Structures of the inactive state of the thermostabilized β1-adrenoceptor have been determined bound to eight different ligands, including full agonists, partial agonists, inverse agonists and biased agonists. Comparison of the structures shows distinct differences within the binding pocket that correlate with the pharmacological properties of the ligands. These data suggest that full agonists stabilize a structure with a contracted binding pocket and a rotamer change of serine (5.46) compared with when antagonists are bound. Inverse agonists may prevent both of these occurrences, whereas partial agonists stabilize a contraction of the binding pocket but not the rotamer change of serine (5.46). It is likely that subtle changes in the interactions between transmembrane helix 5 (H5) and H3/H4 on agonist binding promote the formation of the activated state.

Introduction

GPCRs (G-protein-coupled receptors) comprise a large family of integral membrane proteins that usually bind a diffusible ligand (agonist) that can initiate diverse signalling events through stimulation of heterotrimeric G proteins [1] and through the mitogen-activated protein kinase pathway via arrestin binding [2]. Owing to the diversity of physiological processes that they regulate, GPCRs are important drug targets [3] and therefore there have been intensive efforts to determine their structures. The first high-resolution structure of a GPCR that binds a diffusible ligand was the structure of the β2AR (β2-adrenoceptor) bound to the inverse agonist carazolol, determined in 2007 [4]. Since then, an increasing number of GPCR structures has been determined through the implementation of generic protein engineering and crystallization methodologies [5] and, at the time of writing, the structures of 15 different GPCRs have been deposited in the PDB. A further milestone was reached in 2011 with the structure determination of agonist-bound β2AR in complex with a heterotrimeric G protein [6], which showed how a fully activated GPCR interacts with and activates the G-protein. However, the complexities of GPCR activation are still not fully understood. Why do partial agonists show lower efficacy than full agonists? What is the structural basis for arrestin binding induced upon binding biased agonists? Why do agonists increase the probability of the receptor transitioning into the activated state? Uniquely for a GPCR subfamily, multiple structures of β1AR and β2AR have been determined bound to 14 different ligands, including full agonists, partial agonists, weak partial agonists, inverse agonists and biased agonists. This review focuses primarily on what these structures suggest about how agonists activate β-adrenoceptors.

Structure determination of the β1- and β2-adrenoceptors

Two different methodologies were used to crystallize β2AR and β1AR. In order to obtain crystals of β2AR that diffracted to high resolution, flexible regions of the receptor were deleted and T4L (T4 lysozyme) fused into CL3 (cytoplasmic loop 3), with T4L forming the majority of the crystal contacts [4]. The β1AR was thermostabilized in the inactive (R) state through the inclusion of six thermostabilizing mutations and flexible regions were also deleted, allowing crystals to be grown in short-chain detergents [7]. The avian β1AR was used in preference to human β1AR because it is far more stable [8], but the high sequence identity (76%) between the two receptors makes the avian receptor an excellent model for the determination of ligand–receptor interactions [911]. Thermostabilized β1AR can bind agonists and activate G-proteins [12] and none of the mutations are in the ligand-binding pocket. Comparison between the β1AR and β2AR structures show that there is high overall structural similarity between the transmembrane domains [rmsd (root mean square deviation) 0.6 Å; 1 Å=0.1 nm] and even higher similarity in the positions of the Cα residues around the ligand-binding pocket (rmsd 0.25 Å, 79 atoms) [4,11]. Owing to the great similarity between β1AR and β2AR demonstrated by their respective crystal structures, modes of ligand binding determined in β1AR will probably apply to β2AR, as the only difference in the ligand-binding pocket is the substitution of Phe325 (7.35) in β2AR for Tyr308 (7.35) in β1AR (the numbers in parentheses refer to the Ballesteros–Weinstein nomenclature [13]). There are however important differences in activity between the two receptors, the β2AR is more responsive to agonists and has a higher basal activity than β1AR [14,15], suggesting that the β2AR is the more dynamic receptor. This may at least partly be caused by a difference in the interactions that stabilize transmembrane helix 5 (H5) owing to the presence of the polar residue Thr164 (4.56) in β2AR compared with Val172 (4.56) in β1AR [10].

Structures of β1AR have been determined bound to ligands of diverse efficacy (Figure 1). The differences in chemistry that differentiate agonists and antagonists (i.e. weak partial agonists and inverse agonists) are well characterized; these include an oxymethylene linker acting as a spacer between the head group and β-carbon in antagonists and a more polar head group such as catechol in the case of agonists (Figure 1). All of the structures of β1AR are in the inactive state, even when full agonists are bound to the receptor. It is interesting to note that a structure of β2AR bound to a covalent agonist is also in the inactive state, even though β2AR does not contain any conformationally specific thermostabilizing mutations and β2AR is easier to activate than β1AR [16]. Thus, for β1AR and β2AR, it appears that agonist binding increases the probability of the receptor forming the activated state (R*), but the agonist per se cannot stabilize R*, which requires binding of either a G-protein or a G-protein mimetic. The importance of agonist interactions with serine residues on H5 for the activation of βARs is well known from mutagenesis studies [17,18]. However, precise detailed structural information of ligand–receptor interactions is required to explain how ligands with differing pharmacological profiles alter the dynamics of the βARs. The structures that we have obtained of thermostabilized β1AR are of sufficient resolution (2.1–3.2 Å) to discern with confidence the serine side chain rotamer configurations and the presence of well-ordered water molecules that allows insights into how ligands of different efficacies affect the structure of β1AR.

Ligands used for co-crystallization with β1-adrenergic receptor

Figure 1
Ligands used for co-crystallization with β1-adrenergic receptor

Regions of the ligands that are conserved with the endogenous agonist noradrenaline are highlighted in blue, the oxymethylene linker that is conserved among antagonists is highlighted in red. Head groups are the substituents at the right hand of the ligand; tail regions are to the left of the secondary amine group.

Figure 1
Ligands used for co-crystallization with β1-adrenergic receptor

Regions of the ligands that are conserved with the endogenous agonist noradrenaline are highlighted in blue, the oxymethylene linker that is conserved among antagonists is highlighted in red. Head groups are the substituents at the right hand of the ligand; tail regions are to the left of the secondary amine group.

What are the differences in the binding of full agonists and inverse agonists of the G-protein signalling pathway?

The critical determinants of whether a ligand acts as a full agonist in β1AR appears to be contraction of the ligand-binding pocket and a change in rotamer conformation of Ser215 (5.46) upon formation of a hydrogen bond with the ligand (Table 1 and Figure 2) [10]. Ser211 (5.42) also makes direct contact with the ligand: when a full agonist is bound there is a change in rotamer conformation of Ser211 (5.42) and formation of a hydrogen bond with the ligand, but these changes are not exclusive to the binding of full agonists, as they are also observed in some cases when partial agonists are bound. Ser212 (5.43) has been shown to be important for receptor activation by mutagenesis studies [18], but it does not make direct contact to the ligand [10]. However, Ser212 (5.43) forms a hydrogen bond to Asn310 (6.55), and this residue does interact directly with agonists [10]; this may improve the ability of agonists to activate the receptor, but a hydrogen bond between these two residues can sometimes be observed when antagonists are bound. The 1.1 Å contraction of the binding pocket observed in the β1AR structure with isoprenaline is considerably less than the 2–3 Å inward movement of H5 previously thought necessary to promote high-affinity agonist binding [11,19], because agonists bind with a subtly different pose compared with antagonists (Figure 2B), which also involves the rotation of both Asp121 (3.32) and Asn329 (7.39) side chains closer to H5. When full agonists are bound to the receptor, both Ser211 (5.42) and Ser215 (5.46) side chains are in a conformation that facilitates hydrogen bonding to the catecholamine hydroxy groups (Figure 2B). Inverse agonists and weak partial agonists may form a hydrogen bond with Ser211 (5.42), but they have not been observed to promote rotamer change of Ser215 (5.46) observed in the structures with full agonists.

Key changes in the R-state of β1AR induced by the binding of ligands with differing pharmacological profiles

Figure 2
Key changes in the R-state of β1AR induced by the binding of ligands with differing pharmacological profiles

(A) The structure of β1AR bound to carazolol (PDB 2YCW, chain A), with the receptor monomer in rainbow coloration (N terminus, blue; C terminus, red). Carazolol is shown in space-filling representation (C, yellow; O, red; N, blue). The extracellular surface is at the top of the panel. The dashed box indicates the view of H5 and H7 depicted in panels (B)–(D) and the red arrow shows the view depicted in (E) and (F). (B) Differences in interactions with the ligand between inverse agonist-bound β1AR (coloured) and agonist-bound β1AR (grey) that contain either carazolol (yellow) or isoprenaline (green). (C) Interactions with the partial agonist dobutamine. Features in common with structures with full agonist are highlighted in grey and indicated with green arrows. (D) Interactions with the biased agonist bucindolol bound. Highlighted features (blue) are the additional ligand contacts with H7 [Phe325 (7.35) and Val326 (7.36)] and EL2 (Cys199 and Asp200), with the conformation of Ser211 (5.42) (grey, arrowed) identical to that found in the agonist-bound state. (EG) Selected interactions between H4 and H5 that are different in structures with ligands of different efficacy bound: (E) inverse agonist carvedilol; (F) partial agonist cyanopindolol (LCP structure); (G) full agonist carmoterol. Red spheres represent water molecules W1 and W2, serine rotamer configurations common to structures with full agonists are indicated with an arrow. The extracellular side is at the top of each panel. See Table 1 for the PDB codes for the structures.

Figure 2
Key changes in the R-state of β1AR induced by the binding of ligands with differing pharmacological profiles

(A) The structure of β1AR bound to carazolol (PDB 2YCW, chain A), with the receptor monomer in rainbow coloration (N terminus, blue; C terminus, red). Carazolol is shown in space-filling representation (C, yellow; O, red; N, blue). The extracellular surface is at the top of the panel. The dashed box indicates the view of H5 and H7 depicted in panels (B)–(D) and the red arrow shows the view depicted in (E) and (F). (B) Differences in interactions with the ligand between inverse agonist-bound β1AR (coloured) and agonist-bound β1AR (grey) that contain either carazolol (yellow) or isoprenaline (green). (C) Interactions with the partial agonist dobutamine. Features in common with structures with full agonist are highlighted in grey and indicated with green arrows. (D) Interactions with the biased agonist bucindolol bound. Highlighted features (blue) are the additional ligand contacts with H7 [Phe325 (7.35) and Val326 (7.36)] and EL2 (Cys199 and Asp200), with the conformation of Ser211 (5.42) (grey, arrowed) identical to that found in the agonist-bound state. (EG) Selected interactions between H4 and H5 that are different in structures with ligands of different efficacy bound: (E) inverse agonist carvedilol; (F) partial agonist cyanopindolol (LCP structure); (G) full agonist carmoterol. Red spheres represent water molecules W1 and W2, serine rotamer configurations common to structures with full agonists are indicated with an arrow. The extracellular side is at the top of each panel. See Table 1 for the PDB codes for the structures.

Table 1
Ligand binding pocket properties of β1AR bound to ligands of different efficacies

H-bond, hydrogen bond.

Ligand, PDB (chain)*, (resolution, Å)Efficacy: G-protein (wild-type receptors)Distance Cα Asn329–Ser211 (Å)Distance change (Å)§Ser211 rotamerSer211 interactionSer215 rotamerSer215 interactionSer212–Asn310 interactionTail substituent interactions
Isoprenaline 2Y03(B), (2.85) Full agonist 14.8 −1.1 gauche+ H-bond gauche+ H-bond H-bond – 
Carmoterol 2Y02(B), (2.6) Full agonist β2-specific 14.9 −1.0 gauche+ H-bond gauche+ H-bond H-bond EL2, H3, H7 
Dobutamine 2Y01(B), (2.6) Partial agonist, β1-specific 15.2 −0.7 gauche+ H-bond gauche polar H-bond H2, H7 
Salbutamol 2Y04(A,B), (3.05) Partial agonist, β2-specific 15.0 −0.9 trans H-bond (A), polar (B) gauche polar (A and B) H-bond – 
Bucindolol 4AMI(A), (3.2) Weak partial agonist 15.6 −0.3 gauche+ van der Waals gauche van der Waals H-bond EL2, H3, H7 
Cyanopindolol LCP (2.1) Weak partial agonist 16.1 0.2 gauche+ H-bond gauche van der Waals H-bond – 
Cyanopindolol 2VT4(B), (2.7) Weak partial agonist 15.9 gauche+ H-bond gauche van der Waals Polar – 
Carazolol 2YCW(A), (3.0) Inverse agonist 15.8 −0.1 trans polar gauche van der Waals None – 
Carvedilol 4AMJ(B), (2.3) Inverse agonist 15.9 trans H-bond gauche van der Waals H-bond EL2, H2, H3, H7 
Ligand, PDB (chain)*, (resolution, Å)Efficacy: G-protein (wild-type receptors)Distance Cα Asn329–Ser211 (Å)Distance change (Å)§Ser211 rotamerSer211 interactionSer215 rotamerSer215 interactionSer212–Asn310 interactionTail substituent interactions
Isoprenaline 2Y03(B), (2.85) Full agonist 14.8 −1.1 gauche+ H-bond gauche+ H-bond H-bond – 
Carmoterol 2Y02(B), (2.6) Full agonist β2-specific 14.9 −1.0 gauche+ H-bond gauche+ H-bond H-bond EL2, H3, H7 
Dobutamine 2Y01(B), (2.6) Partial agonist, β1-specific 15.2 −0.7 gauche+ H-bond gauche polar H-bond H2, H7 
Salbutamol 2Y04(A,B), (3.05) Partial agonist, β2-specific 15.0 −0.9 trans H-bond (A), polar (B) gauche polar (A and B) H-bond – 
Bucindolol 4AMI(A), (3.2) Weak partial agonist 15.6 −0.3 gauche+ van der Waals gauche van der Waals H-bond EL2, H3, H7 
Cyanopindolol LCP (2.1) Weak partial agonist 16.1 0.2 gauche+ H-bond gauche van der Waals H-bond – 
Cyanopindolol 2VT4(B), (2.7) Weak partial agonist 15.9 gauche+ H-bond gauche van der Waals Polar – 
Carazolol 2YCW(A), (3.0) Inverse agonist 15.8 −0.1 trans polar gauche van der Waals None – 
Carvedilol 4AMJ(B), (2.3) Inverse agonist 15.9 trans H-bond gauche van der Waals H-bond EL2, H2, H3, H7 
*

Data are shown only for specific monomers (A or B) that are unaffected by distortions at the extracellular end of H7 due to crystal packing interactions. In the case of PDB 2Y04, β1AR with salbutamol bound, neither monomer is distorted. Two slightly different modes of ligand binding are observed in the two monomers, so both are given.

Consensus of activities from numerous determinations at both β1AR and β2AR from published literature.

The Ballesteros–Weinstein numbers for the residues are the following: Ser211 (5.42), Ser212 (5.43), Ser215 (5.46), Asn310 (6.55) and Asn329 (7.39).

§

Contraction of the ligand binding pocket as defined by the distance between Cα atoms Asn329 (7.39) and Ser211 (5.42) with respect to the average distance observed in the four structures of β1AR bound to cyanopindolol, carazolol or carvedilol (15.9 Å).

Additional ligand tail substituent interactions with the receptor may in some cases be responsible for promoting G-protein-independent signalling.

J.L. Miller, T. Warne and C.G. Tate, unpublished work.

Why does the rotamer configuration of Ser215 (5.46) affect the probability of the receptor forming the activated state? One consequence of agonist binding is a change in the interactions between H5 and H3/H4. In the extracellular half of both β1AR and β2AR, H5 has relatively few interactions with H3 and H4. It has been suggested for β2AR that this part of H5 is particularly dynamic and consequently mutagenesis targeting interactions in this region has been used to stabilize both β1AR and β2AR [20,21]. When a full agonist binds to the β1AR, the number of interactions between H5 and H4 is reduced because of the difference in Ser211 (5.42) and Ser215 (5.46) side chain conformation (compare Figures 2E with 2G). When the inverse agonist carvedilol is bound, the interactions between H4 and H5 include those mediated by water molecule W2 that is co-ordinated by hydrogen bonds with the hydroxy groups from Ser211 (5.42) and Ser215 (5.46) and the carbonyl oxygen of Val172 (4.56), and there is also a van der Waals interaction between Ser215 (5.46) and Val172 (4.56). None of these interactions is possible when a full agonist is bound and water molecule W2 is therefore absent.

The crystal structures of β1AR most probably represent one of the lowest energy conformations of the receptor, and we suggest that the rotamer changes of Ser211 (5.42), Ser212 (5.43) and Ser215 (5.46) [and their equivalents: Ser203 (5.42), Ser204 (5.43) and Ser207 (5.46) in the β2AR, see Table 2], and the contraction of the ligand-binding pocket are essential for the efficacy of a full agonist in both β1AR and β2AR. Such changes would affect the dynamics of the receptor and increase the probability of the sliding of H3 and the outward movements of the cytoplasmic ends of H5 and H6 as observed in the R* structures of β2AR [6,22] and R*-like structures of the adenosine A2A receptor [23,24]. In order to appreciate the probable importance of the H4–H5 interface for receptor activation, it is helpful to consider why the basal activity of β1AR is 5-fold lower than for β2AR [15]. At the H4–H5 interface, Thr164 (4.56) in β2AR is equivalent to Val172 (4.56) in β1AR, which results in an additional van der Waals interaction in β1AR compared with β2AR. Crucially, this interaction is also present when a full agonist is bound and this suggests that H5 of β1AR might be less dynamic than H5 of β2AR [10]. The suggestion that different residues at position 4.56 are at least partially responsible for differences in activity between receptor subtypes, is supported by activity measurements on a common, medically important polymorphism of the β2AR, T164I (4.56). The basal activity and response to agonist of the β2AR Ile164 isoform are considerably reduced compared to the β2AR Thr164 isoform, and are similar to those of β1AR [25]. This is consistent with Ile164 (4.56) in β2AR Ile164 forming van der Waals interactions between H4 and H5, similar to those observed in β1AR. It must be emphasized that we are considering only the dynamics of H5 because there is good evidence to support its role in the activation of the β-adrenoceptors. Agonists also make hydrogen bonds to H3, H6 and H7, and these are important in the activation process, but antagonists also interact with these helices in a similar fashion, and it is therefore difficult to define any agonist-specific processes.

Table 2
Ligand binding pocket properties of β2-ARs bound to ligands of different efficacies

H-bond, hydrogen bond.

Ligand, PDB, (resolution, Å)Efficacy: G-protein*Distance Cα Asn312–Ser203 (Å)Distance change (Å)Ser203 rotamerSer203 interactionSer207 rotamerSer207 interactionSer204–Asn293 interactionTail substituent interactions§
FAUC50 3PDS, (3.5) Full agonist (covalent) 14.5 −1.5 gauche+ H-bond gauche+ H-bond H-bond EL2, H2, H3, H7 
BI-167107 3SN6, (3.2) Full agonist 14.6 −1.4 gauche+ H-bond gauche H-bond Polar EL2, H3, H7 
BI-167107 3P0G, (3.5) Full agonist 14.8 −1.2 gauche+ H-bond gauche H-bond Polar EL2, H3, H7 
Alprenolol 3NYA, (3.16) Neutral antagonist 15.7 −0.3 trans van der Waals gauche van der Waals None – 
C18H25NO5 3NY9, (2.84) Inverse agonist 15.8 −0.2 trans Polar gauche van der Waals H-bond – 
Carazolol 2RH1, (2.4) Inverse agonist 16.0 trans H-bond gauche van der Waals None – 
Timolol 3D4S, (2.8) Inverse agonist 15.9 −0.1 trans Polar gauche van der Waals H-bond EL2, H2, H3, H7 
ICI 118,551 3NY8, (2.84) Inverse agonist 16.3 +0.3 trans van der Waals gauche van der Waals None – 
Ligand, PDB, (resolution, Å)Efficacy: G-protein*Distance Cα Asn312–Ser203 (Å)Distance change (Å)Ser203 rotamerSer203 interactionSer207 rotamerSer207 interactionSer204–Asn293 interactionTail substituent interactions§
FAUC50 3PDS, (3.5) Full agonist (covalent) 14.5 −1.5 gauche+ H-bond gauche+ H-bond H-bond EL2, H2, H3, H7 
BI-167107 3SN6, (3.2) Full agonist 14.6 −1.4 gauche+ H-bond gauche H-bond Polar EL2, H3, H7 
BI-167107 3P0G, (3.5) Full agonist 14.8 −1.2 gauche+ H-bond gauche H-bond Polar EL2, H3, H7 
Alprenolol 3NYA, (3.16) Neutral antagonist 15.7 −0.3 trans van der Waals gauche van der Waals None – 
C18H25NO5 3NY9, (2.84) Inverse agonist 15.8 −0.2 trans Polar gauche van der Waals H-bond – 
Carazolol 2RH1, (2.4) Inverse agonist 16.0 trans H-bond gauche van der Waals None – 
Timolol 3D4S, (2.8) Inverse agonist 15.9 −0.1 trans Polar gauche van der Waals H-bond EL2, H2, H3, H7 
ICI 118,551 3NY8, (2.84) Inverse agonist 16.3 +0.3 trans van der Waals gauche van der Waals None – 
*

Consensus of activities from published literature.

The Ballesteros–Weinstein numbers for the residues are the following: Ser203(5.42), Ser204(5.43), Ser207(5.46), Asn293(6.55) and Asn312(7.39).

Contraction of the ligand-binding pocket as defined by the distance between Cα atoms Asn312 (7.39) and Ser203 (5.42) with respect to the distance observed in the structure of β2AR bound to carazolol (16.0 Å).

§

Additional ligand tail substituent interactions with the receptor may in some cases be responsible for promoting G protein-independent signalling.

Activated state (R*), receptor–Gs complex.

Activated state (R*), receptor–Nb80 complex.

How can inverse agonist activity be explained?

Inverse agonists suppress basal signalling activity by preventing the formation of the activated receptor–G-protein complex [26]. Both weak partial agonists and inverse agonists are characterized by an additional spacer between their β-carbon atom and the ligand head group, and also the absence of hydrogen bond donors/acceptors in the equivalent position to the para-hydroxy group in catecholamines (Figure 1). The inverse agonist carazolol binds to β1AR in a manner analogous to an agonist (Figure 2), but there are important differences. The relatively large carbazole head group has an amine group that makes a polar contact with Ser211 (5.42), whereas the β-hydroxyl and amine groups form hydrogen bonds with Asn329 (7.39) and Asp121 (3.32). The presence of the additional spacer between the β-carbon and the head group in carazolol compared with an agonist (Figure 1) would be expected to reduce the probability of the ligand-binding pocket contracting. Additionally, the position of the carbazole head group partially occupies the region to which the hydroxy group of Ser215 (5.46) would rotate if it were in the configuration observed in isoprenaline-bound β1AR. Thus inverse agonists prevent contraction of the binding pocket and the rotamer conformational change of Ser215 (5.46). This is supported by evidence from structures of β2AR bound to the potent inverse agonist ICI 118,551, which is a stronger inverse agonist than carazolol [26,27]. A methyl substituent on the head group of ICI 118,551 projects towards Ser207 (5.46) in β2AR [28] and the conformation (gauche+) seen in structures with full agonists cannot be accommodated due to a steric clash. Perhaps even more significant is the fact that to accommodate the head group of ICI 118,551, H5 has been displaced outwards at the level of Ser207 (5.46) by 0.4–0.9 Å at the Cα atom when compared with other structures of both βARs bound to antagonists [4,9,11,28,29], and the Cα Asn312–Ser203 distance change is +0.3 Å (see Table 2). This expansion of the ligand binding pocket and enhanced occlusion of the Ser207 (5.46) side chain might be expected to attenuate the dynamic nature of H5, and this is entirely consistent with the potent inverse agonist effect of ICI 118,551.

What are the features of the binding of partial agonists?

Partial agonists show a wide variation in chemical structure and efficacies, but the structures of β1AR bound to dobutamine and salbutamol both show a contraction of the binding pocket similar to that seen for full agonists. However, the conformation of Ser215 (5.46) is the same as observed for inverse agonist-bound structures (Table 1). Dobutamine may have the strongest activity of the four partial agonist ligands under consideration, as it is a β1AR-specific heart stimulant [30]. Dobutamine also has a catechol head group similar to the full agonist isoprenaline, but it does not bind in the same manner as isoprenaline because the conformation of Ser215 (5.46) has not changed and is the same as is observed with inverse agonists (Figure 2C). This is probably because dobutamine's catechol head group is shifted slightly upwards in the binding pocket, further from Ser215 (5.46) when compared with the isoprenaline head group. Similar to the partial agonist dopamine [31], dobutamine does not have a β-hydroxy group and this may cause it to adopt a slightly different position in the binding pocket, which affects its interactions with Ser215 (5.46) thus reducing its efficacy. Salbutamol binds in a similar fashion to dobutamine, but both Ser211 (5.42) and Ser215 (5.46) are in the same conformation as observed in inverse agonist-bound structures of β1AR, despite salbutamol forming polar interactions with them. However, there is still a contraction of the binding pocket as observed for full agonists (Table 1).

Both bucindolol and cyanopindolol are weak partial agonists of βARs. They both have an oxymethylene-spacer between the β-hydroxy group and their head groups, and therefore cannot induce a contraction of the binding pocket, and their head groups form van der Waals contacts with Ser215 (5.46) in the same way as inverse agonists do. So why do they show higher efficacy than inverse agonists? The major difference between structures of β1AR with weak partial agonists bound compared with inverse agonists is that the rotamer configuration of Ser211 (5.42) (gauche+) is the same as when full agonists are bound (Table 1). The effect of the gauche+ Ser211 (5.42) rotamer configuration is to affect interactions with water molecule W2 and hence the strength of interaction between H4 and H5 (compare Figures 2e, 2f and 2g). It is not possible for a hydrogen bond to form between Ser211 (5.42) in the gauche+ conformation and water molecule W2, and although W2 is clearly present in high-resolution structures, it is poorly co-ordinated [J.L. Miller, T. Warne, C.G. Tate, unpublished work]. We conclude that a combination of factors including contraction of the binding pocket, variable (van der Waals or polar) contacts with Ser215 (5.46) and the rotamer configuration of Ser211 (5.42) combine to determine the efficacy of partial agonists by subtly influencing the dynamics of the receptor.

How do biased agonists promote interactions with arrestin?

Ligands such as bucindolol and carvedilol stimulate G-protein-independent signalling in βARs by promoting phosphorylation of the C-terminus of the receptor, which then interacts with regulatory proteins such as arrestin [33]. It has been found that carvedilol promotes a pattern of phosphorylation at the C-terminus of the β2AR that is different to the normal pattern of phosphorylation when the receptor is desensitized [34]. There is some evidence from biophysical techniques that biased ligands influence the conformation of H7, which is then thought to have an effect on the flexible C-terminus of the receptor [35,36], a portion of the receptor that has been deleted in all of the crystallized βAR constructs. The structures of β1AR bound to either bucindolol or carvedilol show that the bulky substituents in their tail regions make contacts in the extended ligand-binding pocket to H2, H3, H7 and EL2 (extracellular loop 2) (Figure 2D) [9]. However, the unbiased ligand dobutamine also has an extended tail region, which makes contacts with H2 and H7 [10,37]. The only interaction made uniquely by biased agonists that is not made by unbiased ligands is with EL2. With the limited data available, it is difficult to assign determinants that cause biased agonism and undoubtedly structures of receptors bound to arrestin will also be required to resolve this issue.

Conclusion

βARs are highly dynamic molecules and the crystal structures that have been obtained may represent only a tiny fraction of the possible conformations of the native receptors under physiological conditions. However, there are subtle differences in structures with different classes of ligand that are entirely consistent with their efficacies and these suggest how such ligands may influence subsequent conformational changes and hence receptor signalling.

G-Protein-Coupled Receptors: from Structural Insights to Functional Mechanisms: A Biochemical Society Focused Meeting co-organized with Monash University held at Monash University Prato Centre, Prato, Italy, 12–14 September 2012. Organized and Edited by Bice Chini (CNR Institute of Neuroscience, Italy), Marco Parenti (University of Milano–Bicocca, Italy), David Poyner (Aston University, U.K.) and Mark Wheatley (University of Birmingham, U.K.)

Abbreviations

     
  • β2AR

    β2-adrenoceptor

  •  
  • CL3

    cytoplasmic loop 3

  •  
  • EL2

    extracellular loop 2

  •  
  • GPCR

    G-protein-coupled receptor

  •  
  • H

    transmembrane helix

  •  
  • rmsd

    root mean square deviation

  •  
  • T4L

    T4 lysozyme

  •  
  • W

    water molecule

Funding

Research in the laboratory of T.W. and C.G.T. on GPCRs is funded by the Medical Research Council [grant number MRC U105197215], the Wellcome Trust, the Biotechnology and Biological Sciences Research Council [grant number BB/G003653/1] and by grants from Heptares Therapeutics.

References

References
1
Pierce
 
K.L.
Premont
 
R.T.
Lefkowitz
 
R.J.
 
Seven-transmembrane receptors
Nat. Rev. Mol. Cell Biol.
2002
, vol. 
3
 (pg. 
639
-
650
)
2
Shenoy
 
S.K.
Lefkowitz
 
R.J.
 
Seven-transmembrane receptor signaling through β-arrestin
Sci. Signaling
2005
, vol. 
2005
 pg. 
cm10
 
3
Wise
 
A.
Gearing
 
K.
Rees
 
S.
 
Target validation of G-protein coupled receptors
Drug Discovery Today
2002
, vol. 
7
 (pg. 
235
-
246
)
4
Cherezov
 
V.
Rosenbaum
 
D.M.
Hanson
 
M.A.
Rasmussen
 
S.G.
Thian
 
F.S.
Kobilka
 
T.S.
Choi
 
H.J.
Kuhn
 
P.
Weis
 
W.I.
Kobilka
 
B.K.
Stevens
 
R.C.
 
High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor
Science
2007
, vol. 
318
 (pg. 
1258
-
1265
)
5
Tate
 
C.G.
Schertler
 
G.F.
 
Engineering G protein-coupled receptors to facilitate their structure determination
Curr. Opin. Struct. Biol.
2009
, vol. 
19
 (pg. 
386
-
395
)
6
Rasmussen
 
S.G.
Devree
 
B.T.
Zou
 
Y.
Kruse
 
A.C.
Chung
 
K.Y.
Kobilka
 
T.S.
Thian
 
F.S.
Chae
 
P.S.
Pardon
 
E.
Calinski
 
D.
, et al 
Crystal structure of the β2 adrenergic receptor-Gs protein complex
Nature
2011
, vol. 
477
 (pg. 
549
-
555
)
7
Warne
 
T.
Serrano-Vega
 
M.J.
Tate
 
C.G.
Schertler
 
G.F.
 
Development and crystallization of a minimal thermostabilized G protein-coupled receptor
Protein Expression Purif.
2009
, vol. 
65
 (pg. 
204
-
213
)
8
Serrano-Vega
 
M.J.
Tate
 
C.G.
 
Transferability of thermostabilizing mutations between β-adrenergic receptors
Mol. Membr. Biol.
2009
, vol. 
26
 (pg. 
385
-
396
)
9
Warne
 
T.
Edwards
 
P.C.
Leslie
 
A.G.
Tate
 
C.G.
 
Crystal structures of a stabilized β1-adrenoceptor bound to the biased agonists bucindolol and carvedilol
Structure
2012
, vol. 
20
 (pg. 
841
-
849
)
10
Warne
 
T.
Moukhametzianov
 
R.
Baker
 
J.G.
Nehme
 
R.
Edwards
 
P.C.
Leslie
 
A.G.
Schertler
 
G.F.
Tate
 
C.G.
 
The structural basis for agonist and partial agonist action on a β1-adrenergic receptor
Nature
2011
, vol. 
469
 (pg. 
241
-
244
)
11
Warne
 
T.
Serrano-Vega
 
M.J.
Baker
 
J.G.
Moukhametzianov
 
R.
Edwards
 
P.C.
Henderson
 
R.
Leslie
 
A.G.
Tate
 
C.G.
Schertler
 
G.F.
 
Structure of a β1-adrenergic G-protein-coupled receptor
Nature
2008
, vol. 
454
 (pg. 
486
-
491
)
12
Baker
 
J.G.
Proudman
 
R.G.
Tate
 
C.G.
 
The pharmacological effects of the thermostabilising (m23) mutations and intra and extracellular (β36) deletions essential for crystallisation of the turkey β-adrenoceptor
Naunyn-Schmiedeberg's Arch. Pharmacol.
2011
, vol. 
384
 (pg. 
71
-
91
)
13
Ballesteros
 
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
Methods Neurosci.
1995
, vol. 
25
 (pg. 
366
-
428
)
14
Birnbaumer
 
L.
Levy
 
F.O.
Zhu
 
X.
Kaumann
 
A.J.
 
Studies on the intrinsic activity (efficacy) of human adrenergic receptors. Co-expression of β1 and β2 reveals a lower efficacy for the β1 receptor
Tex Heart Inst. J.
1994
, vol. 
21
 (pg. 
16
-
21
)
15
Engelhardt
 
S.
Grimmer
 
Y.
Fan
 
G.H.
Lohse
 
M.J.
 
Constitutive activity of the human β1-adrenergic receptor in β1-receptor transgenic mice
Mol. Pharmacol.
2001
, vol. 
60
 (pg. 
712
-
717
)
16
Rosenbaum
 
D.M.
Zhang
 
C.
Lyons
 
J.A.
Holl
 
R.
Aragao
 
D.
Arlow
 
D.H.
Rasmussen
 
S.G.
Choi
 
H.J.
Devree
 
B.T.
Sunahara
 
R.K.
, et al 
Structure and function of an irreversible agonist-β2 adrenoceptor complex
Nature
2011
, vol. 
469
 (pg. 
236
-
240
)
17
Liapakis
 
G.
Ballesteros
 
J.A.
Papachristou
 
S.
Chan
 
W.C.
Chen
 
X.
Javitch
 
J.A.
 
The forgotten serine. A critical role for Ser-2035.42 in ligand binding to and activation of the β2-adrenergic receptor
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
37779
-
37788
)
18
Strader
 
C.D.
Candelore
 
M.R.
Hill
 
W.S.
Sigal
 
I.S.
Dixon
 
R.A.
 
Identification of two serine residues involved in agonist activation of the β-adrenergic receptor
J. Biol. Chem.
1989
, vol. 
264
 (pg. 
13572
-
13578
)
19
Katritch
 
V.
Reynolds
 
K.A.
Cherezov
 
V.
Hanson
 
M.A.
Roth
 
C.B.
Yeager
 
M.
Abagyan
 
R.
 
Analysis of full and partial agonists binding to β2-adrenergic receptor suggests a role of transmembrane helix V in agonist-specific conformational changes
J. Mol. Recognit.
2009
, vol. 
22
 (pg. 
307
-
318
)
20
Roth
 
C.B.
Hanson
 
M.A.
Stevens
 
R.C.
 
Stabilization of the human β2-adrenergic receptor TM4–TM3–TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure
J. Mol. Biol.
2008
, vol. 
376
 (pg. 
1305
-
1319
)
21
Miller
 
J.L.
Tate
 
C.G.
 
Engineering an ultra-thermostable β1-adrenoceptor
J. Mol. Biol.
2011
, vol. 
413
 (pg. 
628
-
638
)
22
Rasmussen
 
S.G.
Choi
 
H.J.
Fung
 
J.J.
Pardon
 
E.
Casarosa
 
P.
Chae
 
P.S.
Devree
 
B.T.
Rosenbaum
 
D.M.
Thian
 
F.S.
Kobilka
 
T.S.
, et al 
Structure of a nanobody-stabilized active state of the β2 adrenoceptor
Nature
2011
, vol. 
469
 (pg. 
175
-
180
)
23
Lebon
 
G.
Warne
 
T.
Edwards
 
P.C.
Bennett
 
K.
Langmead
 
C.J.
Leslie
 
A.G.
Tate
 
C.G.
 
Agonist-bound adenosine A(2A) receptor structures reveal common features of GPCR activation
Nature
2011
, vol. 
474
 (pg. 
521
-
525
)
24
Xu
 
F.
Wu
 
H.
Katritch
 
V.
Han
 
G.W.
Jacobson
 
K.A.
Gao
 
Z.G.
Cherezov
 
V.
Stevens
 
R.C.
 
Structure of an agonist-bound human A2A adenosine receptor
Science
2011
, vol. 
332
 (pg. 
322
-
327
)
25
Green
 
S.A.
Rathz
 
D.A.
Schuster
 
A.J.
Liggett
 
S.B.
 
The Ile164 β2-adrenoceptor polymorphism alters salmeterol exosite binding and conventional agonist coupling to Gs
Eur. J. Pharmacol.
2001
, vol. 
421
 (pg. 
141
-
147
)
26
Yao
 
X.J.
Velez Ruiz
 
G.
Whorton
 
M.R.
Rasmussen
 
S.G.
DeVree
 
B.T.
Deupi
 
X.
Sunahara
 
R.K.
Kobilka
 
B.
 
The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex
Proc. Natl. Acad. Sci. U.S.A.
2009
, vol. 
106
 (pg. 
9501
-
9506
)
27
Wisler
 
J.W.
DeWire
 
S.M.
Whalen
 
E.J.
Violin
 
J.D.
Drake
 
M.T.
Ahn
 
S.
Shenoy
 
S.K.
Lefkowitz
 
R.J.
 
A unique mechanism of beta-blocker action: carvedilol stimulates β-arrestin signaling
Proc. Natl. Acad. Sci. U.S.A.
2007
, vol. 
104
 (pg. 
16657
-
16662
)
28
Wacker
 
D.
Fenalti
 
G.
Brown
 
M.A.
Katritch
 
V.
Abagyan
 
R.
Cherezov
 
V.
Stevens
 
R.C.
 
Conserved binding mode of human β2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography
J. Am. Chem. Soc.
2010
, vol. 
132
 (pg. 
11443
-
11445
)
29
Moukhametzianov
 
R.
Warne
 
T.
Edwards
 
P.C.
Serrano-Vega
 
M.J.
Leslie
 
A.G.
Tate
 
C.G.
Schertler
 
G.F.
 
Two distinct conformations of helix 6 observed in antagonist-bound structures of a β1-adrenergic receptor
Proc. Natl. Acad. Sci. U.S.A.
2011
, vol. 
108
 (pg. 
8228
-
8232
)
30
Poldermans
 
D.
Boersma
 
E.
Fioretti
 
P.M.
van Urk
 
H.
Boomsma
 
F.
Man in ‘t Veld
 
A.J.
 
Cardiac chronotropic responsiveness to β-adrenoceptor stimulation is not reduced in the elderly
J. Am. Coll. Cardiol.
1995
, vol. 
25
 (pg. 
995
-
999
)
31
Liapakis
 
G.
Chan
 
W.C.
Papadokostaki
 
M.
Javitch
 
J.A.
 
Synergistic contributions of the functional groups of epinephrine to its affinity and efficacy at the β2 adrenergic receptor
Mol. Pharmacol.
2004
, vol. 
65
 (pg. 
1181
-
1190
)
32
Reference deleted
33
Rajagopal
 
S.
Rajagopal
 
K.
Lefkowitz
 
R.J.
 
Teaching old receptors new tricks: biasing seven-transmembrane receptors
Nat. Rev. Drug Discovery
2010
, vol. 
9
 (pg. 
373
-
386
)
34
Nobles
 
K.N.
Xiao
 
K.
Ahn
 
S.
Shukla
 
A.K.
Lam
 
C.M.
Rajagopal
 
S.
Strachan
 
R.T.
Huang
 
T.Y.
Bressler
 
E.A.
Hara
 
M.R.
, et al 
Distinct phosphorylation sites on the β2-adrenergic receptor establish a barcode that encodes differential functions of β-arrestin
Sci. Signaling
2011
, vol. 
4
 pg. 
ra51
 
35
Granier
 
S.
Kim
 
S.
Shafer
 
A.M.
Ratnala
 
V.R.
Fung
 
J.J.
Zare
 
R.N.
Kobilka
 
B.
 
Structure and conformational changes in the C-terminal domain of the β2-adrenoceptor: insights from fluorescence resonance energy transfer studies
J. Biol. Chem.
2007
, vol. 
282
 (pg. 
13895
-
13905
)
36
Liu
 
J.J.
Horst
 
R.
Katritch
 
V.
Stevens
 
R.C.
Wuthrich
 
K.
 
Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR
Science
2012
, vol. 
335
 (pg. 
1106
-
1110
)
37
Rajagopal
 
S.
Ahn
 
S.
Rominger
 
D.H.
Gowen-McDonald
 
W.
Lam
 
C.M.
Dewire
 
S.M.
Violin
 
J.D.
Lefkowitz
 
R.J.
 
Quantifying ligand bias at seven-transmembrane receptors
Mol. Pharmacol.
2011
, vol. 
80
 (pg. 
367
-
377
)