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Department of Medicine (A.K.S., J.-P.S., R.J.L.), and Department of Biochemistry (A.K.S., J.-P.S., R.J.L.) and Howard Hughes Medical Institute (R.J.L.), Duke University Medical Center, Durham, North Carolina
Received for publication January 31, 2008.
Accepted for publication January 31, 2008.
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Abstract |
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). Now, the recent publication of two crystal structures of the human β2AR once again permits new insights to be gained from comparison of the properties of these two model seven transmembrane-spanning receptors (7TMRs) (Cherezov et al., 2007; Rasmussen et al., 2007; Rosenbaum et al., 2007). The remarkable and unique abundance of rhodopsin in rod outer segments (it constitutes 
90% of the protein in rod outer segment membranes) and its stability led to the determination of its complete amino acid sequence in 1982 by classic Edman degradation and to the appreciation of its seven transmembrane organization (Ovchinnikov, 1982; Hargrave et al., 1983). By comparison, the rarity of the β2AR and essentially all other GPCRs (they need to be purified several hundred thousand-fold from naturally occurring sources to obtain homogeneous preparations) greatly hindered its biochemical study. Nonetheless, sufficient protein was purified so that, based on small stretches of amino acid sequence obtained from the receptor, its gene and cDNA were successfully cloned in 1986 (Dixon et al., 1986). It is remarkable that, in retrospect, it was only then that its close structural relationship with rhodopsin was first appreciated. This, despite the general understanding at the time that both rhodopsin and the β2AR signaled by activation of G proteins (transducin and Gs, respectively). The discovery of the homology between the β2AR and rhodopsin followed rapidly by the cloning of additional adrenergic receptors and others in turn triggered the rapid realization that all GPCRs share a conserved seven transmembrane organization and are members of the same gene family (Dohlman et al., 1991).
The first crystal structure of rhodopsin in its inactive state was reported in 2000 (Palczewski et al., 2000), and the new β2AR structures are the first of any other GPCR to appear. Given the close parallels and centrality of research on these two model 7TMRs, it seems somehow fitting that a comparison of their molecular structures should once again be in the spotlight of molecular pharmacologists.
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Why Did It Take So Long? |
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What Made It Possible? |
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). In addition to truncating part of the flexible C terminus of the β2AR, two parallel approaches were taken to address this issue. First, an antibody fragment (Fab) was generated against a three-dimensional epitope corresponding to the third intracellular loop (Fig. 1A) using hybridoma technology (Day et al., 2007). This approach of cocrystallizing membrane proteins with an antibody fragment was first developed and used successfully in the cytochrome c oxidase structure determination and since then has been used for several other membrane proteins (Hunte and Michel, 2002). The second approach used to reduce the flexibility of the receptor and increase its polar surface area was to replace the third intracellular loop with T4 lysozyme, a highly crystallizable soluble protein (Fig. 1B).
In addition to these protein engineering and antibody approaches, recent developments in membrane protein crystallography were also crucial to the success. In contrast to traditional detergent crystallization of membrane proteins, the β2AR was crystallized in either DMPC/CHAPSO bicelles or monolein lipidic cubic phase with cholesterol as the additive. Both of these methods, which essentially rely on the use of different lipids to present a more native environment to the protein, have been reported to yield well-diffracting crystals for several membrane proteins such as bacteriorhodopsin and the photosynthetic reaction center (Faham and Bowie, 2002). Moreover, because the crystals of the β2AR were small and very radiation-sensitive, microfocused beam lines were used instead of a conventional synchrotron source, for both screening and data collection.
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Why the β2AR Rather Than Others? |
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). As discussed above, one of the major obstacles in crystallizing proteins is obtaining functional and homogenous material, because any heterogeneity hampers the formation of well-ordered crystals. Heterologously produced receptors are often purified using affinity tags genetically fused to the protein. Although such purification schemes may result in reasonably pure receptors as judged by gel analysis, there generally still exists a mixture of both functional and nonfunctional (i.e., denatured) receptor populations, which substantially decreases the chances of crystallization. In this regard, the alprenolol (a β-adrenergic antagonist) affinity column developed for the β2AR (Caron et al., 1979) offers a unique advantage and yields receptor samples with almost 100% specific ligand binding activity and, thus, a very homogenous receptor preparation. It should also be noted that carazolol, used as a ligand in both structures, has exceptionally high affinity for the receptor and very slow dissociation kinetics, thus stabilizing the receptor even under relatively harsh conditions. |
How Similar Is the Structure to That of Rhodopsin? |
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-carbon backbone of the transmembrane region between rhodopsin and β2AR is 1.56 Å, which indicates a very similar arrangement of the transmembrane helices. This feature also supports the previous notion of a conserved activation mechanism (i.e., agonist-induced conformational rearrangement) across this class of transmembrane receptors. Nonetheless, there are structural features in the β2AR that differ significantly from rhodopsin and that thus high-light the existence of receptor-specific patterns in this class of GPCRs. For example, the second extracellular loop of the β2AR contains a previously unanticipated short helix. This helix contains two disulfide bonds which seem to maintain this loop in a constrained state, presumably providing space for the diffusion of ligands to the binding pocket of the receptor (Fig. 1A). This is in contrast with rhodopsin, in which the second extracellular loop contains a buried β-sheet which, together with the interactions of the N terminus with other extracellular loops, forms a cap-like structure that isolates the retinal-binding site in a hydrophobic pocket.
Another interesting difference between rhodopsin and the β2AR structures is the state of the "ionic lock", defined as a network of hydrogen bonding and charge interactions between Arg131 in TM3 and Glu268 in TM6 (Arg135 and Glu247 in rhodopsin). The ionic lock is considered to maintain rhodopsin and presumably other class A GPCRs, including the β2AR, in an inactive conformation (Ballesteros et al., 2001). Indeed the crystal structure of inactive rhodopsin displays an intact ionic lock with a distance of 2.9 Å between Glu247 and Arg135, whereas in the light-activated structure of rhodopsin, the lock is broken with a distance of 4.1 Å between Arg135 and Glu247 (Palczewski et al., 2000; Salom et al., 2006). Both β2AR structures contain an inverse agonist, carazolol, and based on classic receptor theory, one would expect such a ligand to stabilize the receptor in an inactive state, much as covalently attached retinal does for rhodopsin. However, in both β2AR structures, the ionic lock is broken with a distance of 6.2 Å between Arg131 and Glu268 in the β2AR-Fab structure and 10.58 Å between Arg131 and Glu268 in the β2AR-T4 chimera structure (Fig. 1C). Although one cannot exclude the possibility of alteration in some structural features of the β2AR due to binding of Fab or the presence of T4 lysozyme, the fact that similar ionic lock features were seen in both structures speaks against this being an artifact. However, the increased affinity of the β2AR-T4 chimera for agonists compared with the wild-type β2AR also suggests that the receptor is in an "active-like state" and that the broken ionic lock is a genuine feature of the receptor in the conformation that has been crystallized.
Comparison of the ligand binding pockets and the orientation of ligands between rhodopsin and the β2AR reveals another significant difference between these two receptors. Although carazolol in the β2AR and cis-retinal in rhodopsin exhibit similar binding modes, their interaction with the highly conserved "rotamer toggle" (Trp265 in rhodopsin and Trp286 in β2AR) differs. It has been suggested that a change in the rotameric state of this tryptophan in rhodopsin and other class A GPCRs serves as a second activation switch (Shi et al., 2002). Direct interaction of cis-retinal with Trp265 has been proposed to maintain the inactive state of rhodopsin. Although carazolol does not directly interact with Trp286 in the β2AR, it seems to control the rotameric state of Trp286 indirectly via interacting with Phe289 and Phe290. Although these interactions are proposed based on a relatively high-resolution structure of the β2AR, the only direct way to validate these mechanisms would be to determine the structure of the β2AR in an active state.
Another distinctive feature is the different patterns of the two receptors with respect to their oligomeric state in the crystal structure. All of the structures of rhodopsin determined so far exhibit a receptor dimer. On the contrary, the β2AR crystals show monomers or minimal inter-receptor contacts.
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What Does the Structure Teach Us? |
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). Likewise, mutations of Asn312 result in partial or complete loss of ligand affinities (Suryanarayana and Kobilka, 1993). Indeed, both Asp113 and Asn312 are seen to interact with O17 and N19 of carazolol, thus explaining why these mutations are detrimental to ligand binding. Along the same line, Val114 and Phe290 extensively interact with the C8-to-C13 ring of the carbazole moiety of carazolol and form a hydrophobic sandwich with this aryl group. These interactions might explain the loss of affinity for aryl moiety containing antagonists (e.g., alprenolol) and agonists (e.g., epinephrine) upon mutation of Val114 to alanine.
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A feature of many GPCRs is that they can weakly signal even in the absence of ligand, a property referred to as constitutive activity. This has been presumed to be due to spontaneous, albeit scant, isomerization of inactive receptor into the active conformation. Agonists stabilize active conformations of the receptor, thus promoting cellular signaling. Some years ago, it was discovered that the mutation of certain residues in several adrenergic receptors greatly augmented constitutive activity of the receptors (Cotecchia et al., 1990). Spontaneous occurrence of such mutations in a variety of receptors is now known to cause several human diseases (Spiegel, 1998). Mutations leading to enhanced constitutive activity have been thought to abrogate crucial intramolecular interactions between amino acid residues, which normally constrain the receptor to its inactive state, thus somehow mimicking the effects of agonists. Mutagenesis studies had earlier suggested that residues at the cytoplasmic surface of TM helices 3 and 6 form the "ionic lock," discussed above, which is crucially involved in maintaining the inactive state of the receptor. As noted, the crystal structures of the β2AR accord well with these prior studies. For instance, Leu272 in TM6, which was the first residue identified to lead to the constitutively active phenotype of the β2AR (Samama et al., 1993), exhibits interactions with residues toward the cytoplasmic ends of TM3 and TM5 and packing interactions with neighboring Glu268. Therefore, one can speculate that mutation of Leu272 may relieve the constraints at the intracellular side of the helices and promote disruption of the "ionic lock," thus resulting in an "active-like" state of the receptor. More importantly, several residues that lead to constitutively active mutants (e.g., Leu124 and Leu272) and others that lead to uncoupling (uncoupled mutant) (e.g., Asp79, Asn318, Asn322, and Tyr326) are linked through packing interactions (i.e., certain residues that pack against residues in TMs 3 and 6 responsible for constitutive activity) and are also involved in interactions with residues in TM 7, which lead to uncoupling from G proteins. Thus, rearrangement of the side chains of one of these residues can affect the packing and/or orientation of others. It is interesting that these residues are also in close proximity to Trp286, which constitutes the rotamer "toggle switch" in the receptor. Similar to rhodopsin, a cluster of water molecules in this region is also observed that can potentially promote an extended network of hydrogen bonding interactions. It is likely that the water-filled region will impose relatively low steric hindrance on the side chains of amino acids in this region, thus facilitating conformational transitions and repacking. This might be a general feature for the class A GPCRs that allows them to adopt multiple active conformations.
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What Conformation Is the Receptor in? |
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; Rasmussen et al., 2007; Rosenbaum et al., 2007). There are several possible explanations for this unexpected feature. As Rasmussen et al. report, carazolol reduces the basal activity of the receptor only by 50%, which makes it a partial inverse agonist. Therefore, the first possibility is that the broken ionic lock is due to the remaining basal activity of the receptor. In fact, the enhanced agonist affinity of the β2AR-T4 chimera that was observed (Rasmussen et al., 2007) fits with this possibility. The second more intriguing possibility is that the current structures represent an alternative "active" signaling conformation. In this context, it was reported recently that carvedilol, an inverse agonist of the β2AR very similar to carazolol (Fig. 2, A and B), has no detectable G protein signaling properties but is capable of inducing β-arrestin-mediated signaling (Wisler et al., 2007). This finding suggests that the current structures may represent a β-arrestin-coupled signaling state of the receptor. However, further structural and functional analysis will be required to support this interesting possibility. |
What Do the Structures Not Tell Us? |
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). Although the dimeric nature of rhodopsin has been well established both by biochemical and structural data, this issue remains controversial for the β2AR. It has been suggested that dimers or higher-order oligomers of the β2AR may regulate the export of the receptor to the plasma membrane (Salahpour et al., 2004), although receptor dimerization seems not to be essential for G protein activation (Whorton et al., 2007). Although the crystals of β2AR-Fab complex show no inter-receptor contacts, suggesting a receptor monomer, the β2AR-T4 chimera crystals do show lipid-mediated contacts between helixes 1 and 8. However, studies in native membranes have suggested helix 6 as the dimerization interface for the β2AR (Hebert et al., 1996). Again, the possibility that the protein engineering or the Fab interferes with receptor dimerization cannot be ruled out at this stage, and further structural analysis will be required to draw definite conclusions. Furthermore, as discussed above, most of the C terminus of the receptor was truncated to reduce flexibility and heterogeneity. The C terminus is known to be a site of post-translational modification and for interactions with proteins such as β-arrestins, and therefore, the structural organization of this region is likely to carry important information. To visualize this domain, a complex of the receptor with an interacting partner stabilizing this region will probably be necessary. In addition, because the electron density of the N terminus of the receptor is not apparent in the current structures, this region of the molecule is absent from the model, and therefore features of this region remain unknown. Another important piece of information missing from these structures is the structural organization of the third intracellular loop. This loop is crucial for determining the G protein-coupling specificity of the receptors and for forming the binding interface with G proteins and other signaling partners. However in the β2AR-T4 chimera, this loop is replaced by T4 lysozyme, whereas the β2AR-Fab complex contains the Fab bound to this region. |
Future Directions? |
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In this context, a paradigm shift in the field of GPCR research in the last few years has been the discovery that the receptors can signal via G protein-independent mechanisms (e.g., via β-arrestins) (DeWire et al., 2007). Moreover, "biased" ligands have been identified that can direct receptor signaling exclusively through either β-arrestins or G proteins. β-Arrrestin-biased ligands may ultimately represent a novel class of drugs which, like conventional blockers, shut off G protein signaling, but which also initiate the potentially beneficial effects of β-arrestin-mediated signaling. Moreover, mutant β2AR and angiotensin II receptors have been generated, which are totally uncoupled from cognate G proteins but which still lead to extracellular signal-regulated kinase activation via β-arrestins. Determination of the structures of such mutant receptors bound to biased or unbiased ligands should help to clarify the determinants on the receptor and on the ligands that are responsible for such conformational and functional selectivity. This information will be of great value in the design of ever-more selective therapeutic agents.
Another exciting and challenging goal will be the structure determination of GPCRs in complex with other signaling partners such as G proteins, β-arrestins, G protein-coupled receptor kinases, and others. Structural information derived from these signaling complexes will greatly aid the understanding of the interaction interfaces of the receptors and the conformational changes in both receptor and its interacting partners that follow these interactions. Moreover, despite the fact that the signaling mechanisms of GPCRs seem to be highly conserved, receptor-specific features are also likely to exist. Thus, structural information about other class A GPCRs and those of other classes will be necessary to obtain a full understanding of these mechanisms.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: GPCR, G protein-coupled receptor; β2AR, β2-adrenergic receptor; TM, transmembrane; 7TMR, seven transmembrane-spanning receptor.
Address correspondence to: Dr. Robert J. Lefkowitz, Box 3821, Duke University Medical Center, Durham, NC 27710. E-mail: lefko001{at}receptorbiol.duke.edu
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References |
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Abstract
Why Did It Take...
