417) Google Scholar]. Moreover, ligands for a given GPCR can show different efficacy profiles for coupling to distinct signaling pathways [4Kenakin T. Ligand-selective receptor conformations revisited: the promise and the problem.Trends Pharmacol. Sci. 2003; 24: 346-354Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar].Despite advances in the biology and pharmacology of GPCRs, efforts to elucidate the structural basis of this functional plasticity remain limited. So far, only bovine rhodopsin has yielded a high-resolution structure. Nevertheless, both functional studies and low-resolution biophysical studies are providing insights into the structurally dynamic nature of non-rhodopsin GPCRs. Evidence suggests that agonist binding and activation occur through a series of conformational intermediates. Transition to these intermediate states involves the disruption of non-covalent intramolecular interactions that stabilize the basal state of the receptor. Binding of structurally different agonists might entail the disruption of different combinations of these intramolecular interactions, leading to different receptor conformations and differential effects on downstream signaling proteins.The dynamic character of GPCRs is likely to be essential for their physiological functions, and a better understanding of this molecular plasticity might facilitate structure-based drug discovery. Such dynamic behavior, however, makes GPCRs challenging experimental subjects and is an obstacle in obtaining diffraction-quality crystals for high-resolution structure determination. Here, we discuss what is known about the dynamic nature of the structure and mechanism of GPCR activation, focusing on spectroscopic studies of the human β2 adrenergic receptor.Efficacy and conformational statesLigand efficacyWith the exception of rhodopsin, most GPCRs do not behave as bimodal switches. Rhodopsin has almost no detectable basal activity in the absence of light, but can be fully activated by a single photon. Many GPCRs show a considerable amount of basal, agonist-independent activity; in other words, the GPCR can activate its G protein in the absence of an agonist. The activity of receptors can be either increased or decreased by different classes of ligands.The term ‘efficacy’ is used to describe the effect of a ligand on the functional properties of the receptor (for a more complete discussion of efficacy, see Ref. [5Kenakin T. Efficacy at G-protein-coupled receptors.Nat. Rev. Drug Discov. 2002; 1: 103-110Crossref PubMed Scopus (226) Google Scholar]). ‘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 the access of other ligands. On the basis of functional behavior, therefore, GPCRs behave more like rheostats than simple bimodal switches. Ligands can ‘dial in’ almost any level of activity from fully active to fully inactive. Although efficacy can be explained by a simple two-state model of receptor activation, evidence from both functional and biophysical studies supports the existence of multiple, ligand-specific conformational states.Receptor conformationsProteins are often thought of as rigid structures, as in the lock-and-key model of receptor activation where the agonist fits precisely into a complementary pocket in the receptor protein. Proteins, however, are known to be dynamic molecules that show rapid, small-scale structural fluctuations [6Frauenfelder H. et al.The energy landscapes and motions of proteins.Science. 1991; 254: 1598-1603Crossref PubMed Scopus (2581) Google Scholar].An intuitive approach for discussing the dynamic nature of protein conformations is an energy landscape (Figure 1a ), in which a continuum of conformational states ranges from no activity to maximal activity. For the purpose of this discussion, we ignore the denatured states and consider only functional conformational states of native receptors. The basal conformational state can be defined as a low energy state of the receptor in the absence of a ligand. The width of the energy well reflects the conformational flexibility around a particular conformational state. Within this wide energy well, additional substates can be imagined (Figure 1a, inset). The probability that a protein will undergo a transition to another conformational state is a function of the energy difference between the two states and the height of energy barrier between the two states. For a receptor, the energy of ligand binding can be used either to alter the energy barrier between the two states or to change the relative energy levels between the two states, or both.Changing the energy barrier would have an effect on the rate of transition between the two states, whereas changing the energy levels would have an effect on the equilibrium distribution of receptors in the two states. Binding of an agonist or partial agonist would lower the energy barrier and/or reduce the energy of the more active conformation relative to the inactive conformation (Figure 1b). Coupling of the receptor to its G protein could further alter the energy landscape. An inverse agonist would increase the energy barrier and/or reduce the energy of the inactive state conformation relative to the active conformation (Figure 1c).Basal activity and constitutively active mutantsSome GPCRs such as rhodopsin and the follicle-stimulating hormone receptor [7Kudo M. et al.Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop.J. Biol. Chem. 1996; 271: 22470-22478Crossref PubMed Scopus (108) Google Scholar] have little or no detectable basal activity, whereas others such as the cannabinoid receptors show high basal activity [8Sharma S. Sharma S.C. An update on eicosanoids and inhibitors of cyclooxygenase enzyme systems.Indian J. Exp. Biol. 1997; 35: 1025-1031PubMed Google Scholar, 9Nie J. Lewis D.L. Structural domains of the CB1 cannabinoid receptor that contribute to constitutive activity and G-protein sequestration.J. Neurosci. 2001; 21: 8758-8764Crossref PubMed Google Scholar]. Even for receptors with relatively low basal activity, however, constitutively activating mutations (CAMs) can increase this activity [10Parnot C. et al.Lessons from constitutively active mutants of G protein-coupled receptors.Trends Endocrinol. Metab. 2002; 13: 336-343Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar]. Basal activity might reflect the inherent flexibility of a GPCR and its tendency to exist in more than one conformational state in the absence of ligands. It could also reflect a highly constrained state with a relatively high affinity for a G protein.The concept of basal activity and receptor activation can also be considered in terms of an energy landscape (Figure 2). In the absence of agonist, a receptor with low basal activity might be relatively constrained to one inactive conformational state with a deep energy well (Figure 2a). High basal activity might be explained by smaller energy differences between the inactive and active states and a lower energy barrier (Figure 2b), which would increase the probability for spontaneous conformational transitions to the active state. This explanation could also be thought of as a receptor with greater conformational flexibility (fewer conformational constraints). Alternatively, it is possible that a receptor might exist in predominantly one constrained state that has intermediate activity towards its G protein (Figure 2c). Although these two mechanisms might apply to different receptors, there is experimental evidence linking conformational flexibility and structural instability to increased basal activity [11Gether U. et al.Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.J. Biol. Chem. 1997; 272: 2587-2590Crossref PubMed Scopus (261) Google Scholar].Figure 2Theoretical energy landscape of a GPCR with low or high basal activity. (a) Conformational states of a GPCR with low basal activity. (b) Conformational states of a GPCR with high basal activity owing to a low activation energy barrier. (c) Conformational states of a GPCR with high basal activity owing to a more active basal conformation.View Large Image Figure ViewerDownload (PPT)Non-covalent intramolecular interactions define the activity and stability of the basal stateTransmembrane (TM) domains are held in the basal state by intervening loops and non-covalent interactions between side chains. The non-covalent interactions seem to have a greater role in determining the specific basal arrangement of the TM segments relative to each other than do some of the intervening loop structures, as assessed by proteolysis and split receptor studies. For example, co-transfecting a plasmid encoding the amino terminus to TM5 of the β2 adrenoceptor (β2AR), and a plasmid encoding TM6 to the carboxyl terminus of β2AR generates a functional ‘split’ receptor [12Kobilka B.K. et al.Chimeric α2,β2-adrenergic receptors: delineation of domains involved in effector coupling and ligand binding specificity.Science. 1988; 240: 1310-1316Crossref PubMed Scopus (604) Google Scholar], comprising two non-covalently bound receptor fragments. In addition, Schoneberg et al. [13Schoneberg T. et al.Plasma membrane localization and functional rescue of truncated forms of a G protein-coupled receptor.J. Biol. Chem. 1995; 270: 18000-18006Crossref PubMed Scopus (147) Google Scholar] have generated functional M3 muscarinic split receptors with discontinuity in the loop connecting TM3 and TM4, the loop connecting TM4 and TM5, and the loop connecting TM5 and TM6. Similarly, the α2A adrenoceptor can still bind ligands after proteolytic cleavage of its loop structures [14Wilson A.L. et al.The hydrophobic tryptic core of the porcine α2-adrenergic receptor retains allosteric modulation of binding by Na+, H+, and 5-amino-substituted amiloride analogs.J. Biol. Chem. 1990; 265: 17318-17322Abstract Full Text PDF PubMed Google Scholar].It might be predicted that disrupting one of the stabilizing intramolecular interactions would favor either a more active conformation or denaturation of the receptor. This prediction is consistent with the observation that the extent of basal activity for GPCRs can be markedly enhanced by single-point mutations in various structural domains [10Parnot C. et al.Lessons from constitutively active mutants of G protein-coupled receptors.Trends Endocrinol. Metab. 2002; 13: 336-343Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar]. Mutations that disrupt intramolecular interactions would increase the ‘flexibility’ of the protein (movement of TM domains relative to each other) and thus the probability that the receptor can assume an active conformation.Some of the best-characterized examples of CAMs are those that disrupt the highly conserved (D/E)R(Y/W) amino acid sequence, present in 72% of GPCRs belonging to the rhodopsin family (http://lmc.uab.cat/gmos/). In rhodopsin, there is a network of interactions between Glu1343.49 and Arg1353.50 at the cytoplasmic end of TM3, and between Glu2476.30 and Thr2516.34 at the cytoplasmic end of TM6 [15Palczewski K. et al.Crystal structure of rhodopsin: A G protein-coupled receptor.Science. 2000; 289: 739-745Crossref PubMed Scopus (4992) Google Scholar] (note that the position of residues are followed by the Ballesteros general number [16Ballesteros J.A. 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. 1995; 25: 366-428Crossref Scopus (2419) Google Scholar] in the form superscript X.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 50.) This network, known as the ‘ionic lock’ is one of the non-covalent interactions that stabilize the receptor in the basal state. Disruption of this network by mutating Glu1343.49 to glutamine leads to constitutive activity in opsin [17Kim J.M. et al.Structure and function in rhodopsin: rhodopsin mutants with a neutral amino acid at E134 have a partially activated conformation in the dark state.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14273-14278Crossref PubMed Scopus (103) Google Scholar]. Experimental evidence indicates that Glu1343.49 is protonated during activation of rhodopsin, demonstrating that disruption of this network is part of the normal activation process [18Arnis S. et al.A conserved carboxylic acid group mediates light-dependent proton uptake and signaling by rhodopsin.J. Biol. Chem. 1994; 269: 23879-23881Abstract Full Text PDF PubMed Google Scholar].Amino acids constituting this ionic lock are conserved in other GPCRs, and mutations of the acidic amino acid have been reported to increase basal activity in various GPCRs, including β2AR [19Rasmussen S.G. et al.Mutation of a highly conserved aspartic acid in the β2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6.Mol. Pharmacol. 1999; 56: 175-184Crossref PubMed Scopus (193) Google Scholar], the H2 histamine receptor [20Alewijnse A.E. et al.The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H2 receptor.Mol. Pharmacol. 2000; 57: 890-898PubMed Google Scholar], the α1b adrenoceptor [21Scheer A. et al.Constitutively active mutants of the α1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation.EMBO J. 1996; 15: 3566-3578Crossref PubMed Scopus (358) Google Scholar, 22Scheer A. et al.The activation process of the α1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 808-813Crossref PubMed Scopus (197) Google Scholar] and the angiotensin (AT1) receptor [23Gaborik Z. et al.The role of a conserved region of the second intracellular loop in AT1 angiotensin receptor activation and signaling.Endocrinology. 2003; 144: 2220-2228Crossref PubMed Scopus (103) Google Scholar]. My co-workers and I have recently used fluorescence spectroscopy to monitor movement of the cytoplasmic end of TM3 relative to TM6 in β2AR [24Yao X. et al.Coupling ligand structure to specific conformational switches in the β2-adrenoceptor.Nat. Chem. Biol. 2006; 2: 417-422Crossref PubMed Scopus (297) Google Scholar]. We observed an agonist-induced conformational change similar to that seen on light activation of rhodopsin [25Farrens D.L. et al.Requirement of rigid-body motion of transmembrane helices for light activation of rhodopsin.Science. 1996; 274: 768-770Crossref PubMed Scopus (1104) Google Scholar]. This conformational change was observed on binding of almost all agonists and partial agonists [24Yao X. et al.Coupling ligand structure to specific conformational switches in the β2-adrenoceptor.Nat. Chem. Biol. 2006; 2: 417-422Crossref PubMed Scopus (297) Google Scholar]. We also observed that β2AR shows higher basal activity when the pH is reduced from 7.5 to 6.5, presumably owing to the disruption of the ionic lock and/or other intramolecular interactions as a result of protonation of an acidic amino acid [26Ghanouni P. et al.The Effect of pH on β2 adrenoceptor function. Evidence for protonation-dependent activation.J. Biol. Chem. 2000; 275: 3121-3127Crossref PubMed Scopus (91) Google Scholar].It might be predicted that mutations that cause enhanced basal activity by disrupting intramolecular interactions could also lead to structural instability. Mutation of Leu2726.34 to alanine at the cytoplasmic end of TM6 in β2AR results in increased basal activity [27Samama P. et al.A mutation-induced activated state of the β2-adrenergic receptor. Extending the ternary complex model.J. Biol. Chem. 1993; 268: 4625-4636Abstract Full Text PDF PubMed Google Scholar] and biochemical instability [11Gether U. et al.Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.J. Biol. Chem. 1997; 272: 2587-2590Crossref PubMed Scopus (261) Google Scholar]. Purified Leu272 → Ala β2AR denatures 2–3 times faster than wild-type receptor [11Gether U. et al.Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.J. Biol. Chem. 1997; 272: 2587-2590Crossref PubMed Scopus (261) Google Scholar]. The increase in basal activity observed in native β2AR at reduced pH is also associated with a higher rate of denaturation [26Ghanouni P. et al.The Effect of pH on β2 adrenoceptor function. Evidence for protonation-dependent activation.J. Biol. Chem. 2000; 275: 3121-3127Crossref PubMed Scopus (91) Google Scholar]. Instability has also been reported in constitutively active mutants of the β1 adrenoceptor [28McLean A.J. et al.Generation and analysis of constitutively active and physically destabilized mutants of the human β1-adrenoceptor.Mol. Pharmacol. 2002; 62: 747-755Crossref PubMed Scopus (19) Google Scholar] and the H2 histamine receptor [20Alewijnse A.E. et al.The effect of mutations in the DRY motif on the constitutive activity and structural instability of the histamine H2 receptor.Mol. Pharmacol. 2000; 57: 890-898PubMed Google Scholar]. Of note, ligands (both agonists and antagonists) can stabilize the receptor against denaturation and act as biochemical chaperones [11Gether U. et al.Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.J. Biol. Chem. 1997; 272: 2587-2590Crossref PubMed Scopus (261) Google Scholar, 28McLean A.J. et al.Generation and analysis of constitutively active and physically destabilized mutants of the human β1-adrenoceptor.Mol. Pharmacol. 2002; 62: 747-755Crossref PubMed Scopus (19) Google Scholar, 29Petaja-Repo U.E. et al.Ligands act as pharmacological chaperones and increase the efficiency of δ opioid receptor maturation.EMBO J. 2002; 21: 1628-1637Crossref PubMed Scopus (232) Google Scholar], suggesting that they form stabilizing bridges between TM segments.Agonist binding and activationAgonists disrupt stabilizing intramolecular interactionsThe energy of agonist binding is used to change the energy landscape by altering the network of stabilizing intramolecular interactions to favor an active conformation. Figure 3 shows two possible ways in which ligands might disrupt intramolecular interactions and thereby influence the arrangement of TM domains.Figure 3Possible mechanisms by which agonist binding disrupts intramolecular interactions that stabilize the inactive state. (a) The agonist binds directly to amino acids involved in stabilizing the inactive state. (b) Agonist binding stabilizes a new set of intramolecular interactions.View Large Image Figure ViewerDownload (PPT)First, agonists might effect a conformational change by simply disrupting existing intramolecular interactions (Figure 3a), thereby favoring a new set of interactions that stabilize a new conformational state. This effect is analogous to a mutation that produces high basal activity. The angiotensin AT1 receptor provides an example of an agonist binding to and displacing stabilizing interactions. Experimental evidence suggests that Asn1113.35 interacts with Asn2957.46 in TM7 of the angiotensin AT1 receptor to stabilize the inactive state [30Balmforth A.J. et al.The conformational change responsible for AT1 receptor activation is dependent upon two juxtaposed asparagine residues on transmembrane helices III and VII.J. Biol. Chem. 1997; 272: 4245-4251Crossref PubMed Scopus (105) Google Scholar]. Consistent with this interpretation, mutation of Asn1113.35 to alanine leads to constitutive activity [31Groblewski T. et al.Mutation of Asn111 in the third transmembrane domain of the AT1A angiotensin II receptor induces its constitutive activation.J. Biol. Chem. 1997; 272: 1822-1826Crossref PubMed Scopus (147) Google Scholar]. Asn1113.35 also seems to interact with Tyr4 of the agonist angiotensin [32Noda K. et al.The active state of the AT1 angiotensin receptor is generated by angiotensin II induction.Biochemistry. 1996; 35: 16435-16442Crossref PubMed Scopus (141) Google Scholar]; therefore, angiotensin binding might disrupt the interaction between Asn1113.35 and Asn2957.46.Second, agonists might serve as bridges that create new interactions between TM domains that stabilize a more active state (Figure 3b). For example, catecholamines can disrupt the ionic lock of β2AR without directly interacting with amino acids involved in forming the ionic lock [24Yao X. et al.Coupling ligand structure to specific conformational switches in the β2-adrenoceptor.Nat. Chem. Biol. 2006; 2: 417-422Crossref PubMed Scopus (297) Google Scholar].It is likely that a combination of the mechanisms shown in Figure 3 is operable for any given ligand, particularly for larger ligands such as peptide agonists. Notably, in both models shown in Figure 3, there is no pre-existing agonist-binding site in the basal state of the receptor. Spontaneous conformational transitions are needed to expose the amino acids that form the binding site.Structurally different agonists disrupt distinct combinations of stabilizing intramolecular interactionsEvidence from both cell-based and biophysical studies suggests that structurally different agonists (and partial agonists) of a given GPCR can induce distinct conformational states, rather than simply altering the equilibrium between two states (inactive and active) [3Azzi M. et al.β-Arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 11406-11411Crossref PubMed Scopus (417) Google Scholar, 4Kenakin T. Ligand-selective receptor conformations revisited: the promise and the problem.Trends Pharmacol. Sci. 2003; 24: 346-354Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar]. Moreover, studies in HEK293 cells show that, for β2AR, an inverse agonist that inhibits basal signaling through Gs and adenylyl cyclase is a partial agonist of arrestin-mediated activation of the ERK signaling pathway [4Kenakin T. Ligand-selective receptor conformations revisited: the promise and the problem.Trends Pharmacol. Sci. 2003; 24: 346-354Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar].Over the past several years, my co-workers and I have developed biophysical approaches that detect agonist-induced conformational changes in purified β2AR. We’ve compared the ability of a set of structurally related agonists (Figure 4) to induce conformational changes in different assays. These experiments provide evidence that different agonists disrupt distinct networks of stabilizing intramolecular interactions.Figure 4Ligands of the β2 adrenoceptor. Agonists, partial agonists and inverse agonists are shown.View Large Image Figure ViewerDownload (PPT)Molecular switches: the ionic lock and the rotamer toggle switchFor the purpose of discussion, we define ‘molecular switches’ as non-covalent intramolecular interactions that exist in the basal state of a GPCR and that must be disrupted to achieve an active state. For a given GPCR, there are likely to be several molecular switches. Two that have been proposed to exist in β2AR are the ionic lock and the rotamer toggle switch. As discussed earlier, the ionic lock consists of the (D/E)R(W/Y) sequence at the cytoplasmic end of TM3 and an acidic amino acid at the cytoplasmic end of TM6 (Figure 5a ). The ionic lock is highly conserved among the rhodopsin family of GPCRs. These amino acids form a stabilizing network of non-covalent intramolecular interactions that retain the cytoplasmic ends of TM3 and TM6 in an inactive conformation.Figure 5Fluorescence spectroscopy of disruption of the ionic lock in β2AR. (a) Model of TM3 (red) and TM6 (blue) from β2AR, highlighting the amino acids that comprise the ionic lock at the cytoplasmic end of these TM segments. (b) Close-up view of the ionic lock and the modifications made to monitor conformational changes in this region. Ala271 was mutated to cysteine (C271) and Ile135 was mutated to tryptophan (W135). C271 was labeled with monobromobimane in purified β2AR. On activation, W135 moves closer to bimane on C271 and quenches fluorescence. (c) Emission spectrum of bimane on C271 before and after activation by the agonist isoproterenol. (d) Effect of different ligands on disruption of the ionic lock, as determined by bimane fluorescence. The partial agonists dopamine (DOP) and salbutamol (SAL) are as effective at disrupting the ionic lock as the full agonists norepinephrine (NE) and isoproterenol (ISO). Only catechol (CAT) has no effect on the ionic lock. Data adapted from Ref. [24Yao X. et al.Coupling ligand structure to specific conformational switches in the β2-adrenoceptor.Nat. Chem. Biol. 2006; 2: 417-422Crossref PubMed Scopus (297) Google Scholar].View Large Image Figure ViewerDownload (PPT)Another molecular switch, known as a ‘rotamer toggle switch’, has been proposed to be involved in activation of the amine and opsin receptor families [33Shi L. et al.β2 Adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch.J. Biol. Chem. 2002; 277: 40989-40996Crossref PubMed Scopus (324) Google Scholar]. This switch involves a change in the bend of TM6 at the highly conserved residue Pro2886.50. In β2AR, the aromatic catechol ring of catecholamines would interact directly with the aromatic residues of the rotamer toggle switch, Trp2866.48 and Phe2906.52. Monte Carlo simulations suggest that rotamer configurations of Cys2856.47, Trp2866.48 and Phe2906.52 – the residues that comprise the rotamer toggle switch – are coupled and modulate the bend angle of TM6 around the highly conserved proline kink at Pro2886.50, leading to movement of the cytoplasmic end of TM6 on receptor activation [33Shi L. et al.β2 Adrenergic receptor activation. Modulation of the proline kink in transmembrane 6 by a rotamer toggle switch.J. Biol. Chem. 2002; 277: 40989-40996Crossref PubMed Scopus (324) Google Scholar].Recent biophysical experiments on purified β2AR suggest that these two switches can be activated independently of each other, and that agonists differ in the
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