Abstract
Rearrangement of transmembrane domains (TMs) 3 and 5 after agonist binding is necessary for stabilization of the active state of class A G protein-coupled receptors (GPCRs). Using site-directed mutagenesis and functional assays, we provide the first evidence that the TAS(I/V) sequence motif at positions 3.37 to 3.40, highly conserved in aminergic receptors, plays a key role in the activation of the histamine H1 receptor. By combining these data with structural information from X-ray crystallography and computational modeling, we suggest that Thr3.37 interacts with TM5, stabilizing the inactive state of the receptor, whereas the hydrophobic side chain at position 3.40, highly conserved in the whole class A GPCR family, facilitates the reorientation of TM5. We propose that the structural change of TM5 during the process of GPCR activation involves a local Pro5.50-induced unwinding of the helix, acting as a hinge, and the highly conserved hydrophobic Ile3.40 side chain, acting as a pivot.
Introduction
G protein-coupled receptors (GPCRs) transduce sensory signals of external origin such as photons, odors, or pheromones and endogenous signals, including biogenic amines, (neuro)peptides, proteases, glycoprotein hormones, and ions, to the cytoplasmic side of the cell membrane (Kristiansen, 2004). The mechanism by which binding of these highly diverse chemical signals triggers a set of conformational rearrangements of the transmembrane (TM) segments near the G-protein binding domains remains largely unknown. Nevertheless, comparison of the structure of inactive rhodopsin (Li et al., 2004) with the recent crystal structure of the ligand-free opsin (Park et al., 2008), which contains several distinctive features of the presumed active state, leads to conclude that during the process of GPCR activation, among other changes, TM3 rotates clockwise (viewed from the intracellular side), the intracellular part of TM6 tilts outwards by 6 to 7 Å, TM5 approaches TM6, and Arg3.50 (see Materials and Methods for the general numbering scheme) within the (D/E)R(Y/W) motif in TM3 adopts an extended conformation pointing toward the protein core (Park et al., 2008). Notably, as revealed in the original publication of the opsin crystal structure, these conformational changes disrupt the ionic interaction between Arg3.50 with negatively charged side chains at positions 3.49 and 6.30 [Smit et al. (2007) and Rosenbaum et al. (2009) discuss conserved amino acids involved in GPCR activation] and facilitate the interactions between the highly conserved Tyr5.58 and Lys5.66 in TM5 and Arg3.50 in TM3 and Glu6.30 in TM6, respectively. These structural changes strongly suggest that both TMs 3 and 5 play a central role in stabilizing the active state of GPCRs and, therefore, in the process of GPCR activation.
In this study, we have combined the latest structural insights in GPCR structure, computational modeling, and site-directed mutagenesis to study the role of the TAS(I/V) motif at positions 3.37 to 3.40 in TM3, highly conserved in aminergic receptors, in the process of receptor activation. Our results suggest that Thr3.37 interacts with TM5 in the inactive state of the receptor. In addition, we provide the first evidence that the hydrophobic side chain at position 3.40, highly conserved in the whole class A of GPCRs, plays a key role in activation. Mutation of Ile3.40 to either alanine or glycine (i.e., removing the bulky side chain at this position) abolishes the constitutive activity of the histamine H1 receptor (H1R), the effect of constitutive-activity increasing mutations, and the histamine-induced receptor activation.
Materials and Methods
Materials.
Gifts of mianserin hydrochloride (Organon NV, Oss, The Netherlands), pcDEF3 (Dr. J. Langer; Goldman et al., 1996), and the cDNA encoding the human histamine H1R (Dr. H. Fukui; Fukui et al., 1994) are greatly acknowledged. pNF-κB-Luc was obtained from Stratagene (La Jolla, CA), ATP disodium salt, bovine serum albumin, chloroquine diphosphate, DEAE-dextran (chloride form), histamine dihydrochloride, mepyramine (pyrilamine maleate), and polyethylenimine were purchased from Sigma-Aldrich (St. Louis, MO). d-Luciferin was obtained from Duchefa Biochemie BV (Haarlem, The Netherlands), glycerol from Riedel-de-Haen (Seelze, Germany), and Triton X-100 from Fluka (Buchs, Switzerland). Cell culture media, penicillin, and streptomycin were obtained from Invitrogen (Carlsbad, CA). Fetal bovine serum was obtained from Intergro B.V. (Zaandam, the Netherlands). Cell culture plastics were obtained from Corning Life Sciences (Lowell, MA). [3H]Mepyramine (30 Ci/mmol) was purchased-from MP Biomedicals (Solon, OH).
Cell Culture and Transfection.
COS-7 African green monkey kidney cells were maintained at 37°C in a humidified 5% CO2/95% air atmosphere in Dulbecco's modified Eagle's medium containing 50 IU/ml penicillin, 50 μg/ml streptomycin, and 5% (v/v) fetal bovine serum. COS-7 cells were transiently transfected using the DEAE-dextran method as described previously (Bakker et al., 2001).
Site-Directed Mutagenesis.
Single-point mutant human histamine H1Rs T3.37A, T3.37E, I3.40A, and I3.40G and double mutant human histamine H1Rs S3.36T/I3.40A, S3.36T/I3.40G, I6.40K/I3.40A, and I6.40S/I3.40A were created using a polymerase chain reaction-based mutagenesis approach. All constructs were subcloned into the expression vector pcDEF3 and were verified by DNA sequencing.
Reporter-Gene Assay.
Reporter-gene assays were performed essentially as described previously (Bakker et al., 2001). In brief, cells transiently cotransfected with pNF-κB-Luc (125 μg/1.107 cells) and pcDEF3 encoding wild-type (WT) or mutant H1Rs (25 μg/1.107 cells) were seeded in 96-well white plates (Corning Life Sciences) in serum-free culture medium and incubated with drugs. After 48 h, cells were assayed for luminescence by aspiration of the medium and the addition of 25 μl/well luciferase assay reagent [0.83 mM ATP, 0.83 mM d-luciferin, 18.7 mM MgCl2, 0.78 μM Na2H2P2O7, 38.9 mM Tris, pH 7.8. 0.39% (v/v) glycerol, 0.03% (v/v) Triton X-100, and 2.6 μM dithiothreitol]. After 30 min, luminescence was measured for 3 s/well in a Victor2 plate reader (PerkinElmer Life and Analytical Sciences, Waltham, MA). Structural rearrangements during GPCR activation probably occur on a millisecond time scale and, therefore, this assay cannot assess the influence of the mutations on these fast processes. However, we show that specific mutations disrupt receptor activation, probably by stabilizing nonfunctional conformations. We are able to detect these types of disruptions by measuring the changes in receptor signaling in the reporter-gene assay. This assay has been shown to be comparable with the classic Gq-linked generation of inositol phosphates (Bakker et al., 2001).
Histamine H1R Binding Studies.
Histamine H1R binding studies were performed essentially as described previously (Bakker et al., 2001). In brief, transfected COS-7 cells used for radioligand binding studies were harvested after 48 h and homogenized in 50 mM ice-cold Na2/potassium phosphate buffer, pH 7.4 (binding buffer). The COS-7 cell homogenates were incubated for 30 min at 30°C in binding buffer in 200 μl with 3 nM [3H]mepyramine. Nonspecific binding was determined in the presence of 1 μM mianserin. The incubations were stopped by rapid dilution with 3 ml of ice-cold binding buffer. The bound radioactivity was separated by filtration through Whatman GF/C filters that had been treated with 0.3% polyethyleneimine. Filters were washed twice with 3 ml of binding buffer, and the radioactivity retained on the filters was measured by liquid scintillation counting.
Analytical Methods.
Protein concentrations were determined according to Bradford's method (Bradford, 1976), using bovine serum albumin as a standard. Binding and functional data were evaluated by a nonlinear, least-squares curve-fitting procedure using GraphPad Prism 4 (GraphPad Software, Inc., San Diego, CA).
Computational Model of the Histamine H1R.
A model of the TM domains 1 to 7 of the histamine H1R was built by homology modeling using the crystal structure of the β2-adrenergic receptor (Protein Data Bank code 2RH1) (Rosenbaum et al., 2007) as template. Building this homology model was straightforward except for the highly conserved NPxxYxn = 5,6F motif at the junction between TM7 and the intracellular helix 8. Notably, this junction is one residue shorter in the β2-adrenergic receptor (n = 5) than in rhodopsin (n = 6). As a result, Tyr7.53 is pointing toward TM2 in rhodopsin (Li et al., 2004) and toward TM6 in the β2-adrenergic receptor (Rosenbaum et al., 2007). The fact that the histamine H1R contains six amino acids (n = 6) between both aromatic side chains led us to model the junction between TM7 and helix 8 as in rhodopsin (Protein Data Bank code 1GZM). Modeler 9v1 (Martí-Renom et al., 2000) was used to add intracellular loops 1 to 2 and extracellular loops 1 to 3 using the structure of the β2-adrenergic receptor as template. Internal water molecules 506, 519, 528, 529, 532, 534, 537, 543, 546, and 548 that mediate a number of key interhelical interactions (Rosenbaum et al., 2007) and are probably conserved in family A GPCRs (Pardo et al., 2007) were explicitly included in the model.
Distortion of TM5.
The program HELANAL (Bansal et al., 2000) was used to calculate the residue unit twist angle of TM5, a structural parameter that describes local helical uniformity. This parameter is interpreted as follows: an ideal α-helix, with approximately 3.6 residues per turn, has a twist angle of approximately 100° (360°/3.6); a closed helical segment, with <3.6 residues per turn, possesses a twist >100°; whereas an open helical segment, with >3.6 residues per turn, possesses a twist <100°.
Nomenclature of Side Chain Conformation.
The side chain conformations have been categorized into gauche− (g−: 0°<χ1<120°), trans (t: 120°< χ 1<240°), or gauche+ (g+: 240°< χ 1<360°) depending on the value of the χ1 torsional angle.
Numbering Scheme of GPCRs.
Residues are identified by the general numbering scheme of Ballesteros and Weinstein (1995) that allows easy comparison among residues in the 7TM segments of different receptors.
Sequence Analysis.
Family-specific analyses of GPCR sequence conservation were performed using the GMoS program (http://lmc.uab.cat/gmos).
Results
A Proline-Induced Unwinding in the Structure of TM5 of Class A GPCRs.
GPCRs possess a highly conserved Pro5.50 in TM5, present in 77% of the rhodopsin-like sequences, absent only in melanocortin, glycoprotein hormone, lysosphingolipid, prostanoid, and cannabinoid receptors. Usually, in proline-containing α-helices, the steric clash between the pyrrolidine ring of proline and the backbone carbonyl oxygen in the preceding turn induces a bend angle of approximately 20° in the helical structure (Deupi et al., 2004). However, a detailed analysis of the structures of bovine (Li et al., 2004) and squid (Murakami and Kouyama, 2008) rhodopsin, adenosine A2A receptor (Jaakola et al., 2008), and the β1- (Warne et al., 2008) and β2- (Rosenbaum et al., 2007) adrenergic receptors has allowed us to detect and quantify a peculiar distortion of TM5 in which a local opening of the helix (>3.6 residues/turn, twist <100°, see Materials and Methods) at the 5.45 to 5.48 turn (Fig. 1B) partially removes the steric clash between the pyrrolidine ring of Pro5.50 and the backbone carbonyl oxygen at position 5.46 (i-4 in Fig. 1, C–H). This local opening of TM5 (proline-unwinding, in contrast to proline-kink) also modifies the relative orientation of the side chains at the extracellular side, including residues involved in neurotransmitter binding such as 5.42, 5.43, and 5.46 (Ballesteros et al., 2001b; Deupi et al., 2007). Other membrane proteins also feature this type of helical wide turns (Riek et al., 2008).
Role of TM3 in Stabilizing the Proline-Unwinding of TM5.
The analysis of the currently available GPCR crystal structures in the present study allows us to propose a common mechanism by which this unusual conformation of TM5 is stabilized. In bovine rhodopsin, the backbone carbonyl oxygen at position 5.46 is stabilized by a hydrogen bond interaction with Glu3.37 (at the interatomic distance of 2.8 Å) and a van der Waals interaction with Leu3.40 (3.2 Å) (Fig. 1, A and C) (Li et al., 2004; Deupi et al., 2007). Adenosine A2A receptor preserves similar interactions through Gln3.37 (3.2 Å) and Ile3.40 (3.9 Å) (Fig. 1G) (Jaakola et al., 2008). It is noteworthy that squid rhodopsin replaces Glu3.37 of bovine rhodopsin by phenylalanine but presents a discrete water molecule linking the backbone carbonyls at positions 3.37 (3.0 Å) and 5.46 (3.3 Å) (Murakami and Kouyama, 2008) while maintaining the van der Waals interaction between the hydrophobic Ile3.40 and the carbonyl oxygen at position 5.46 (3.2 Å) (Fig. 1D). Notably, the recent X-ray structure of opsin (Park et al., 2008) shows that during the process of rhodopsin activation, the hydrogen bond interaction between Glu3.37 and the backbone carbonyl (4.9 Å) is disrupted, as previously suggested by NMR measurements of rhodopsin containing 13C-labeled histidine (Patel et al., 2005), whereas Leu3.40 has moved slightly away (3.5 Å) (Figs. 1F and 2B). This effect correlates with the minor, but significant, clockwise rotation (viewed from the intracellular side) of TM3 during the process of receptor activation (see Discussion). The release of the strong packing of Leu3.40 with the 5.45 to 5.48 turn does not result in a change in the local opening of TM5 (Fig. 1B) but in a localized decrease in the bend around Pro5.50 (Fig. 1A). In the β1- (Warne et al., 2008) and β2- (Rosenbaum et al., 2007) adrenergic receptors, Ile3.40 also stabilizes the carbonyl oxygen at position 5.46, similarly to adenosine and opsin receptors, whereas, in this case, the shorter Thr3.37 (compared with glutamic acid or glutamine) interacts with the side chain rather than with the backbone carbonyl, of Ser5.46 (Figs. 1, E and H, and 2C).
To investigate the role of the amino acids at positions 3.37 and 3.40 in GPCR activation, we have created and studied several H1R mutants. We have previously used the H1R as a model system for the study of class A GPCR activation (Bakker et al., 2008). The H1R belongs to the aminergic subfamily of Class A GPCRs and, like the β1- and β2-adrenergic receptors, features Thr3.37 and Ile3.40 in TM3. Moreover, the H1R contains a polar Asn5.46 amino acid at position 5.46, which is involved in histamine binding (Leurs et al., 1994), linking this GPCR subdomain directly to the agonist binding site.
Influence of Thr3.37 and Ile3.40 in Agonist-Induced Activation of the Human Histamine H1R.
To test the role of the amino acids at positions 3.37 and 3.40, we engineered mutants in which Thr3.37 of the histamine H1R was substituted by either alanine (T3.37A) to remove the hydrogen bonding capability at this position, or glutamic acid (T3.37E) to mimic the bovine rhodopsin sequence, and Ile3.40 was replaced by either alanine (I3.40A) or glycine (I3.40G), removing the bulky hydrophobic side chain and, thus, the van der Waals interaction with the carbonyl group at position 5.46. Figure 3D shows the environment of Thr3.37 in the β2-based molecular model of the histamine H1R (see Materials and Methods). Thr3.37 forms a hydrogen bond with Asn5.46, the binding partner for the imidazole ring of histamine (Leurs et al., 1994), as observed for the Thr3.37…Ser5.46 interaction in the structure of the β2-adrenergic receptor (Fig. 1H). Wild-type, T3.37A, and T3.37E mutant H1Rs were well expressed in COS-7 cells at approximately 10 pmol/mg protein as determined by radioligand binding analysis (Table 1). The function of the WT H1R was evaluated in an NF-κB reporter-gene assay as reported previously (Jongejan et al., 2005; Bakker et al., 2008), and the activation of NF-κB was increased up to 10-fold (Emax = 1002 ± 34%) when stimulated with histamine (Fig. 3, A and B). The fact that the functional potency of histamine at WT H1R exceeds its binding affinity is well in accordance with data published previously (Jongejan et al., 2005; Bakker et al., 2008). Removal of the interaction of Thr3.37 with Asn5.46, by mutating residue Thr3.37 to alanine resulted in a 0.6 log unit decrease of the binding affinity of histamine (pKi of 5.3 for the WT H1R versus 4.7 for the T3.37A H1R) (Table 1), as measured by [3H]mepyramine displacement. A lower constitutive signaling of the T3.37A H1R mutant compared with that of WT H1R was observed (Fig. 3, A and B). However, the basal signaling could still be increased by histamine, albeit at higher concentrations (pEC50 = 4.5 ± 0.2) (Table 1) (Supplemental Fig. 1). Mutation of residue Thr3.37 to glutamic acid (i.e., making this region similar to bovine rhodopsin) did not influence the binding affinity of histamine (Table 1), possibly because Glu3.37 maintains both the interaction with Asn5.46 (Fig. 3E), as in the WT H1R (Fig. 3D), and with the carbonyl group at position 5.46, as in bovine rhodopsin (Fig. 1C). Notably, the T3.37E mutant H1R hardly shows any constitutive activity (Fig. 3, A and B). Moreover, the T3.37E mutant H1R is activated very poorly by histamine (pEC50 > 3.2). Thus, we propose that the additional constraint between Glu3.37 and the backbone carbonyl at position 5.46, introduced in the T3.37E mutation, impedes the activation of the mutant H1R by locking a local structure that cannot be overcome by the agonist histamine.
Removal of the bulky hydrophobic Ile3.40 side chain by substitution to alanine or glycine also results in significant effects on both the constitutive activity of the receptor and the process of histamine-induced receptor activation (Fig. 3, A and C). It is noteworthy that these effects are not due to a loss of binding affinity for histamine. As shown in Table 1, both I3.40A and I3.40G mutant H1Rs are well expressed in COS-7 cells and bind histamine with comparable affinity compared with WT H1R. The fact that removal of the Ile3.40 side chain abolished both the constitutive and histamine-induced activity strongly suggests that this hydrophobic side chain plays a key role in the mechanism of activation. Based on these results and on the recent insights into GPCR structure (Park et al., 2008; Scheerer et al., 2008), we propose that the hydrophobic side chain at position 3.40, which is strongly packed against TM5 (Fig. 2), acts as a pivot and the Pro5.50-induced unwinding acts as a hinge in the crucial reorientation of TM5 upon activation, which ultimately facilitates the interactions of Tyr5.58 and Lys5.66 with Arg3.50 and Glu6.30, respectively (Fig. 2). Notably, mutations at position 3.40 in rhodopsin are related to retinitis pigmentosa, poor retinal binding, misfolding, reduced expression, decreased time in the MII state, and reduced transducin activation (Madabushi et al., 2004).
To substantiate this hypothesis, we performed two sets of additional experiments. We combined activating point mutations leading to agonist-independent constitutive activity with the inactivating I3.40A/G mutation with the aim to assess their compensatory consequences on receptor function. We have chosen the previously reported S3.36T mutation (Jongejan et al., 2005) in the ligand binding site and the I6.40K/S mutations (Bakker et al., 2008) near the cytoplasmic site, both leading to a large increase in constitutive activity (see Discussion). The S3.36T mutation induces the transition of Trp6.48 toward TM5 (Jongejan et al., 2005), considered to be the initial stage of the activation process (see below), as observed in the electron microscopy density map of metarhodopsin I (Ruprecht et al., 2004) and in solid-state NMR measurements of metarhodopsin II (Crocker et al., 2006). Thus, we combined the activating S3.36T point mutation with the inactivating I3.40A/G mutation to generate the S3.36T/I3.40A and S3.36T/I3.40G double mutant H1Rs. The double mutant H1Rs are well expressed in COS-7 cells and have similar affinity for histamine compared with wild-type H1R (Table 1). It is noteworthy that both double mutants show lack of constitutive activity and cannot be activated by histamine, resembling the single I3.40A/G mutant H1R (Fig. 4). On the other hand, the I6.40S and I6.40K mutations induce constitutive receptor activation by triggering the conformational change of Asn7.49 toward Asp2.50 (Urizar et al., 2005; Bakker et al., 2008). The I6.40S /I3.40A and I6.40K /I3.40A double mutant H1Rs show similar affinities for histamine binding (Table 1), but lack constitutive activity and cannot be activated by histamine (Fig. 4). These data reinforce the importance of Ile3.40 in the process of receptor activation.
Discussion
A number of studies have provided convincing evidence that GPCRs coexist in different conformations (Kobilka and Deupi, 2007). GPCRs are maintained within an ensemble of inactive conformations through noncovalent interactions between side chains, mostly located in the TM segments (Smit et al., 2007; Rosenbaum et al., 2009) but also at the extracellular surface (Bokoch et al., 2010). Disruption of these stabilizing interactions results in gain-of-function mutations. An evolutionary trace analysis has led to the identification of a putative common signal transduction process for class A GPCRs, in which key residues are grouped into a network of interactions extending from a trigger region near the binding pocket to a coupling region in the cytoplasmic region, connected by an intermediate linking core (Madabushi et al., 2004). This analysis detected Leu3.40 of rhodopsin in the trigger region as a structurally and/or functionally important residue.
The fact that the histamine H1R shows considerable agonist-independent, constitutive signaling, which can be strongly reduced by the inverse agonist mepyramine (Bakker et al., 2001), provides an unique opportunity to approach the mechanism of receptor activation. In this study, we used site-directed mutagenesis of the H1R to study the proposed role of the hydrophobic side chain at position 3.40, highly conserved in the whole class A of GPCRs, in GPCR activation. Mutation of Ile3.40 to either alanine or glycine, removing the bulky side chain, abolished the constitutive activity of the receptor, the effect of constitutive-activity increasing mutations, and the histamine-induced receptor activation, but not the binding of histamine. These data strongly suggest that Ile3.40 is not involved in the initial binding step but participates in the subsequent signal propagation upon histamine binding. In the inactive state (Fig. 2, A and C) the hydrophobic side chain at position 3.40 (green surface) is located between the pyrrolidine ring of Pro5.50 and the carbonyl oxygen at position 5.46, inducing or stabilizing the local opening of TM5 (Fig. 1B). However, the molecular mechanism by which this residue facilitates receptor activation is not straightforward. The structure of opsin (Park et al., 2008) and previous substituted-cysteine accessibility studies in the β2-adrenergic receptor (Gether et al., 1997) have shown a clockwise rotation (viewed from the intracellular side) of TM3 during the process of receptor activation. Rotation of TM3 moves the bulky side chain at position 3.40, located between Pro5.50 and the 5.46 carbonyl, away from these moieties (Fig. 2B), resulting in a localized decrease of the bend around Pro5.50 (Fig. 1A).
Fluorescence spectroscopy experiments used to monitor agonist-induced conformational changes in the β2-adrenergic receptor provide evidence of a sequential binding model through discrete intermediate conformational states (Swaminath et al., 2004). In an initial step, agonist binding induces relocation of the extracellular side of TM5 to facilitate binding of the hydroxyl groups of the catechol moiety of β2-adrenergic receptor agonists (Kobilka, 2004). We propose that this movement is transduced as a set of structural rearrangements toward the intracellular side of TM5 with the assistance of Ile3.40 acting as a pivot, ultimately enabling the interactions of Tyr5.58 with Arg3.50 and Lys5.66 with Glu6.30 in the cytoplasmic side of the receptor (Fig. 5, right arrow). In support of this hypothesis, the T3.37E mutation in the H1R, which adds an additional constraint between TMs 3 and 5 (Fig. 3E), prevents the transmission of the activation signal through TM5 (Fig. 3B) but does not affect the binding of histamine.
In addition to this activation pathway through TM5, amino acids located in TMs 3 and 6 also participate in other routes of receptor activation (Fig. 5, top left arrow). For instance, agonist binding triggers the Trp6.48 rotamer toggle switch from the inactive g+ (pointing toward TM7) to the active t (toward the binding pocket) conformation (Shi et al., 2002; Ruprecht et al., 2004; Crocker et al., 2006; Holst et al., 2010) through the formation of specific hydrogen bonds (López-Rodríguez et al., 2005). Notably, the rotamer toggle switch of Trp6.48 occurs in a concerted manner with the side chain at position 3.36 from the inactive t (toward the binding pocket) to the active g+ (toward TM7) conformation (Jongejan et al., 2005). To assess whether these pathways of receptor activation are independent or not, we have combined the inactivating I3.40A/G mutations (Figs. 4) with the activating S3.36T point mutation, which is also located in the trigger region and leads to agonist-independent constitutive activity (Jongejan et al., 2005). The absence of both constitutive activity and histamine-induced activation in these double mutant receptors, despite normal histamine binding, led us to conclude that the movement of TM5 (via the Ile3.40 pivot) is essential to stabilize the activation pathway by means of the Trp6.48/Ser3.36 rotamer toggle switch.
The signal triggered by the Trp6.48/Ser3.36 toggle switch is transmitted toward intracellular microdomains (Smit et al., 2007; Rosenbaum et al., 2009). In particular, Asn7.49 and Tyr7.53 (in the NPxxY motif of TM7) undergo conformational changes (Urizar et al., 2005; Park et al., 2008), ultimately leading to the disruption of the ionic interaction between Arg3.50 and the adjacent Asp/Glu3.49 (the DRY motif of TM3) (Scheer et al., 1996), and an additional Asp/Glu6.30 near the cytoplasmic end of TM6 (Ballesteros et al., 2001a) (Fig. 5, bottom left arrow). Disruption of these ionic interactions permits Arg3.50 to adopt an extended conformation (Bakker et al., 2008; Park et al., 2008), pointing toward the protein core and to interact with the highly conserved Tyr5.58 and Tyr7.53 in TMs 5 and 7 (Park et al., 2008). These conformational changes open a cavity between TMs 3, 5, and 6 where the G protein can bind (Scheerer et al., 2008). We have shown previously that mutation of the highly conserved hydrophobic side chain at position 6.40 to glutamic acid, glycine, alanine, arginine, lysine, or serine results in highly constitutively active receptors for which almost no additional histamine-induced receptor activation can be detected (Bakker et al., 2008). These substitutions at position 6.40 seem to modify the conformation of the key Asn7.49, Tyr7.53, and Arg3.50 side chains, located in the coupling region near the G protein binding site (Bakker et al., 2008). Notably, adding the I3.40A mutation, in the trigger region, to the constitutively active I6.40S/K mutant receptors decreases constitutive activity and impedes histamine-induced receptor activation (Fig. 4). These results suggest a key role of Ile3.40 in transmitting the activating signal from the binding pocket to the cytoplasmic side and in stabilizing the conformational changes of TM5 at the intracellular side of the receptor, probably by means of Tyr5.58.
Triggering specific pathways along the activation process might be an explanation for the observed collateral efficacy of GPCR agonists. Our data provide further insight in the molecular mechanisms of GPCR activation, and we propose that one of such additional pathways consists in the agonist-induced relocation of the extracellular side of TM5 toward the transmembrane bundle. The activation-signal is then further propagated toward the cytoplasmic region through the fulcrum/lever mechanism described above, in which Pro5.50 acts as a hinge and the highly conserved hydrophobic Ile3.40 side chain acts as a pivot.
Authorship Contributions
Participated in research design: Sansuk, Bakker, Jongejan, Pardo, and Leurs.
Conducted experiments: Sansuk, Torrecillas, and Nijmeijer.
Performed data analysis: Sansuk, Deupi, Torrecillas, and Nijmeijer.
Wrote or contributed to the writing of the manuscript: Sansuk, Deupi, Bakker, Pardo, and Leurs.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the Ministerio de Educación y Ciencia [Grant SAF2010-22198-C02-02]; the Instituto de Salud Carlos III [Grant RD07/0067/0008]; and the Ministerio de Educación y Ciencia through the Ramon y Cajal program (to X.D.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.066068.
-
ABBREVIATIONS:
- GPCR
- G protein-coupled receptor
- H1R
- H1 receptor
- WT
- wild type
- TM
- transmembrane
- t
- trans
- g+
- gauche+
- g−
- gauche−
- NF-κB
- nuclear factor κB.
- Received May 5, 2010.
- Accepted November 16, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
References
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