Research Article

GPCR Engineering Yields High-Resolution Structural Insights into β2-Adrenergic Receptor Function

See allHide authors and affiliations

Science  23 Nov 2007:
Vol. 318, Issue 5854, pp. 1266-1273
DOI: 10.1126/science.1150609


The β2-adrenergic receptor (β2AR) is a well-studied prototype for heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) that respond to diffusible hormones and neurotransmitters. To overcome the structural flexibility of the β2AR and to facilitate its crystallization, we engineered a β2AR fusion protein in which T4 lysozyme (T4L) replaces most of the third intracellular loop of the GPCR (“β2AR-T4L”) and showed that this protein retains near-native pharmacologic properties. Analysis of adrenergic receptor ligand-binding mutants within the context of the reported high-resolution structure of β2AR-T4L provides insights into inverse-agonist binding and the structural changes required to accommodate catecholamine agonists. Amino acids known to regulate receptor function are linked through packing interactions and a network of hydrogen bonds, suggesting a conformational pathway from the ligand-binding pocket to regions that interact with G proteins.

The adrenergic receptors make up a class of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) that play a central role in mediating the effects of catecholamine hormones. In contrast to rhodopsin, which is expressed at very high levels in photoreceptor cells and has the ligand retinal covalently bound (1), other GPCRs such as the β2-adrenergic receptor (β2AR) generally express at low levels, bind diffusible ligands, and exhibit greater functional and structural plasticity (2). The β2AR was the first non-rhodopsin GPCR to be cloned and has been one of the most extensively studied members of this large receptor family.

To obtain high-resolution structural information on the β2AR, we increased its proteolytic stability and crystallizability by eliminating the C-terminal tail and replacing most of the third intracellular loop (ICL3) with the protein T4 lysozyme (T4L). The optimized β2AR-T4L protein was crystallized in lipidic cubic phase, as described in the companion paper by Cherezov et al. (3), and the resulting 2.4 Å resolution crystal structure reveals the interface between the receptor and the ligand carazolol, a partial inverse agonist (4). Analysis of mutagenesis data in light of the structure clarifies the roles of different amino acids in inverse-agonist binding and implies that rearrangement of the binding pocket accompanies agonist binding. In addition, the structure reveals how mutations known to cause constitutive activity or uncoupling of agonist binding and G protein activation are distributed between the ligand-binding pocket and the cytoplasmic surface of the protein, such that changes in side chains due to interaction with the ligand can be transmitted through the structure to the site of G protein interaction.

β2AR-T4L: A crystallizable GPCR fusion protein. The conformational complexity that makes GPCRs versatile signaling molecules may contribute to the difficulty of crystallizing these proteins (2). Despite substantial efforts, we were unable to grow diffraction-quality crystals from purified, homogeneous wild-type (WT) β2AR. This was probably due to conformational variability of the flexible ICL3 and C terminus, as well as the relatively small polar surface available for crystal contacts. Fluorescence resonance energy transfer experiments show that the C-terminal portion of the β2AR is in an extended conformation (5), a property that may be important for the role of this region in interacting with signaling and scaffold proteins, but one that would probably interfere with crystal-lattice formation. ICL3, which links the cytoplasmic ends of helices V and VI, is functionally important both for the specificity of receptor–G protein interactions and for G protein activation. In the β2AR, this domain is susceptible to proteolysis; however, proteolytic cleavage does not lead to dissociation of the transmembrane segments linked by the ICL3. In fact, β2AR helices I to V and helices VI and VII behave as independent folding domains that can be expressed on separate plasmids and assemble to form a functional “split” receptor (6). Therefore, we speculated that ICL3 links two domains with a relatively dynamic interface, which could be important for function but may also contribute to greater instability.

Because the majority of membrane protein crystals form through contacts of the nonmembranous portions of the molecule, we sought to improve the chances of β2AR crystallization by replacing ICL3 with a well-structured, soluble domain that might aid in the formation of lattice contacts. The initial criteria for choosing the inserted soluble protein were that the N and C termini would approximate the predicted distance between the cytoplasmic ends of helix V and helix VI and that the protein would crystallize under a variety of conditions. T4L is a small, stable protein that fulfills these criteria (7). The N and C termini of WT T4L are 10.7 Å apart in Protein Data Bank (PDB) structure 2LZM (8), compared with a distance of 15.9 Å between the carbonyl carbon of residue 2285.63 (9) and the amide nitrogen of residue 2416.24 in the high-resolution structure of rhodopsin (PDB 1U19) (10).

DNA encoding the T4L protein (Cys54 → Thr54, Cys97 → Ala97) (11) was initially cloned into the human β2AR gene, guided by a comparison of ICL3 length and sequence among class A GPCRs (12): residues 2345.73 to 2596.21 of the β2AR were replaced by residues 2 to 164 of T4L (construct “E3” in Fig. 1A). In addition, the receptor was truncated at position 365, which aligns approximately with the position of the rhodopsin C terminus. Although these modifications resulted in a receptor that was expressed efficiently in Sf9 cells, further optimization was carried out to reduce the length of the junction between the receptor and the T4L termini (13). Several candidate constructs are illustrated in Fig. 1A, and selected immunofluorescence images of transfected, permeabilized human embryonic kidney (HEK) 293 cells are shown in Fig. 1B. Relative to the initial construct, we could remove three residues from the cytoplasmic end of helix V, three from the C-terminal end of T4L, and three from the N terminus of helix VI, all without losing substantial cell-surface expression. The final construct used for crystallization trials (β2AR-T4L) has residues 2315.70 to 2626.24 of the β2AR replaced by amino acids 2 to 161 of T4L (D1 in Fig. 1A). Similar reduction of flexibility through minimization of linker length has been important in previous crystallization studies on soluble fusion proteins (14).

Fig. 1.

Design and optimization of the β2AR-T4L fusion protein. (A) The sequence of the region of the β2AR targeted for insertion of a crystallizable domain is shown, and the positions of the junctions between the receptor and T4L (red) for various constructs are indicated. The sequences that were initially replaced or removed are faded. Red lines are shown after every tenth residue. ECL, extracellular loop. (B) Immunofluorescence images of HEK293 cells expressing selected fusion constructs. (Left) M1 anti-FLAG signal corresponding to antibody bound to the N terminus of the receptor. (Right) Same signal merged with blue emission from 4′,6′-diamidino-2-phenylindole (nuclear staining for all cells). Plasma membrane staining is observed in the positive control, D3, and D1, whereas C3 and D5 are retained in the endoplasmic reticulum.

Functional properties ofβ2AR-T4L. We measured saturation binding of [3H]DHA to the β2AR-T4L, as well as competition binding of the inverse agonist ICI-118,551 and several agonists (Fig. 2A, fig. S1, and table S1). The results show that β2AR-T4L has WT affinity for the antagonist [3H]DHA and the inverse agonist ICI-118,551, whereas the affinity for both agonists (e.g., isoproterenol, epinephrine, formoterol) and a partial agonist (salbutamol) is two-to-threefold higher relative to WT β2AR. Higher agonist binding affinity is a property associated with constitutively active mutants (CAMs) of GPCRs. CAMs of the β2AR also exhibit elevated basal, agonist-independent activation of the stimulatory G protein for adenylyl cyclase (Gs) and typically have lower expression levels and reduced stability (15, 16). β2AR-T4L exhibits binding properties of a CAM, but it expresses at levels exceeding 1 mg/liter of Sf9 cell culture, is more resistant to trypsin proteolysis than the WT β2AR (fig. S2), and retains binding activity in detergent at 37°C, as well as the WT receptor (fig. S3).

Fig. 2.

Functional characterization of β2AR-T4L. (A) Affinity competition curves for adrenergic ligands binding to β2AR-T4L and WT β2AR. Binding experiments on membranes isolated from Sf9 insect cells expressing the receptors were performed as described (13). (B) β2AR-T4L is still able to undergo ligand-induced conformational changes. Bimane fluorescence spectra (excitation at 350 nm) of detergent-solubilized β2AR-T4L and WT β2AR truncated at 365, labeled under conditions that selectively modify Cys2656.27 (13), were measured after incubating the unliganded receptors with compounds for 15 min at room temperature. The cartoon illustrates that the observed changes in fluorescence can be interpreted as a movement of the bimane probe from a more buried, hydrophobic environment to a more polar, solvent-exposed position. cps, counts per second.

β2AR-T4L did not couple to Gs, as expected, because of the replacement of ICL3 by T4L. To assess whether the fused protein alters receptor function at the level of its ability to undergo conformational changes, we used a covalently attached fluorescent probe as a reporter for ligand-induced structural changes. Fluorophores attached at Cys2656.27, at the cytoplasmic end of helix VI, detect agonist-induced conformational changes that correlate with the efficacy of the agonist toward G protein activation (1720). Detergent-solubilized β2AR365 (WT receptor truncated at 365) and β2AR-T4L were each labeled with monobromobimane, which has been used previously to monitor conformational changes of the β2AR (21). The addition of the agonist isoproterenol to purified β2AR365 induces a decrease in fluorescence intensity and a shift in the wavelength at maximum intensity (λmax) for the attached bimane probe (Fig. 2B and table S2). These changes in intensity and λmax are consistent with an agonist-induced increase in polarity around bimane. A smaller change is observed with the partial agonist salbutamol, whereas the inverse agonist ICI-118,551 had little effect. For the β2AR-T4L, there are subtle differences in the baseline spectrum of the bimane-labeled fusion protein, as might be expected if the environment around Cys2656.27 is altered by T4L. However, the full agonist isoproterenol induces a qualitatively similar decrease in intensity and rightward shift in λmax. Thus, the presence of the fused T4L does not prevent agonist-induced conformational changes. The partial agonist salbutamol induced larger responses in β2AR-T4L than were observed in WT β2AR, and there was a small increase in fluorescence in response to the inverse agonist ICI-118,551. These properties are observed in CAMs (15, 22) and are consistent with the higher affinities for agonists and partial agonists exhibited by β2AR-T4L. Therefore, we conclude that the T4L fusion induces a partial constitutively active phenotype in the β2AR, probably caused by changes at the cytoplasmic ends of helices V and VI.

Comparison between b2AR-T4L and b2AR-Fab structures. The β2AR-T4L fusion strategy is validated by a comparison of its structure to that of WT β2AR complexed with a Fab that recognizes a three-dimensional epitope consisting of the N- and C-terminal ends of ICL3, determined at an anisotropic resolution of 3.4 Å/3.7 Å (23). Figure 3A illustrates the similarity between the fusion and antibody complex approaches to β2AR crystallization, in that both strategies rely on attachment (covalent or noncovalent, respectively) of a soluble protein partner between helices V and VI. A major difference between the two structures is that the extracellular loops and the carazolol ligand could not be modeled in the β2AR-Fab complex, whereas these regions are resolved in the structure of β2AR-T4L. Nonetheless, it is clear that the T4L insertion does not substantially alter the receptor. Superposition of the two structures (fig. S4) illustrates that the transmembrane helices of the receptor components are very similar (root mean square deviation = 0.8 Å for 154 common modeled transmembrane Cα positions versus 2.3 Å between β2AR-T4L and the 154 equivalent residues in rhodopsin), especially when the modest resolution of the Fab complex is taken into account.

Fig. 3.

(A) Side-by-side comparison of the crystal structures of the β2AR-T4L fusion protein and the complex between β2AR365 and a Fab fragment. The receptor component of the fusion protein is shown in blue (with modeled carazolol as red spheres), whereas the receptor bound to Fab5 is yellow. (B) Differences in the environment surrounding Phe2646.26 (shown as spheres) for the two proteins. (C) Analogous interactions to the ionic lock between the E(D)RY motif and Glu2476.30 seen in rhodopsin (right panel, purple) are broken in both structures of the β2AR (left panel, blue and yellow as above). PyMOL (43) was used for the preparation of all figures.

There is one major difference between the Fab-complex and chimeric-receptor structures that can be attributed to the presence of T4L. The cytoplasmic end of helix VI is pulled outward as a result of the fusion to the C terminus of T4L, which alters the packing of Phe2646.26 at the end of helix VI (Fig. 3B). In the Fab-complex β2AR, interactions between Phe2646.26 and residues in helix V, helix VI, and ICL2 may be important in maintaining the β2AR in the basal state. The loss of these packing interactions in β2AR-T4L could contribute to the higher agonist binding affinity characteristic of a CAM.

An unexpected difference between the structure of rhodopsin and the β2AR-T4L involves the sequence E/DRY (24) found at the cytoplasmic end of helix III in 71% of class A GPCRs. In rhodopsin, Glu1343.49 and Arg1353.50 form a network of hydrogen bond and ionic interactions with Glu2476.30 at the cytoplasmic end of helix VI. These interactions have been referred to as an “ionic lock” that stabilizes the inactive state of rhodopsin and other class A members (25). However, the arrangement of the homologous residues is considerably different in β2AR-T4L: Arg1313.50 interacts primarily with Asp1303.49 and a sulfate ion rather than with Glu2686.30, and the distance between helix III and helix VI is greater than in rhodopsin (Fig. 3C). This difference might be explained by the interaction between Glu2686.30 and Arg8 of T4L; however, the arrangement of Asp1303.49 and Arg1313.50 and the distance between helix III and helix VI is very similar to that observed in the β2AR-Fab structure. Although the presence of an antibody or T4L at the ICL3 region could potentially affect the arrangement of these residues, the fact that similar ionic-lock structures were obtained using two different approaches suggests that a broken ionic lock may be a genuine feature of the carazolol-bound state of the receptor.

Ligand binding to the β2AR. The β2AR-T4L fusion protein was purified and crystallized in complex with the inverse agonist carazolol. Carazolol stabilizes the β2AR against extremes of pH and temperature, perhaps related to its unusually high binding affinity (equilibrium dissociation constant Kd < 0.1 nM) and slow dissociation kinetics (half-time t1/2 ∼ 30 hours) (fig. S5). The interactions between carazolol and β2AR-T4L are depicted schematically in Fig. 4. The carbazole ring system is oriented roughly perpendicular to the plane of the membrane, and the alkylamine chain (atoms 15 to 22 in the model) is nearly parallel to the heterocycle (Fig. 5, A and B). Carazolol was modeled into the electron density (3) as the (S)-(–) isomer because of the higher affinity of this enantiomer, despite the fact that a racemic mixture of the ligand was used in crystallization. Asp1133.32, Tyr3167.43, and Asn3127.39 present a constellation of polar functional groups to the alkylamine and alcohol moieties of the ligand, with the Asp1133.32 and Asn3127.39 side chains forming close contacts (<3 Å) with the O17 and N19 atoms of carazolol (Figs. 4 and 5, A and B). Asp1133.32 was one of the first β2AR residues shown to be important for ligand binding; the D113N mutation causes a complete loss of detectable affinity for antagonists (26) and a decrease in the potency of agonists toward cell-based G protein activation by over four orders of magnitude (27). Likewise, mutations of Asn3127.39 perturb β2AR binding to agonists and antagonists: Changes to nonpolar amino acids (Ala or Phe) reduce affinities to undetectable levels, whereas retention of a polar functionality (Thr or Gln) gives partial affinity (28). On the opposite end of the ligand near helix V, N7 of the carbazole heterocycle forms a hydrogen bond with the side-chain hydroxyl of Ser2035.42. Mutations of Ser2035.42 specifically decrease β2AR affinity toward catecholamine agonists and aryloxyalkylamine ligands with nitrogen-containing heterocycles such as pindolol (29) and, by implication, carazolol. Thus, the polar interactions between carazolol and the receptor observed in the crystal structure agree with the known biochemical data. The contribution of Tyr3167.43 to antagonist and agonist affinity remains to be tested; this residue is conserved as Tyr in all sequenced adrenergic receptor genes (12).

Fig. 4.

Schematic representation of the interactions between β2AR-T4L and carazolol at the ligand binding pocket. The residues shown here have at least one atom within 4 Å of the ligand in the 2.4 Å resolution crystal structure.

Fig. 5.

Ligand binding pocket of β2AR-T4L with carazolol bound. (A) Residues within 4 Å of the ligand are shown as sticks, with the exception of A200, N293, F289, and Y308. Residues that form polar contacts with the ligand (distance cutoff: 3.5 Å) are shown in green, other residues are gray, and carazolol is yellow (in all panels, oxygens are red and nitrogens are blue). (B) Same as (A), except that the ligand is oriented with its amine facing out of the page. W109 is not shown. (C) Packing interactions between carazolol and all residues within 5 Å of the ligand. The view is from the extracellular side of the membrane. Carazolol is shown as yellow spheres, and receptor residues are shown as sticks within van der Waals dot surfaces. Val1143.33, Phe1935.32, and Phe2906.52 are red, and all other residues are gray. (D) Model of (–)-isoproterenol (magenta sticks) in the ligand binding pocket observed in the crystal structure. A model of the agonist with optimal bond lengths and angles was obtained from the PRODRG server (44), and the dihedral angles were adjusted to the values observed in the homologous atoms of bound carazolol (16 to 22 in Fig. 4). The one remaining unaccounted dihedral in (–)-isoproterenol was adjusted in order to place the catechol ring in the same plane as the C16–C15–O14 plane in carazolol. Residues known to specifically interact with agonists are shown as green sticks.

Figure 5C shows the tight packing between carazolol and surrounding amino acids that buries 790 Å2 of surface area from solvent; specific contacts are depicted schematically in Fig. 4. Notable among the hydrophobic residues contacting carazolol are Val1143.33, Phe2906.52, and Phe1935.32. The side chain of Val1143.33 from helix III makes multiple contacts with the C8-to-C13 ring of the carbazole heterocycle, and Phe2906.52 from helix VI forms an edge-to-face aromatic interaction with the same ring. As a result, these two amino acids form a hydrophobic “sandwich” with the portion of the aryl moiety that is common to many adrenergic antagonists. The mutation of Val1143.33 to Ala was shown to decrease β2AR affinity toward the antagonist alprenolol by one order of magnitude, as well as to lower affinity for the agonist epinephrine 300-fold (30). Phe1935.32 is different from other carazolol contact residues in that it is located on the second extracellular loop, in the path of hormone accessibility to the binding pocket. This amino acid contributes more buried surface area than any other residue to the interface between β2AR-T4L and carazolol (table S3). Therefore, Phe1935.32 is likely to contribute substantially to the energy of β2AR-carazolol complex formation, and the position of this residue on the extracellular side of the binding site may allow it to act as a gate that contributes to the unusually slow dissociation of the ligand (fig. S5).

Analysis of the binding pocket provides insights into the structural basis for pharmacologic selectivity between the β2AR and closely related adrenergic receptors such as the β1AR. The affinities of these two receptors for certain ligands, such as ICI-118,551, betaxolol, and RO363 (31), differ by up to 100-fold. Curiously, all of the amino acids in the carazolol binding pocket are conserved between the β1AR and β2AR [fig. S6 in (13)]. The majority of the 94 amino acid differences between the β1AR and β2AR are found in the cytoplasmic and extracellular loops. Whereas residues that differ in the transmembrane segments generally face the lipid bilayer, eight residues lie at the interface between helices and may influence helix packing. The structural basis for pharmacologic differences between β1AR and β2AR must, therefore, arise from amino acid differences in the entrance to the binding pocket or subtle differences in the packing of helices. Evidence for the latter comes from chimeric-receptor studies (32) in which successive exchange of helices between β1AR and β2ARs led to a gradual change in affinity for the β2AR selective ICI-118,551 and the β1AR selective betaxolol.

As discussed above, β2AR-T4L shows CAM-like properties with respect to agonist binding affinities, suggesting that the unliganded β2AR-T4L may exist in a more active conformation than the WT β2AR. Nevertheless, as shown in Fig. 2B, β2AR-T4L can be stabilized in an inactive conformation by an inverse agonist. Because β2AR-T4L was crystallized with bound carazolol, a partial inverse agonist, the structure most likely represents an inactive state. This is consistent with the similarity of the β2AR-T4L and β2AR-Fab5 carazolol-bound structures. To assess whether conformational changes are required to accommodate catecholamines, a model of isoproterenol was placed in the binding site such that common atoms (16 to 22 in Fig. 4) were superimposed onto the analogous carazolol coordinates in the crystal structure (Fig. 5D). Residues Ser2045.43 and Ser2075.46 are critical for catecholamine binding and activation of the β2AR, with Ser2045.43 hydrogen bonding to the meta-hydroxyl and Ser2075.46 to the para-hydroxyl of the catechol ring, respectively (33). In our model, the catechol hydroxyls of isoproterenol face the appropriate Ser residues on helix V, but the distances are too long for hydrogen bonding (6.8 Å from the meta-hydroxyl oxygen to the side-chain oxygen of Ser2045.43 and 4.8 Å from the para-hydroxyl oxygen to the side-chain oxygen of Ser2075.46). In addition, Asn2936.55 and Tyr3087.35, two residues expected to form selective interactions with agonists based on the literature (34, 35), are too distant to form productive polar or hydrophobic contacts with the modeled isoproteronol molecule. These observations suggest that agonist binding requires changes in the binding site relative to the carazolol-bound structure, unless common structural components of agonists and inverse agonists bind in a considerably different manner.

Structural insights into β2AR activation. Biophysical studies provide evidence that conformational changes associated with activation of the β2AR are similar to those observed for rhodopsin (2, 18, 21, 36). Yet the highly efficient process of light activation of rhodopsin through the cis-trans isomerization of covalently bound retinal is very different from activation of the β2AR and other GPCRs by diffusible hormones and neurotransmitters (2). Despite representing a static picture of the inverse agonist–bound state, the crystal structure of β2AR-T4L may still provide clues as to how agonist binding is translated into structural changes in the cytoplasmic domains of receptor. Agonist binding occurs at the extracellular ends of helices III, IV, V, and VII, and G protein activation is mediated by the cytoplasmic ends. Whereas the structure is open at the extracellular face to form the ligand binding pocket, the helices are more closely packed in the intracellular half of the receptor. This close packing implies that isolated rigid-body movement of any of these helices is unlikely and that conformational changes can be accomplished only by rearrangement of the side chains forming the network of interactions between the helices. Biophysical studies show that structurally different agonists stabilize distinct active states (17, 20), suggesting that different ligands could stabilize different combinations of side-chain rearrangements.

Analysis of mutations that affect β2AR function provides insights into structural rearrangements that are likely to occur during receptor activation. Figure 6A illustrates the location of amino acids for which mutations lead to elevated basal, agonist-independent activity (CAMs), as well as amino acids for which mutations impair agonist activation [uncoupling mutations (UCMs)]. Residues for which CAMs have been described are likely to be involved in interactions that maintain the receptor in the inactive conformation. These amino acids are centrally located on helices III and VI. In contrast, positions in which UCMs have been observed are likely to form intramolecular interactions that stabilize the active state. A cluster of UCMs is found at the cytoplasmic end of helix VII. Neither CAMs nor UCMs are directly involved in agonist binding. Although the CAMs and UCMs are not directly connected in sequence, it is evident from the structure that they are linked through packing interactions, so that movements in one will probably affect the packing of others. For example, Fig. 6A (right panel) shows all amino acids with atoms within 4 Å of the two centrally located CAMs, Leu1243.43 (37) and Leu2726.34 (38). Several amino acids that pack against these CAMs also interact with one or more UCMs. Trp2866.48 lies at the base of the binding pocket. It has been proposed that agonist binding leads to a change in the rotameric state of Trp2866.48, with subsequent changes in the angle of the helical kink formed by Pro2886.50 (39). It is likely that an agonist-induced change in the rotameric state of Trp2866.48 will be linked to changes in side chains of CAMs and UCMs through packing interactions and propagated to the cytoplasmic ends of the helices and the associated intracellular loops that interact with G proteins and other signaling molecules.

Fig. 6.

Packing interactions in the β2AR that are likely to be modulated during the activation process. (A) (Left) Residues previously demonstrated to be CAMs (16, 25, 3739, 45) or UCMs (26, 41, 4649) are shown as van der Waals spheres mapped onto a backbone cartoon of the β2AR-T4L structure. (Right) Residues that are found within 4 Å of the CAMs Leu1243.43 and Leu2726.34 are shown as yellow spheres or dot surfaces. A vertical cross section through the structure illustrates that these surrounding residues connect the CAMs on helices III and VI with the UCMs on helix VII through packing interactions. (B) In both β2AR-T4L (blue) and rhodopsin (purple), a network of ordered water molecules is found at the interface between the transmembrane helices at their cytoplasmic ends. (C) Network of hydrogen bonding interactions between water molecules and β2AR-T4L residues (side chains shown as blue sticks), notably three UCMs on helix VII (orange) and one on helix II (yellow). In (B) and (C), only Ballesteros-Weinstein numbers (9) are used to identify amino acids.

In the structures of both rhodopsin and the β2AR, a cluster of water molecules lies near the most highly conserved class A GPCR residues (Fig. 6B). It has been proposed that these water molecules may play a role in the structural changes involved in receptor activation (40). Figure 6C shows the network of potential hydrogen bonding interactions that link Trp2866.48 with conserved amino acids extending to the cytoplasmic ends of helices. UCMs have been identified for four amino acids linked by this network: Asp792.50, Asn3187.45, Asn3227.49, and Y3267.53 (41). This relatively loose-packed, water filled region is probably important in allowing conformational transitions, as there will be fewer steric restraints to side-chain repacking. Future structures of the agonist-bound state of the β2AR will help to clarify the precise rearrangements that accompany activation of the receptor.

Although crystallization of β2AR-T4L in the presence of other ligands remains an exciting prospect, future efforts to obtain structures of the catecholamine-bound receptor will be challenging because of the relatively low affinity of these compounds in the absence of G protein and their chemical lability. Moreover, biophysical studies indicate that agonist binding and activation is a multistep process involving distinct conformational intermediates (2, 17, 1921, 42), which produces structural heterogeneity even at saturating concentrations of agonist. It is therefore possible that structural elucidation of the active state of the β2AR will be possible only through crystallization of the complex between agonist, β2AR, and Gs.

Supporting Online Material

Materials and Methods

Figs. S1 to S6

Tables S1 to S3


References and Notes

View Abstract

Navigate This Article