Conformational changes involved in receptor activation

Activation of GPCRs for 'diffusible' ligands is initiated by binding of the agonist ligand to the receptor molecule. This binding event is thought to trigger a cascade of structural changes in the receptor molecule that are capable of inducing activation of the associated G protein and subsequent stimulation of a broad variety of intracellular signal transduction pathways. Ultimate understanding of this activation mechanism requires techniques that can provide insight into the character of the physical changes which occur upon agonist binding to the receptor and accompany the transition of the receptor from the inactive to the active state. One obvious goal is to obtaining high-resolution crystal structures of GPCRs in both inactive and active states. However, due to the still prevailing difficulties in obtaining sufficient material for X-ray crystallography the only available high-resolution structure of a full-length GPCR is the structure described above of rhodopsin in its inactive dark state (Palczewski etal. 2000).

It is interesting to note that a structure of the large extracellular domain, which characterizes Family 3 receptors and contains their ligand binding domain, has been solved for the metabotrobic glutamate receptor-1 (see Chapter 4.3). The structure was solved in both the agonist-bound state and in two ligand-free conformational states of which one resembled the agonist bound state and the other 'inactive state' represented a conformation markedly different from the agonist bound active state (Kunishima et al. 2000). Although the structures provided no information about the transmembrane domains, they did show the first direct structural glimpse of what might distinguish an active state from an inactive state in at least for a Family C GPCR. Remarkably, the extracellular domain was crystallized as a dimer with a large dimeric interface (Kunishima et al. 2000). Comparison of the inactive state with the active state revealed major changes at the dimeric interface and it was suggested that such changes are highly critical for the receptor activation process (Kunishima et al. 2000). It remains unknown, however, how the signal is transmitted from the large extracellular domains to the transmembrane region of the protein.

Studies in Family 1 receptors performed during recent years have provided insight into conformational changes in the transmembrane region that characterizes transition from an inactive state to an active state. In particular the application of biophysical techniques have proven highly useful and allowed direct analysis of conformational changes in the receptor molecule (Gether 2000; Hubbell etal. 2000). Importantly, these techniques also allow insight into the dynamics of the receptor activation process in contrast to crystal structures that despite a high degree of structural information still only represents single pictures at fixed conformations. The majority of the initial biophysical studies were carried out in rhodopsin. There are abundant natural sources of rhodopsin and the inherent stability of the rhodopsin molecule makes it possible to produce and purify relatively large quantities of recombinant protein. Several spectroscopic techniques have been applied to rhodopsin including Fourier Transform Infrared Resonance Spectroscopy (FTIR) (Rothschild et al. 1983; GarciaQuintana etal. 1995), Surface Plasmon Resonance (SPR) spectroscopy (Salamon etal. 1994), tryptophan UV-absorbance spectroscopy (Lin and Sakmar 1996) and Electron Paramagnetic Resonance Spectroscopy (EPR) (Farahbakhsh etal. 1995; Altenbach etal. 1996,1999a,b). All approaches have consistently provided evidence for a significant conformational rearrangement accompanying transition of rhodopsin to metarhodopsin II. Using tryptophan UV-absorbance spectroscopy, Lin and Sakmar (1996) were able to obtain the first direct evidence that photoactivation may involve relative movements of TM3 and 6. Thus, mutation of tryptophans in TM3 and 6 eliminated the spectral differences in the UV absorbance spectra that distinguished rhodopsin from metarhodopsin II (Lin and Sakmar 1996).

In a series of elegant studies, the use of EPR spectroscopy in combination with multiple cysteine substitutions has led to further insight into the character of conformational changes accompanying photoactivation ofrhodopsin (reviewed in Hubbell etal. 2000). Site-directed labelling of single cysteines inserted at the cytoplamic side of the transmembrane helices with sulfhydryl-specific nitroxide spin labels provided evidence for movements of particularly the cytoplasmic termination of TM6 upon light-induced activation of rhodopsin (Farahbakhsh et al. 1995; Altenbach et al. 1996, 1999a,k; Farrens et al. 1996; Langen et al. 1999). To investigate the character of the conformational changes, Khorana, Hubbell and coworkers have taken advantage of the magnetic dipole interaction between two nitroxide spin labels causing spectral line broadening if the two probes are less than 25 A apart (Farrens et al. 1996). Pairs of sulfhydryl-reactive spin labels were incorporated into a series of double cysteine mutants enabling measurement of changes in relative distance between TM3 and TM6 (Farrens etal. 1996). While the movement ofTM3 was interpreted as relatively small, the data pointed to a significant rigid-body movement of TM6 in a counterclockwise direction (as viewed from the extracellular side) and a movement of the cytoplasmic end of TM6 away from TM3 (Farrens et al. 1996). Importantly, these movements of TM6 in rhodopsin upon photoactivation have also been additionally documented by site-selective fluorescent labelling of cysteine inserted at the cytoplasmic termination of the helix (Dunham and Farrens 1999).

The first direct structural analysis of conformational changes in a GPCR activated by a dif-fusable ligand was performed in the p2 adrenergic receptor (Chapter 3.2) using fluorescence spectroscopic techniques (Gether etal. 1995, 1997a,b; Ghanouni etal. 2001b; Jensen etal. 2001). The spectroscopic technique that initially was applied utilized the sensitivity of many fluorescent molecules to the polarity of their local molecular environment (Gether et al. 1995). The sulfhydryl reactive fluorophore IANBD (N,N'-dimethyl-N (iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-diamine) was used to label free cysteine residues in purified detergent solubilized ß2 adrenergic receptor (Gether et al. 1995). Exposure of the IANBD-labelled receptor to agonist led to a reversible and dose-dependent decrease in emission consistent with movements of the fluorophore to a more hydrophilic environment following binding of the full agonist isoproterenol (Gether et al. 1995). Subsequent analysis of a series of mutant ß2 receptors with one, two, or three of the natural cysteines available for fluorescent labelling showed that IANBD bound to Cys1253.44 in TM3 and Cys2856.47 in TM6 (Fig. 3.2) were responsible for the observed changes in fluorescence

Fig. 3.2 'Snake-diagram' of the p2 adrenergic receptor. Cys1 253 44 and Cys285647 were identified as major labelling sites for the environmentally, cysteine-reactive fluorophore IANBD reporting agonist-induced conformational changes (Gether etal. 1997b). His269631, Lys270632, Ala271633, and Leu272634 were one by one mutated to cysteines in a background mutant with a reduced number of reactive cysteines (p2AR-Cys-min) (Jensen etal. 2001). Subsequent fluorescent labelling of these residues allowed identification of conformational changes at the cytoplasmic side of TM6 (Jensen etal. 2001). Asp130349, Arg131350, and Glu2686 30 are believed to form an ionic lock that is disrupted during receptor activation (Ballesteros et al. 2001a). The positions of highlighted residues are indicated by their generic number followed by their number according to the Ballesteros--Weinstein nomenclature (Ballesteros and Weinstein 1995). In this scheme the most conserved residue in each helix is given the number 50, and each residue is numbered according to its position relative this conserved residue. For example, 3.49 indicates a residue in TM3, one residue aminoterminal to Arg3.50, the most conserved residue in this helix.

Fig. 3.2 'Snake-diagram' of the p2 adrenergic receptor. Cys1 253 44 and Cys285647 were identified as major labelling sites for the environmentally, cysteine-reactive fluorophore IANBD reporting agonist-induced conformational changes (Gether etal. 1997b). His269631, Lys270632, Ala271633, and Leu272634 were one by one mutated to cysteines in a background mutant with a reduced number of reactive cysteines (p2AR-Cys-min) (Jensen etal. 2001). Subsequent fluorescent labelling of these residues allowed identification of conformational changes at the cytoplasmic side of TM6 (Jensen etal. 2001). Asp130349, Arg131350, and Glu2686 30 are believed to form an ionic lock that is disrupted during receptor activation (Ballesteros et al. 2001a). The positions of highlighted residues are indicated by their generic number followed by their number according to the Ballesteros--Weinstein nomenclature (Ballesteros and Weinstein 1995). In this scheme the most conserved residue in each helix is given the number 50, and each residue is numbered according to its position relative this conserved residue. For example, 3.49 indicates a residue in TM3, one residue aminoterminal to Arg3.50, the most conserved residue in this helix.

(Gether et al. 1997b) suggesting that movements of TM3 and 6 might occur during receptor activation (Gether et al. 1997b). It was, however, difficult to assess the precise character of the movements based on labelling of only two single sites with a rather flexible fluorescent reporter molecule. Therefore, a new set of experiments was carried out to achieve further insight into movements associated with receptor activation (Jensen et al. 2001). It was decided to focus on the cytoplasmic end of TM6 for two major reasons. First, the evidence for TM6 movements in response to light-activation of rhodopsin, was based on spectroscopic analysis of mutants which contained cysteine residues in this particular region ofTM6, labeled with either nitroxide spin labels or fluorescent probes (Altenbach etal. 1996; Farrens et al. 1996; Dunham and Farrens 1999). Labelling of the p2 adrenergic receptor in this region with a molecular reporter of conformational changes would thus allow a more direct comparison between rhodopsin and the p2 adrenergic receptor. Second, many mutagenesis-based studies have indicated the importance of the cytoplasmic region of TM6 in receptor activation and G protein coupling (Strader et al. 1994; Wess 1998); nonetheless, its precise role is still not fully understood and conformational changes at the cytoplasmic side of TM6 had not been described for receptors activated by diffusable ligands (Gether

2000). Accordingly, four residues in the predicted cytoplasmic region of TM6 of the p2 adrenergic receptor were substituted with cysteines in a mutant p2 adrenergic receptor containing a reduced number of endogenous cysteines (|32AR-Cys-min) (Jensen et al. 2001) (Fig. 3.2). Fluorescence spectroscopy analysis of the purified and site-selectively IANBD labelled mutants suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding (Jensen et al. 2001). Whereas evidence for only a minor change in the molecular environment was obtained for positions 2696.31 and 270632, the full agonist isoproterenol (ISO) caused clear dose-dependent and reversible increases in fluorescence emission at positions 271633 and 272634 (Fig. 3.3; Jensen et al. 2001). The magnitude of the responses correlated with the efficacy of the used agonist suggesting that the observed changes are relevant for receptor activation (Jensen et al.

2001). In contrast to Cys2856.47, which is situated in a highly hydrophobic environment in the middle of the membrane, the cysteines inserted at the cytoplasmic side of TM6 reside in a very complex hydrophobic/hydrophilic environment (Figs 3.2 and 3.3). To take this into consideration when attempting to interpret the spectroscopic data a new computational method was developed. Notably, the available computational simulation methods had not incorporated the complexity of the mixed hydrophobic-hydrophilic region and only recently, abi-phasiclipid-water solvent continuum model was developed (Ballesteros etal. 1998). The results of the simulations, illustrated by the most preferred conformations for each of the four IANBD-derivatized cysteine residues, are shown in context of a molecular model in Fig. 3.3D (viewed from the extracellular side).

A simple rotation of TM6 was sufficient to explain the data based on IANBD labelling of Cys2856.47 (Gether et al. 1997b). However, the observation that IANBD at all four inserted cysteines residues most likely moves into a more hydrophobic environment upon agonist binding suggests that the helical movement is more complex and likely involves a rigid-body movement of the cytoplasmic part of TM6 away from TM3 similar to what was found for rhodopsin (Farrens et al. 1996). As seen from the side (Fig. 3.3G), TM6 is predicted to form a kinked a-helix due to the presence of a highly conserved proline (Pro2876.50) (Ballesteros and Weinstein 1995). The rhodopsin structure (Palczewski et al. 2000), which are believed to represent the inactive state of the receptor, indicates that the cytoplasmic part of TM6 below the proline kink is almost perpendicular to the plane of the membrane, whereas the

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Fig. 3.3 Characterization of agonist-induced conformational changes at the cytoplasmic side of TM6 in the p2 adrenergic receptor. His269631, Lys270632, Ala271633, and Leu272634 at the bottom of TM6 (Fig. 3.2) were one by one mutated to cysteines in a background mutant with a reduced number of reactive cysteines (p2AR-Cys-min) (Jensen et al. 2001). The mutants were expressed in Sf-9 insect cells, purified and labeled with the environmentally, sulfhydryl reactive fluorophore, IANBD. Panels a, b, c, e, and f show time course experiments where fluorescence intensity is measured over time in response to indicated concentrations of (-)isoproterenol (ISO)

part above the proline kink is tilted approximately 25° (Unger et al. 1997). A rigid body movement of the cytoplasmic part of TM6 away from TM3, and thus the receptor core, will result in large changes in the axial positioning of all four IANBD-labelled substituted cysteines. In the inactive conformation the IANBD moieties would be predicted to reside in the polar headgroup region (Fig. 3.3G). However, if the cytoplasmic part of TM6 is moved away from the receptor core all four IANBD labelled residues are brought upwards and outwards allowing them to penetrate further into the more hydrophobic region of the membrane/detergent micelles and away from the more hydrophilic polar headgroups as well as from the predicted more hydrophilic interior of the receptor protein (illustrated by the hypothetical active structure in Fig. 3.3G, right panel, where the cytoplasmic part of TM6 with the IANBD moieties attached is tilted arbitrarily away from the receptor core).

The importance of TM6 movements relative to TM3 is also supported by the possibility of inhibiting activation of rhodopsin by generation of bis-His metal ion binding sites between

Fig. 3.3 (Continued) followed by 10-4M (-)alprenolol (ALP). Excitation was 481 nm and emission was measured at 530 nm. Fluorescence in the individual traces was normalized to the fluorescence observed just before addition of ligand (Jensen et al. 2001). In p2AR-Cys-min, ISO causes a decrease in fluorescence that can be reversed by ALP. This decrease is reported by IANBD bound to Cys1 253 44 and Cys285647 (Gether etal. 1997b). In Cys271 and Cys272, ISO causes ALP-reversible increases in fluorescence intensity consistent with movement of the fluorophore to a more hydrophobic environment. In Cys269 and Cys270, ISO causes no apparent change in fluorescence intensity. A likely interpretation is that ISO induces an increase that counterbalances the decrease observed in the control (p2AR-Cys-min). Panel d shows an extracellular view of a receptor model with an illustrative set of the preferred conformations of the IANBD side chain covalently attached to the four substituted cysteines (color-coded). Note that IANBD attached to Cys271633 (yellow) and Cys272634 (purple) are facing the interior of the TM helix bundle, while IANBD attached to Cys269631 (blue) and Cys270632 (red) are oriented towards the lipid membrane. The preferred conformations of IANBD were determined from computational simulations as described (Jensen et al. 2001). Panel G shows the proposed conformations of the inactive and active states of the p2AR. The inactive conformation of the receptor (left panel) is characterized by a highly kinked TM6 helix (blue) with the cytoplasmic end in close proximity to TM3 and the helix bundle. An illustrative set of the preferred conformations of the IANBD moiety covalently attached to the four substituted cysteines at the cytoplasmic side of TM6 is shown. The hypothetical active conformation (right panel) of the receptor in which the cytoplasmic side of TM6 is moved away arbitrarily from the helix bundle and upwards towards the hydrophobic region, marked by straight lines. This putative rearrangement of TM6 moves all four IANBD labelled residues upwards and outwards allowing them to penetrate further into the more hydrophobic region of the membrane/detergent micelles and away from the more hydrophilic polar headgroups as well as from the predicted more hydrophilic interior of the receptor protein. The movement can explain the observed shift for all four IANBD labelled cysteines towards a less polar environment upon receptor activation (Jensen et al. 2001). Note that the movement of the cytoplasmic part of TM6 is shown to occur around the conserved proline kink but could as well involve a rigid body movement of the entire helix. However, our previous simulation of the TM6 helix indicated the possibility that the kink in the TM6 helix induced by Pro287650could behave as a flexible hinge, which can modulate the movement of the cytoplasmic side of TM6 helix relative to the extracellular region (Gether et al. 1997b). (Figure modified from Jensen etal., 2001, J Biol Chem 276, 9279-90). (See Plate 3.)

the cytoplasmic ends of these TM domains (Sheikh et al. 1996). The recent application of a disulfide cross-linking strategy to the M3 muscarinic receptor, another Family 1 member, has moreover suggested that significant movements occur in the G protein coupling domain at the cytoplasmic side of TM6, although the movements predicted by the authors are somewhat distinct from those predicted in the biophysical studies (Ward et al. 2001). Altogether, the striking agreement between the data obtained in rhodopsin and in the p2 adrenergic receptor strongly indicates that the activation mechanism in many aspects is similar among at least Family 1 GPCRs. It is important to note that the established importance of TM6 does not exclude that movements of other domains may contribute to receptor activation. New evidence from EPR spectroscopy studies in rhodopsin suggest that movements of the cytoplasmic portion of TM7 relative to TM1 and of TM2 relative to the so-called intracellular helix 8 (the horizontal extension of TM7) may also occur in response to photoactivation (Altenbach et al. 2001a,b). The possible importance of TM7 in receptor activation is indirectly supported by the finding that activating metal-ion binding sites can be generated between TM3 and 7 in both the p2 adrenergic receptor and in the neurokinin-1 receptor (Elling et al. 1999; Holst et al. 2000).

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