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J. Biol. Chem., Vol. 279, Issue 46, 48024-48037, November 12, 2004
Structural Mimicry in Class A G Protein-coupled Receptor Rotamer Toggle SwitchesTHE IMPORTANCE OF THE F3.36(201)/W6.48(357) INTERACTION IN CANNABINOID CB1 RECEPTOR ACTIVATION*![]() || || || || ¶
From the
Received for publication, June 15, 2004 , and in revised form, August 19, 2004.
In this study, we tested the hypothesis that a CB1 TMH3-4-5-6 aromatic microdomain, which includes F3.25(190), F3.36(201), W5.43(280), and W6.48(357), is centrally involved in CB1 receptor activation, with the F3.36(201)/W6.48(357) interaction key to the maintenance of the CB1-inactive state. We have shown previously that when F3.36(201), W5.43(280), and W6.48(357) are individually mutated to alanine, a significant reduction in ligand binding affinity is observed in the presence of WIN 55,212-2 and SR141716A but not CP55,940 and anandamide. In the work presented here, we report a detailed functional analysis of the F3.36(201)A, F3.25(190)A, W5.43(280)A, and W6.48(357)A mutant receptors in stable cell lines created in HEK cells for agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTP S) binding and GIRK1/4 channel current effects in Xenopus oocytes where the mutant proteins were expressed transiently. The F3.36(201)A mutation showed statistically significant increases in ligand-independent stimulation of GTP S binding versus wild type CB1, although basal levels for the W6.48(357)A mutant were not statistically different from wild type CB1. F3.36(201)A demonstrated a limited activation profile in the presence of multiple agonists. In contrast, enhanced agonist activation was produced by W6.48(357)A. These results suggest that a F3.36(201)/W6.48(357)-specific contact is an important constraint for the CB1-inactive state that may need to break during activation. Modeling studies suggest that the F3.36(201)/W6.48(357) contact can exist in the inactive state of CB1 and be broken in the activated state via a 1 rotamer switch (F3.36(201) trans, W6.48(357) g+) (F3.36(201) g+, W6.48(357) trans). The F3.36(201)/W6.48(357) interaction therefore may represent a "toggle switch" for activation of CB1.
The cannabinoid CB1 receptor belongs to the class A rhodopsin-like family of G protein-coupled receptors (GPCRs)1 (see helix net, Fig. 1) (2). The availability of high resolution crystal structures of rhodopsin (Rho) (3, 4) (Protein Data Bank accession code 1GZM [PDB] ) and the availability of biophysical data on the conformational changes that occur when rhodopsin and other class A receptors are activated (68) have greatly aided the study of structure-function relationships in class A GPCRs. Ballesteros et al. (9) have proposed that "structural mimicry" may occur in GPCRs such that different amino acids or alternate microdomains in class A receptors (e.g. the amine receptors) can support similar deviations from the regular -helical structure seen in Rho, thereby resulting in similar tertiary structures. In the work presented here, we provide evidence that "structural (functional) mimicry" by alternate microdomains may also support the core function of signaling activation through transmembrane helix conformational change in the class A family.
There is a growing body of evidence in the literature that activation of GPCRs is accompanied by rigid domain motions and rotations of transmembrane helices (TMHs) 3 and 6 (68). At their intracellular ends, TMHs 3 and 6 in Rho are constrained by an E3.49(134)/R3.50(135)/E6.30 (247) salt bridge that limits the relative mobility of the cytoplasmic ends of TMH3 and TMH6 in the inactive state (3) and acts like an "ionic lock" (10, 11). During activation, P6.50 of the highly conserved CWXP motif in TMH6 of GPCRs may act as a flexible hinge, permitting TMH6 to straighten upon activation, moving its intracellular end away from TMH3 and upwards toward the lipid bilayer (12).
Khorana and co-workers (13) have reported that even in the dark (inactive) state of Rho, only some strong constraints exist, whereas the majority of the molecule experiences conformational flexibility. Therefore, light activation of Rho does not require the breaking and forming of thousands of specific contacts within nanoseconds, rather only a few specific contacts restricting the inactive state, including indole side chain contacts of tryptophan residues, need to break on activation. These changes can then be transmitted through the entire membrane protein because of its dynamic plasticity. One of the tryptophan residues that Khorana and co-workers (13) have reported to be restricted is W6.48(265). In the dark (inactive) state of Rho, the
In the class A cationic neurotransmitter receptors, a highly conserved cluster of aromatic amino acids is found on TMH6 that faces the binding site crevice bracketing W6.48 (F6.44, W6.48, F6.51, and F6.52) (16). Shi et al. (16) have proposed that an aromatic at 6.52 (F6.52) in the
Restriction of W6.48 by a TMH6 aromatic cluster is not possible in the cannabinoid receptors, as the CB1 receptor has leucines at 6.44, 6.51, and 6.52. Instead, the CB1 receptor contains a microdomain of aromatic residues that face into the ligand-binding pocket in the TMH3-4-5-6 region, including F3.25(190), F3.36(201), W4.64(256), Y5.39(276), W5.43(280), and W6.48(357) (Fig. 2). In work reported here, we suggest that the F3.36(201)/W6.48(357) interaction may act as a mimic of the 11-cis-retinal/W6.48 interaction in the Rho dark state and may serve as the "toggle switch" for CB1 activation, with F3.36(201)
Molecular Modeling Amino Acid Numbering SystemIn the discussion of receptor residues below, the amino acid numbering scheme proposed by Ballesteros and Weinstein (17) is used. In this numbering system, the most highly conserved residue in each transmembrane helix (TMH) is assigned a locant of 0.50. This number is preceded by the TMH number and followed in parentheses by the sequence number. All other residues in a TMH are numbered relative to this residue. In this numbering system, for example, the most highly conserved residue in TMH2 of the mouse CB1 receptor is D2.50(164). The residue that immediately precedes it is A2.49(163). Fig. 1 serves as a reference for this numbering system in mouse CB1.
Definition of Rotameric State of Conformational Memories Studies of TMH6 and the W6.48A Mutant TMH6 In order to explore the consequences of the W6.48A mutation upon the conformation of TMH6, we used the conformational memories (CM) method (18), a method that employs multiple Monte Carlo/simulated annealing random walks and the Amber* force field. Conformational memories has been shown to converge in a very practical number of steps and to be capable of overcoming energy barriers efficiently. By using CM, the conformational properties of a helix can be fully characterized by the free energy of each of the conformations that the helix can adopt, and this property includes not only the intrinsic energy of each conformational state but also the probability that the helix will adopt each particular conformation relative to all other ones accessible in an equilibrated thermodynamic ensemble. The calculation is performed in two phases. In the first phase, repeated runs of Monte Carlo/simulated annealing are carried out to map the entire conformational space of the helix. In the second phase, new Monte Carlo/simulated annealing runs are performed only in the populated regions identified in the first phase of the calculation.
WT TMH6 Versus TMH6 W6.48A MutantThe CB1 TMH6 (from residue 6.30 to residue 6.57, DIRLAKTLVLILVVLIICWGPLLAIMVY) and the TMH6 W6.48A mutant (DIRLAKTLVLILVVLIICAGPLLAIMVY) were built using MacroModel (19). In the Rho 2.8-Å crystal structure (3),
All calculations were performed using a distance-dependent dielectric. Each TMH6 was first minimized using the Amber* forcefield in MacroModel (19). For WT CB1 TMH6 73 torsion angles were allowed to vary during the CM runs, whereas 71 torsion angles were allowed to vary in the CB1 TMH6 W6.48(357)A mutant run. These included all helix backbone Exploratory PhaseIn the exploratory phase, a random walk was used to identify the region of conformational space that is populated for each torsion angle studied. Starting at a temperature of 2070 K, 20,000 steps were applied to the rotatable bonds with cooling in 18 steps to a final temperature of 310 K. Trial conformations were generated at each temperature by randomly picking three torsion angles from the set of 73 (71 in the W6.48A mutant) and changing each angle by a random value within the range set in the calculation (see above). After each step, the generated trial conformation was either accepted or rejected using the Metropolis criterion. This calculation was repeated for a total of 100 cycles. Accepted conformations were used to map the conformational space of TMH6 by creating "memories" of values for each torsion angle that were accepted. Biased Annealing PhaseIn the second phase of the CM calculation, the only torsion angle moves attempted were those that would keep the angle in the "populated conformational space" mapped in the exploratory phase. The biased annealing phase began at a temperature of 722 K cooling to 310 K in 8 steps. 100 structures were written out at 310 K. Analysis of OutputFinally, the output of 100 structures at 310 K was clustered using X-Cluster in MacroModel (19). This program reorders the structures according to their root mean square deviation and groups the structures into families of similar conformers. The resulting 100 structures from CM were also analyzed using the program, ProKink (20). This program, which is embedded in the Simulaid Conversion program,2 was used to calculate the face shift, wobble, and bend angles of each helix. Statistically significant differences between the face shift, wobble, and bend angles of the R and R* CB1 WT versus W6.48A R and R* helix families were evaluated in the two-sample independent t test computed using OriginPro version 7 (Origin Lab Corp.).
Models of CB1 R and R* StatesIn the present study, the literature on GPCR activation discussed above was used to generate an R* CB1 TMH bundle from a model of the inactive (R) CB1 receptor based on the 2.8-Å crystal structure of rhodopsin (3). The creation of these two forms of CB1 is described briefly below. Model of Inactive State (R) Form of CB1A model of the R form of CB1 was created using the 2.8-Å crystal structure of bovine Rho (3). First, the sequence of the mouse CB1 receptor (22) (see Fig. 1) was aligned with the sequence of bovine Rho using the same highly conserved residues as alignment guides that were used initially to generate our first model of CB1 (23). TMH5 in CB1 lacks the highly conserved proline in TMH5 of Rho. Therefore, the sequence of CB1 in the TMH5 region was aligned with that of Rho as described previously using its hydrophobicity profile (23). The mouse CB1 sequence (22) is 97.7% identical to the human CB1 sequence (2) overall and 100% identical within the transmembrane regions. The mouse sequence is one residue longer (473 residues) than the human sequence (472 residues) due to an additional residue in the N terminus.
Initial helix ends for mouse CB1 were chosen in analogy with those of Rho (3). With the exception of TMH1, these helix ends were found to be within one turn of the helix ends originally calculated by us and reported in 1995 (23). Two changes dictated by the CB1 sequence were made in the helix ends. The shortness of the E1 loop region in CB1 necessitated starting TMH3 at 3.23 (N3.23(188) to R3.56(221)). The break in helicity caused by the GWNC sequence motif on the extracellular end of TMH4 necessitated that TMH4 end at 4.62 instead of 4.66 (as is found in Rho). Changes to the general Rho structure that were necessitated by sequence divergences included the absence of helixkinking proline residues in TMH1 and TMH5, the lack of a GG motif in TMH2, as well as the presence of extra flexibility in TMH6 (24). Our recent conformational memories study of TMH6 in CB1 revealed that TMH6 in CB1 has high flexibility due to the small size of residue 6.49 (a glycine) immediately preceding Pro 6.50. The conformer selected from our CM results for inclusion in the CB1 R bundle (Pro kink angle = 53.1°) was chosen so that R3.50(215) and D6.30(339) could form a salt bridge at the intracellular ends of TMH3 and -6 in the CB1 TMH bundle. An analogous salt bridge has been shown to be an important stabilizer of the inactive state of the
Model of Active (R*) Form of CB1Based upon experimental results for rhodopsin and the Preparation of HelicesEach helix of the model was capped as the acetamide at its N terminus and as the N-methyl amide at its C terminus. Ionizable residues in the first turn of either end of the helix were neutralized, as were any lipid facing charged residues. Ionizable residues were considered charged if they appeared anywhere else in the helix. Energy Minimization, Unoccupied Receptor StatesThe energy of the CB1 R or CB1 R* TMH bundle complex was minimized using the AMBER* united atom force field in Macromodel 6.5 (Schrödinger Inc., Portland, OR). A distance-dependent dielectric, 8.0-Å extended non-bonded cut-off (updated every 10 steps), 20.0-Å electrostatic cut-off, 4.0-Å hydrogen bond cut-off, and explicit hydrogens on sp2 carbons were used. The first stage of the calculation consisted of 2000 steps of Polak-Ribier conjugate gradient (CG) minimization in which a force constant of 225 kJ/mol was used on the helix backbone atoms in order to hold the TMH backbones fixed, while permitting the side chains to relax. The second stage of the calculation consisted of 100 steps of CG in which the force constant on the helix backbone atoms was reduced to 50 kJ/mol in order to allow the helix backbones to adjust. Stages one and two were repeated with the number of CG steps in stage two incremented from 100 to 500 steps until a gradient of 0.001 kJ/(mol·Å2) was reached. This same protocol was followed for the W6.48(357)A TMH bundle.
Assessment of Aromatic Stacking InteractionsResidues were designated here as participating in an aromatic stacking interaction if subject rings had centroid to centroid distances (d) between 4.5 and 7.0Å. These interactions were further classified as "tilted t" arrangements if 30°
Assessment of Cation- Ligand Conformations and Docking PositionsThe binding site conformations and anchoring interactions inside the receptor used for each ligand discussed here are based on our recently published work (1).
Mutation Studies Mutagenesis, Cell Culture, and Radioligand BindingThe cell lines used in this study have been described previously (1). Site-directed mutations were introduced into mouse CB1 in pcDNA3 at the designated sites using the QuikChange mutagenesis technique (Stratagene, La Jolla, CA). Stable cell lines were created by transfection of wild type or mutant CB1pcDNA3 into HEK 293 cells by the LipofectAMINE reagent (Invitrogen) and cultured as described previously (30). Cell lines containing moderate to high levels of receptor mRNA, assessed by Northern analysis, were tested for receptor binding and signal transduction properties. Receptor binding was determined as described previously (1). Cell lines with the most similar receptor expression profile, as ascertained by Bmax values, were chosen for further analysis (Table I).
[35S]GTP S Binding AssayThe basal activity and the ability of various cannabinoids to stimulate the wild type CB1 receptor or the mutant receptors were tested with [35S]GTP S binding. Cells were harvested in phosphate-buffered saline containing 1 mM EDTA and centrifuged at 500 x g for 5 min. The cell pellet was homogenized and centrifuged at 50,000 x g for 10 min at 4 °C. The pellet was resuspended in buffer composed of (mM): Tris-HCl 50, EDTA 1, and MgCl2 3, pH 7.4, to yield protein concentration of 24 mg/ml. Membrane preparations were aliquoted and stored at -80 °C. Binding was initiated by the addition of 20 µg of membrane protein into glass tubes containing 0.1 nM [35S]GTP S, 10 µM GDP in GTP S binding buffer (50 mM Tris-HCl, 100 mM NaCl, 3 mM MgCl2, 0.2 mM EGTA, 0.1% bovine serum albumin, pH 7.4). Nonspecific binding was assessed in the presence of 20 µM unlabeled GTP S. Binding assays were performed for 90 min at 30 °C with various concentrations of WIN 55212-2, CP55,940, anandamide, and SR141716A in a total volume of 500 µl. Free and bound radioligands were separated by a rapid filtration through Whatman GF/C filters. Filters were shaken for 1 h in 6 ml of scintillation fluid (Fisher), and radioactivity was determined by a liquid scintillation counter.
Expression in Oocytes and RecordingsThis technique was performed as described previously (31). Briefly, 0.013 pg of GIRK1 and GIRK4 and 25 ng of CB1 (wild type or mutant) cRNAs were co-injected using a micromanipulator (Drummond Scientific Co., Broomall, PA) into Xenopus laevis oocytes (Xenopus One, Dexter, MI). Recordings were performed after 79 days of incubation in 0.5x L-15 media (Sigma) supplemented with L-glutamine and antibiotics. For recordings, the eggs were placed in a chamber (total volume 200 ml) and perfused at 4 ml/min with LK (2 mM KCl, 96 mM NaCl, 2 mM CaCl2, 1.8 mM MgCl2, and 5 mM HEPES, pH 7.5), HK (96 mM KCl, 2 mM NaCl, 2 mM CaCl2, 1.8 mM MgCl2, and 5 mM HEPES, pH 7.5), or HK plus drug. Bovine serum albumin (3 µM) was added to all drug solutions to minimize absorption of cannabinoid compounds to the perfusion system. Oocytes were impaled with two microelectrodes filled with 3 M KCl and were voltage-clamped at reported voltages using an Axon GeneClamp amplifier (Axon Instruments, Foster City, CA). Currents were filtered at 10 Hz, collected, and analyzed using a Macintosh Centris 650 containing a 16-bit analog-digital interface board and voltage-clamp software running under the IGOR graphics environment (Wavemetrics, Lake Oswego, OR). Oocytes were voltage clamped at -80 mV and superfused in a low potassium (LK) solution containing 96 mM Na+ and 2 mM K+. When a high potassium (HK) solution containing 96 mM K+ and 2 mM Na+ was exchanged for LK, an inward current was produced and termed IHK. IHK represents the basal activity of GIRK1/4 channels. This current reached a plateau with Statistical AnalysesThe EC50 and Emax values were calculated by unweighted least squares nonlinear regression of log concentration values versus percent effect. Significant differences were determined (GraphPad Prism) using analysis of variance or the unpaired Student's t test, where suitable. Bonferroni-Dunn post-hoc analyses were conducted when appropriate. p values <0.05 defined statistical significance.
The TMH3-4-5-6 Aromatic Microdomain at the CB1 Receptor; Comparison of R and R* State Models in the Absence of Ligand CB1 R StateFig. 2A illustrates key features of the CB1 TMH bundle model in the inactive (R) state in the TMH3-4-5-6 region. One of the significant features of this model is a salt bridge between K3.28(193) and D6.58(367) (N-O distance = 2.6 Å; N-H-O angle = 159°) (51). This salt bridge is made possible by the profound flexibility in TMH6 due to the presence of G6.49 in the CWXP motif of TMH6 (25). The TMH3-4-5-6 region of the R bundle in the absence of ligand is characterized by a W6.48(357)/F3.36(201)/W5.43(280)/Y5.39(276)/W4.64(256) aromatic cluster in which W6.48(357) stacks with F3.36(201) (d = 5.3 Å, = 90°), whereas F3.36(201) stacks with W5.43(280) (d = 5.6 Å, = 40°). W5.43(280) also has an off-set parallel stack with Y5.39(276) (d = 5.9 Å, = 0°), whereas Y5.39(276) stacks with W4.64(256) (d = 6.5 Å, = 90°) (see "Experimental Procedures" for definitions of d and ).
Residue 3.25 (190) does not stack with the other aromatic residues in the TMH3-4-5-6 region but still appears to be an important residue. In the R bundle, F3.25(190) is located directly extracellular to K3.28 and is close enough to K3.28 to be able to form a cation-
CB1 R* StateFig. 2B illustrates key features of the CB1 TMH bundle model in the active (R*) state in the TMH3-4-5-6 region. The conformational changes that occur upon receptor activation result in rotations of TMH3 and -6 as well as a change in the conformation of TMH6 (by moderation of its proline kink angle) (68, 10). In our models, both W6.48(357) and F3.36(201) undergo a change in their
As stated above, in the R* state, the rotation of TMH3 upon activation causes K3.28(193) to point toward TMH2/TMH7, and because F3.25(190) is one turn above K3.28(193), it also now faces the TMH2/TMH7 region. The
Ligand Binding
Functional Analysis of Mutant Receptors
F3.36A(201)The F3.36A(201) mutation produced a significant reduction in agonist-dependent activation produced by WIN 55,212-2 (Fig. 3, A and B, and Tables II and III). This could be observed at the level of direct receptor-G protein stimulation (GTP
We next examined whether observed reductions in function were the result of a constitutively activated receptor. The CB1 receptor has been shown to have a high level of agonist-independent activation (i.e. constitutive activity) in transfected cell lines where the reporter system is a G protein-activated ion channel or an intracellular kinase (33, 34). The ability of CB1 to activate G proteins in the absence of exogenously applied agonist has also been shown for native CB1 receptors in human and rat brain (35, 36). Kearn and co-workers (37) have estimated that in WT CB1, the receptor populations are 70% inactive state (R) and 30% active state (R*).
Agonist-independent activation of GPCRs can be assessed by measuring basal turnover of [35S]GTP
Modeling studies also suggest that the F3.36(201)A mutant should be constitutively active, if it is assumed that changes in the TMH3-4-5-6 aromatic cluster influence the state preference for the receptor. The loss of aromaticity at 3.36 (i.e. in the F3.36(201)A mutation) would result in the inability of W6.48(357) to participate in the extended aromatic cluster to which it belongs in WT CB1. Specifically, this mutation would reduce the aromatic cluster present in the R state of WT CB1 from W6.48(357)/F3.36(201)W5.43(280)/Y5.39(276)/W4.64(256) to W5.43(280)/Y5.39(276)/W4.64(256) (Fig. 2A). However, the F3.36(201)A mutation would not have an effect on the R* state, as F3.36(201) is not part of the aromatic cluster in the R* state of CB1 (Fig. 2B). Thus, the W6.48(357)/W5.43(280)/Y5.39(276)/W4.64(256) cluster present in the R* state would be unchanged by the F3.36(201)A mutation. The net result of the F3.36(201)A mutation, therefore, should be a de-stabilization of the inactive state of CB1, leading to increased constitutive activity.
W5.43(280)AThe EC50 values reported in Tables II and III show that the potency of WIN 55,212-2 at the W5.43(280)A mutant was reduced 245- and 16.8-fold when this receptor was studied by using GTP
The full effect of WIN 55,212-2, however, at the W5.43(280)A mutant receptor could be achieved at higher concentrations. The substantial reductions in potency, in two cell systems, may be due in part to the decreased affinity of WIN 55,212-2 at W5.43(280)A. The affinity of WIN 55,212-2 at W5.43(280)A was reduced 16.8-fold in HEK cells, whereas the affinity of CP55,940 and anandamide was unaffected (Table I) (1). Because the affinities of CP55,940 and anandamide were unaffected at this mutation, the functional responses produced by these ligands were tested at W5.43(280)A to determine whether the effects observed in the presence of WIN 55,212-2 were primarily a result of a reduction in affinity at the mutant receptor. As shown in Fig. 4, C and D, there was a substantial reduction in the potency of both anandamide and CP55,940 at W5.43(280)A. It is clear in Table I, however, that the Bmax of the W5.43(280)A mutant was statistically lower than that of WT CB1. Lower receptor expression levels can produce a rightward shift in plots of response versus log[ligand] compared with a system in which a greater amount of receptor protein is expressed, despite the fact that the ligand has equal affinity in both systems. Thus, the reduced expression level of the W5.43(280)A mutant compared with WT CB1 may explain the rightward shifts in Fig. 4, C and D, seen for anandamide and CP55940 (40). There were no significant changes in agonist-independent activation of W5.43(280)A in either cell system tested (Figs. 7 and 8 and Table IV).
W6.48A(357)The EC50 values reported in Tables II and III show that the potency of WIN 55,212-2 at the W6.48(357)A mutant was reduced 12- and 1.8-fold when this receptor was studied using GTP
Most interestingly, the maximum stimulation of GTP S activity produced by WIN 55,212-2 at W6.48(357)A was significantly enhanced compared with WT CB1 (Fig. 5A and Table II). To determine whether this effect was specific for aminoalkylindoles, the bicyclic cannabinoid CP55,940 was also tested at W6.48(357)A. As observed with WIN 55,212-2, CP55,940 also produced enhanced stimulation of GTP S activity at W6.48(357)A compared with WT CB1 (Fig. 5D). The enhanced activity of WIN 55,212-2 at W6.48(357)A was not observed when GIRK channel activity was measured (Fig. 5B). There were no significant changes in agonist-independent activation of W6.48(357)A in either cell system tested (Figs. 7 and 8 and Table IV). Consistent with unchanged constitutive activity, the response to the inverse agonist at W6.48(357)A was not different from the response of WT CB1 to the inverse agonist (Fig. 5E). Modeling studies also suggest that the W6.48(357)A mutant should not be constitutively active, if it is assumed that changes in the TMH3-4-5-6 aromatic cluster influence the state preference for the receptor. The W6.48A mutation will affect the TMH3-4-5-6 aromatic cluster in both the R and R* states. In the R state, the W6.48A mutation will result in the loss of one aromatic stacking interaction, i.e. the W6.48(357)/F3.36(201)/W5.43(280)/Y5.39(276)/W4.64(256) cluster in R will become a F3.36(201)/W5.43(280)/Y5.39(276)/W4.64(256) cluster in the mutant. In the R* state, the W6.48(357)/W5.43(280)/Y5.39(276)/W4.64(256) cluster present in the R* state will become a W5.43(280)/Y5.39(276)/W4.64(256) cluster in the mutant. Because aromatic stacking in both the R and R* states will be equally impacted, it is reasonable to expect that the W6.48A mutant will not produce a change in basal levels for the W6.48A mutant relative to WT CB1, and this is what was seen experimentally. F3.25(190)AWe previously reported that F3.25(190) is not part of the binding site of WIN 55,212-2, SR141716A, or CP55,940 but is a part of the anandamide-binding pocket (1). When F3.25(190) was mutated to an alanine, a 6-fold reduction in binding affinity was observed in the presence of anandamide, but no changes were observed in the presence of WIN 55,212-2, SR141716A, and CP55,940 (Table I). When GIRK channel activity was measured there was a significant reduction in both the potency and efficacy of anandamide at F3.25(190)A but no change in the presence of WIN 55,212-2 (Fig. 6, A and B, and Table III).
This result correlates with the binding data and receptor modeling. The effect seen with anandamide is likely to be related in part to the decreased affinity of anandamide for the F3.25(190)A mutant, as F3.25(190) is part of the anandamide-binding site (1). Anandamide stimulation of GTP S turnover, in HEK cells transfected with F3.25(190)A, could not be measured because the partial agonist nature of the endogenous ligand results in a nonsignificant amount of GTP S stimulation (41). Therefore, the findings with anandamide and the GIRK channel activity could not be compared with GTP S stimulation because anandamide did not produce a significant functional response in HEK cells. There was a 6.2-fold decrease in the potency of WIN 55,212-2 when receptor activation was assessed by measuring GTP S activity.
These agonists do not exhibit affinity changes from WT in the F3.25(190)A mutant. However, the effects observed on potency and/or efficacy reported here for WIN 55212-2 at the F3.25(190)A mutant may be related to the general role F3.25(190) plays in positioning K3.28 in the TMH bundle. As is clear from Fig. 2, F3.25(190) is just above K3.28 in the inactive state of CB1 and has a cation- There were no significant changes in agonist-independent activation of F3.25(190)A in either cell system tested (Fig. 7 and Fig. 8 and Table IV). F3.25(190) is not part of the TMH3-4-5-6 aromatic cluster that characterizes the R and R* states of CB1 (see Fig. 2). Therefore, the mutation of this aromatic residue to a nonaromatic residue would not be expected to affect basal levels.
Toggle Switch Residues
Conformational Memories Study of WT TMH6 Versus W6.48A TMH6 TMH6 in the class A GPCRs contains the highly conserved sequence motif, CWXP. This proline containing region of TMH6 is thought to act as a flexible hinge during GPCR activation (44). Proline residues are known to perturb the structure of helices by introducing a kink between the segments preceding (pre-proline helix) and following the proline residue (post-proline helix). The distortion of the helical structure results from the avoided steric clash between the ring of the proline at position (i) and the backbone carbonyl at position (i - 4), as well as the elimination of helix backbone hydrogen bonds for the carbonyls at positions (i - 3) and (i - 4). Both the departure from the ideal helical pattern and the reduction in H-bond stabilization contribute to the observed flexibility of a proline-containing -helix. Table V lists the results of ProKink analyses of WT TMH6 CM output and the W6.48A TMH6 CM output. This analysis yields values not only for the bend angle of this proline-containing helix but also the wobble angles and face shifts for these helices. The bend angle is the angle between the two parts when the helix is kinked along its axis. The wobble angle is the angle that defines the orientation of the post-proline helix in three-dimensional space, with respect to the pre-proline helix. The face shift measures the distortion that causes a twisting of the helix "face" in such a way that amino acids that used to be on the same side (face) of the helix are shifted and are on different sides of the helix as a result of the bend (20).
The results of conformational memories calculations on WT CB1 and the W6.48A mutant are summarized in Table V. In each case, X-cluster identified two major clusters of conformers. The first cluster contained TMH6s with large bend angles, whereas the second cluster contained TMH6s with straighter helices (i.e. smaller bend angles). Because TMH6 is more kinked in the inactive state (R) and straightens in the activated state (R*), these two clusters are labeled R and R* in Table V (12). The ProKink program (20) was used to analyze each helix within each cluster in terms of bend angle, wobble angle, and face shift and to compute averages and standard deviations for each cluster. For WT CB1, cluster 1 contained 40 members with an average proline bend (kink) angle of 75.9 ± 1.0°, whereas cluster 2 contained 51 members with average bend angle of 33.7 ± 1.1°. The TMH6 used in our CB1 R model was selected from the more bent cluster, cluster 1, whereas the TMH6 used in our CB1 R* model was selected from the straighter cluster, Cluster 2 (see "Experimental Procedures"). For the TMH6 W6.48A mutant, cluster 1 contained 19 members with an average proline bend (kink) angle of 74.0 ± 1.0°, whereas cluster 2 contained 72 members with an average proline bend angle of 36.8 ± 1.2°. At the 0.01 level, the difference of population means for the kink, wobble, and face shift angles for the W6.48A mutant reported in Table V were not significantly different from the corresponding measures in WT CB1, except for the R* wobble angle. Here the W6.48A R* wobble angle (-105.6 ± 3.4°) was found to be significantly different from the WT R* wobble angle (-120.5 ± 4.6°) at the 0.01 level. Fig. 10 illustrates the steric consequence of this 15° difference in wobble angle. In the R to R* transition, the salt bridge between R3.50 and D/E6.30 is thought to be broken via a conformational change in TMH6 mediated by the flexible hinge region (CWXP motif) of TMH6. Fig. 10 shows that D6.30 in the W6.48A mutant is capable of pulling further away from the intracellular end of TMH3 and R3.50 than is D6.30 in WT TMH6.
Mutation of the toggle switch residues, F3.36(201) and W6.48(357), yielded perhaps the most interesting results of all aromatic microdomain mutations reported here. Mutation of F3.36(201) resulted in elevated basal signaling (increased constitutive activity) and reduced ligand efficacies. Mutation of W6.48(357) resulted in unaltered basal signaling but greatly enhanced ligand efficacies. These results are discussed below.
F3.36(201)A MutationF3.36(201) is revealed here to be a key residue both for ligand binding and for CB1 activation. One of the significant results in the work reported here is that although WT CB1 and the F3.36(201)A mutant have statistically equivalent protein expression levels in HEK293 cells (see Table I), an F3.36(201)A mutation results in a statistically significant higher level of ligand-independent activation of CB1 (i.e. higher basal levels, increased constitutive activity) as assessed by [35S]GTP
The W6.48(357)/F3.36(201) Toggle Switch in WT CB1In recently published Monte Carlo/stochastic dynamics calculations on the inactive state of CB1, we found that one of the most persistent aromatic stacking interactions in the inactive state of CB1 is the F3.36(201)/W6.48(357) interaction (46). Models of the CB1 inactive (R) and active (R*) TMH bundles illustrated here in Fig. 9 show that in the inactive state residues W6.48(357)(
The conformational changes that occur upon receptor activation result in rotations of TMH3 and -6, as well as a change in the conformation of TMH6 (by moderation of its proline kink angle) (68,12). In the light-activated state of Rho, the -ionone ring moves away from TMH F and toward TMH D where it resides close to A4.58(169) (14). This movement releases the constraint on W6.48, making it possible for W6.48(265) to undergo a conformational change. For the CB1 receptor, counterclockwise (from the extracellular side) rotations of TMH3 and -6 concomitant with activation (26, 32) would move F3.36(201) and W6.48(357) past each other, with W6.48(357) moving toward the viewer in Fig. 11 and F3.36(201) moving away from the viewer (also see Fig. 9). Our modeling studies of the CB1 TMH bundle suggest that these rotations cannot take place without a rotamer change for both W6.48(357) and F3.36(201) due to steric clashing. In our models, activation is accompanied by a 1 change in W6.48(357) from g+ trans and a 1 change in F3.36(201) from trans g+ (see above and Fig. 9). The W6.48(357)/F3.36(201) interaction may act as the toggle switch for CB1 activation, with W6.48(357) 1 g+/F3.36(201) 1 trans representing the inactive and W6.48(357) 1 trans/F3.36(201) 1 g+ representing the active state of CB1 (see Fig. 9). If this is true, then mutation of F3.36(201) to a smaller residue, a residue that is no longer a steric block to conformational change in W6.48, would be expected to increase ligand-independent activation of CB1, and this is what is seen here experimentally. Whereas the F3.36(201)A mutation leads to a high degree of constitutive activity as is indicated in Fig. 7, this mutation also results in the reduced efficacy for WIN and CP. This reduction cannot be explained simply by reduced ligand affinity, as the affinity of CP is not affected by this mutation, but its efficacy is affected. It is possible that because the WT CB1 receptor itself exhibits a high level of constitutive activity (3335) and because the F3.36(201)A mutant exhibits an increase in constitutive activity relative to WT CB1, the population of F3.36(201)A mutant receptors is already shifted heavily toward R*. This should not interfere with agonist binding, as agonists have higher affinity for the R* state. However, this would leave fewer receptors for ligands to activate, resulting in a reduced Emax. This explanation is consistent with that of Milligan (45) in a recent review concerning constitutive activity of GPCRs. Milligan (45) noted that "a number of GPCRs do seem to have significant levels of constitutive activity when expressed in cell lines; in some cases, ligand-induced stimulation of activity is relatively small compared with the signal in the absence of ligand" (45). Alternatively, the profound effect on ligand-dependent activation seen here for the F3.36(201)A mutation may indicate that aromaticity at residue 3.36 is central to the CB1 agonist-induced activation process and, consequently, that mutation to a nonaromatic residue impairs the function of this mutant CB1. W6.48(357)A MutationThe W6.48(357)A mutation did not result in a statistically significant change in basal levels relative to WT CB1 (see Fig. 7). Our CB1 modeling studies have shown that when W6.48(357) is mutated to alanine, helix packing allows F3.36(201) to interact with both A6.48 and L6.51. This interaction would serve the same function as F3.36(201) serves in WT CB1, i.e. effectively helping to stabilize TMH6 in its inactive state conformation. Therefore, these results suggest that basal levels should not change between WT CB1 and the W6.48(357)A mutant. Looking at this from another perspective, consideration of the changes in the aromatic microdomain as a result of this mutation also suggests that aromatic stacking in both the R and R* states will be equally impacted by the W6.48(357)A mutation (see "Results"), it is reasonable then to expect that the W6.48(357)A mutant will not produce a change in basal levels relative to WT CB1, and this is what was seen experimentally.
Although the W6.48(357)A mutation had no effect on basal levels, it had noticeable effects on ligand-induced activation, such that the efficacy of every ligand tested was enhanced. One of the steps that has been proposed to occur during GPCR activation is the breaking of an ionic lock (salt bridge) between R3.50 and E/D6.30 that constrains the intracellular ends of TMH3 and TMH6 to remain close in the inactive state (see Fig. 9, left) (10). GPCR activation appears to open a cleft at the cytoplasmic face of a GPCR. Meng and Bourne (47) have described activation as a "blossoming open" of the receptor at its intracellular end. This opening permits G protein docking and interaction with the cytoplasmic domains of the receptor. In Rho, upon light activation, receptor residues in the IC-2 and IC-3 loops have been cross-linked with the C terminus of the G
This phenomenon of dramatic enhancement of Emax values upon mutation of residue W6.48(357) has been seen previously in the CCK-B gastrin receptor (49). Blaker and co-workers (49) found that whereas a W6.48(346)A mutation did not affect basal inositol phosphate production, this mutation affected the functional activity of PD-135,158 (from 20 ± 1% for WT CCK-B to 43 ± 5% in the W6.48(346)A mutant). An enhancement of Emax has also been seen previously for mutations at other loci. For example, in the
ConclusionsModeling, mutation, and functional studies undertaken to test the importance of the TMH3-4-5-6 aromatic microdomain in ligand recognition and in the conformational changes that accompany activation of CB1 suggest that a F3.36(201)/W6.48(357)-specific contact is an important constraint for the CB1-inactive state that may need to break during activation. Modeling studies suggest that the F3.36(201)/W6.48(357) contact can exist in the inactive state of CB1 and be broken in the activated state via a
* This work has been supported by National Institutes on Drug Abuse Grants DA09978, DA05274 (to M. E. A.), DA00489, and DA03934 (to P. H. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|| Current address: University of North Carolina, Greensboro, NC 27402. ¶ To whom correspondence should be addressed: Forbes Norris ALS/MDA Research Center, 2351 Clay St, Suite 416, California Pacific Medical Center Research Institute, San Francisco, CA 94115. Tel.: 415-600-3607; Fax: 415-563-7325; E-mail: mabood{at}cooper.cpmc.org.
1 The abbreviations used are: GPCR, G protein-coupled receptor; CB, cannabinoid; TMH, transmembrane helix; GIRK, G protein-coupled inwardly rectifying potassium; Rho, rhodopsin; WT, wild type; GTP
2 M. Mezei, personal communication.
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