The local environment at the cytoplasmic end of TM6 of the mu opioid receptor differs from those of rhodopsin and monoamine receptors: introduction of an ionic lock between the cytoplasmic ends of helices 3 and 6 by a L6.30(275)E mutation inactivates the mu opioid receptor and reduces the constitutive activity of its T6.34(279)K mutant

Activation of rhodopsin and monoamine G protein-coupled receptors (GPCRs) has been proposed to involve in part the disruption of a conserved E6.30-R3.50 ionic interaction between transmembrane segments (TMs) 3 and 6. However, this interaction does not occur in the opioid receptors, which have L275 at 6.30. On the basis of our findings that mutations of T6.34(279) to K and D produced, respectively, a constitutively active and an inactive form of the mu opioid receptor, we previously suggested that the functional role of the 6.30(275) residue could be assumed by T6.34(279), but the interplay between residues at positions 6.30 and 6.34 remained unresolved. In this study, we examined the effects of introducing an E in position 6.30(275) of the wild type (WT) and of the T6.34(279) mutants of the mu opioid receptor to compare the participation of the 6.30 locus in molecular events during activation in this receptor with its role in other GPCRs. The L6.30(275)E and the L6.30(275)E/T6.34(279)D mutants displayed no constitutive activity and could not be activated by the agonist DAMGO or morphine. The L6.30(275)E/T6.34(279)K mutant had some constitutive activity, but much less than the T6.34(279)K mutant, and could be activated by both agonists. The rank order of affinity for the agonist DAMGO is as follows: T6.34(279)K > WT congruent with L6.30(275)E/T6.34(279)K > L6.30(275)E congruent with T6.34(279)D > L6.30(275)E/T6.34(279)D; however, all constructs have a similar affinity for the antagonist [(3)H]diprenorphine. These data are interpreted in the context of interactions with the conserved R3.50(165) in TM3. When L6.30(275) is mutated to E, the favorable E6.30(275)-R3.50(165) interaction stabilizes an inactive state, as in rhodopsin, and hence reduces the activities of T6.34(279) mutants. Thus, the mu opioid receptor is shown to be different from rhodopsin and monoamine GPCRs, of which the WTs with native E6.30 can be activated, and the 6.34D or 6.34K mutants display enhanced constitutive activities. Our molecular modeling results suggest that some specific differences in local geometry at the cytoplasmic ends of TM5 and TM6 may account in part for the observed differences in the molecular mechanisms of receptor activation.     

Structural features of the inactive and active states of the melanin-concentrating hormone receptors: insights from molecular simulations

Comparative molecular dynamics simulations of both subtypes 1 and 2 of the melanin-concentrating hormone receptor (MCHR1 and MCHR2, respectively) in their free and hormone-bound forms have been carried out. The hormone has been used in its full-length and truncated forms, as well as in 16 mutated forms. Moreover, MCHR1 has been simulated in complex with T-226296, a novel orally active and selective antagonist. The comparative analysis of an extended number of receptor configurations suggests that the differences between inactive (i.e., free and antagonist-bound) and active (i.e., agonist-bound) states of MCHRs involve the receptor portions close to the E/DRY and NPxxY motifs, with prominence to the cytosolic extensions of helices 2, 3, 6, and 7. In fact, the active forms of these receptors share the release of selected intramolecular interactions found in the inactive forms, such as that between R3.50 of the E/DRY motif and D2.40, and that between Y7.53 of the NPxxY motif and F7.60. Another feature of the active forms of both MCHRs is the approach of "helix 8" to the cytosolic extension of helix 3. These features of the active forms are concurrent with the opening of a cleft at the cytosolic end of the helix bundle. For both MCHRs, the agonist-induced chemical information transfer from the extracellular to the cytosolic domains is mediated by a cluster of aromatic amino acids in helix 6, following the ligand interaction with selected amino acids in the extracellular half of the receptor.     

Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.     

Structure and pH sensitivity of the transmembrane segment 3 of rhodopsin

Activation of G protein-coupled receptors (GPCRs) originates in ligand-induced protein conformational changes that are transmitted to the cytosolic receptor surface. In the photoreceptor rhodopsin, and possibly other rhodopsin-like GPCRs, protonation of a carboxylic acid in the conserved E(D)RY motif at the cytosolic end of transmembrane helix 3 (TM3) is coupled to receptor activation. Here, we have investigated the structure of synthetic peptides derived from rhodopsin TM3. Polarized FTIR spectroscopy reveals a helical structure of a 31-mer TM3 peptide reconstituted into PC vesicles with a large tilt of 40-50 degrees of the helical axis relative to the membrane normal. Helical structure is also observed for the TM3 peptide in detergent micelles and depends on pH, especially in the C-terminal sequence. In addition, the fluorescence emission of the single tyrosine of the D(E)RY motif in the TM3 peptide exhibits a pronounced pH sensitivity that is abolished when Glu is replaced by Gln, demonstrating that protonation of the conserved Glu side chain affects the structure in the environment of the D(E)RY motif of TM3. The pH regulation of the C-terminal TM3 structure may be an intrinsic feature of the E(D)RY motif in other class I receptors, allowing the coupling of protonation and conformation of membrane-exposed residues in full-length GPCRs.     

Constitutive activation of A(3) adenosine receptors by site-directed mutagenesis

The objective of this study was to create constitutively active mutant human A(3) adenosine receptors (ARs) using single amino acid replacements, based on findings from other G protein-coupled receptors. A(3) ARs mutated in transmembrane helical domains (TMs) 1, 3, 6, and 7 were expressed in COS-7 cells and subjected to agonist radioligand binding and phospholipase C (PLC) and adenylyl cyclase (AC) assays. Three mutant receptors, A229E in TM6 and R108A and R108K in the DRY motif of TM3, were found to be constitutively active in both functional assays. The potency of the A(3) agonist Cl-IB-MECA (1-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) in PLC activation was enhanced by at least an order of magnitude over wild type (EC(50) 951 nM) in R108A and A229E mutant receptors. Cl-IB-MECA was much less potent (>10-fold) in C88F, Y109F, and Y282F and mutants or inactive following double mutation of the DRY motif. The degree of constitutive activation was more pronounced for the AC signaling pathway than for the PLC signaling pathway. The results indicated that specific locations within the TMs proximal to the cytosolic region were responsible for constraining the receptor in a G protein-uncoupled conformation.     

The third intracellular loop stabilizes the inactive state of the neuropeptide Y1 receptor

Constitutively active G-protein-coupled receptors (GPCRs) can signal even in the absence of ligand binding. Most Class I GPCRs are stabilized in the resting conformation by intramolecular interactions involving transmembrane domain (TM) 3 and TM6, particularly at loci 6.30 and 6.34 of TM6. Signaling by Gi/Go-coupled receptors such as the Neuropeptide Y1 receptor decreases already low basal metabolite levels. Thus, we examined constitutive activity using a biochemical assay mediated by a Gi/Gq chimeric protein and a more direct electrophysiological assay. Wild-type (WT-Y1) receptors express no measurable, agonist-independent activation, while mu-opioid receptors (MOR) and P2Y12 purinoceptors showed clear evidence of constitutive activation, especially in the electrophysiological assay. Neither point mutations at TM6 (T6.30A or N6.34A) nor substitution of the entire TM3 and TM6 regions from the MOR into the Y1 receptor increased basal WT-Y1 activation. By contrast, chimeric substitution of the third intracellular loop (ICL3) generated a constitutively active, Y1-ICL3-MOR chimera. Furthermore, the loss of stabilizing interactions from the native ICL3 enhanced the role of surrounding residues to permit basal receptor activation; because constitutive activity of the Y1-ICL3-MOR chimera was further increased by point mutation at locus 6.34, which did not alter WT-Y1 receptor activity. Our results indicate that the ICL3 stabilizes the Y1 receptor in the inactive state and confers structural properties critical for regulating Y receptor activation and signal transduction. These studies reveal the active participation of the ICL3 in the stabilization and activation of Class I GPCRs.     

Agonist-induced conformational changes in bovine rhodopsin: insight into activation of G-protein-coupled receptors

Activation of G-protein-coupled receptors (GPCRs) is initiated by conformational changes in the transmembrane (TM) helices and the intra- and extracellular loops induced by ligand binding. Understanding the conformational changes in GPCRs leading to activation is imperative in deciphering the role of these receptors in the pathology of diseases. Since the crystal structures of activated GPCRs are not yet available, computational methods and biophysical techniques have been used to predict the structures of GPCR active states. We have recently applied the computational method LITiCon to understand the ligand-induced conformational changes in β₂-adrenergic receptor by ligands of varied efficacies. Here we report a study of the conformational changes associated with the activation of bovine rhodopsin for which the crystal structure of the inactive state is known. Starting from the inactive (dark) state, we have predicted the TM conformational changes that are induced by the isomerization of 11-cis retinal to all-trans retinal leading to the fully activated state, metarhodopsin II. The predicted active state of rhodopsin satisfies all of the 30 known experimental distance constraints. The predicted model also correlates well with the experimentally observed conformational switches in rhodopsin and other class A GPCRs, namely, the breaking of the ionic lock between R135^3.50 at the intracellular end of TM3 (part of the DRY motif) and E247^6.30 on TM6, and the rotamer toggle switch on W265^6.48 on TM6. We observe that the toggling of the W265^6.48 rotamer modulates the bend angle of TM6 around the conserved proline. The rotamer toggling is facilitated by the formation of a water wire connecting S298^7.45, W265^6.48 and H211^5.46. As a result, the intracellular ends of TMs 5 and 6 move outward from the protein core, causing large conformational changes at the cytoplasmic interface. The predicted outward movements of TM5 and TM6 are in agreement with the recently published crystal structure of opsin, which is proposed to be close to the active-state structure. In the predicted active state, several residues in the intracellular loops, such as R69, V139^3.54, T229, Q237, Q239, S240, T243 and V250^6.33, become more water exposed compared to the inactive state. These residues may be involved in mediating the conformational signal from the receptor to the G protein. From mutagenesis studies, some of these residues, such as V139^3.54, T229 and V250^6.33, are already implicated in G-protein activation. The predicted active state also leads to the formation of new stabilizing interhelical hydrogen-bond contacts, such as those between W265^6.48 and H211^5.46 and E122^3.37 and C167^4.56. These hydrogen-bond contacts serve as potential conformational switches offering new opportunities for future experimental investigations. The calculated retinal binding energy surface shows that binding of an agonist makes the receptor dynamic and flexible and accessible to many conformations, while binding of an inverse agonist traps the receptor in the inactive state and makes the other conformations inaccessible.     

Mutagenesis and peptide analysis of the DRY motif in the alpha2A adrenergic receptor: evidence for alternate mechanisms in G protein-coupled receptors

In G protein-coupled receptors (GPCRs), a conserved aspartic acid in the DRY motif at the cytoplasmic end of helix 3 regulates the transition to the active state, while the adjacent arginine is crucial for G protein activation. To examine the functions of these two residues, we made D130I and R131Q mutations in the alpha2A adrenergic receptor (AR). We demonstrate that, unlike other GPCRs, the alpha2A AR is not constitutively activated by the D130I mutation, although the mutation increases agonist affinity. While the R131Q mutation severely disrupts function, it decreases rather than increasing agonist affinity as seen in other GPCRs. We then investigated the molecular effects of the same mutations in a peptide model and showed that Arg131 is not required for peptide-mediated G protein activation. These results indicate that the alpha2A AR does not follow the conventional GPCR mechanistic paradigm with respect to the function of the DRY motif.     

High affinity agonistic metal ion binding sites within the melanocortin 4 receptor illustrate conformational change of transmembrane region 3

We created a molecular model of the human melanocortin 4 receptor (MC4R) and introduced a series of His residues into the receptor protein to form metal ion binding sites. We were able to insert micromolar affinity binding sites for zinc between transmembrane region (TM) 2 and TM3 where the metal ion alone was able to activate this peptide binding G-protein-coupled receptor. The exact conformation of the metal ion interactions allowed us to predict the orientation of the helices, and remodeling of the receptor protein indicated that Glu100 and Ile104 in TM2 and Asp122 and Ile125 in TM3 are directed toward a putative area of activation of the receptor. The molecular model suggests that a rotation of TM3 may be important for activation of the MC4R. Previous models of G-protein-coupled receptors have suggested that unlocking of a stabilizing interaction between the DRY motif, in the cytosolic part of TM3, and TM6 is important for the activation process. We suggest that this unlocking process may be facilitated through creation of a new interaction between TM3 and TM2 in the MC4R.     

Structural insight into the binding complex: β-arrestin/CCR5 complex

The chemokine receptor 5 (CCR5) belongs to the superfamily of serpentine G protein-coupled receptors (GPCRs). The DRY motif (Asp, Arg, Tyr) of the intracellular loop 2 (ICL2), which is highly conserved in the GPCRs has been shown to be essential for the stability of folding of CCR5 and the interaction with β-arrestin. But the molecular mechanism by which it recognizes and interacts with β-arrestin has not been elucidated. In the present study, we described the active state of the β-arrestin structure using normal mode analysis and characterized the binding cleft of CCR5-ICL2 with β-arrestin using SABRE© docking tool and molecular dynamics simulation. Based on our computational results, we proposed a mode of binding between the ICL2 loop of CCR5 and β-arrestin structure, and modeled the energetically stable β-arrestin/CCR5 complex. In view of CCR5’s importance as a therapeutic target for the treatment of HIV, this observation provides novel insight into the β-arrestin/CCR5 pathway. As a result, the current computational study of the detailed β-arrestin/CCR5 binding complex could provide the rationale for the development of next generation of HIV peptide inhibitors as therapeutic agents.     

Tissue distribution and functional analyses of the constitutively active orphan G protein coupled receptors, GPR26 and GPR78

GPR26 and GPR78 are orphan GPCRs (oGPCRs) that share 51% amino acid sequence identity and are widely expressed in selected tissues of the human brain as well as the developing and adult mouse brain. Investigation of the functional activity of GPR26 and GPR78 via expression in HEK293 cells showed that both proteins are constitutively active and coupled to elevated cAMP production. Accordingly, in yeast, GPR26 demonstrated apparent agonist-independent coupling to a chimeric Gpa1 protein in which the 5 C-terminal amino acids were from Galphas. A comparison of the proteins revealed an atypical glutamine residue in GPR78 in place of the conserved arginine residue (R3.50) in the so-called DRY box. Site-directed mutants R3.50 in GPR26 were constructed and retained their constitutive activity suggesting that these 2 receptors activate G proteins in a manner that is distinct from other group 1 GPCRs.     

β-Arrestin Recruitment and G Protein Signaling by the Atypical Human Chemokine Decoy Receptor CCX-CKR

Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric G_i proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21, and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate G_i signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21, and CCL25 in the presence of the G_i inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive G_i proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.     

Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties

From computational simulations of a serotonin 2A receptor (5-HT(2A)R) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT(2A)R, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.     

The effects of charge-neutralizing mutation D6.30N on the functions of CB1 and CB2 cannabinoid receptors

Charge-neutralizing mutation D6.30N of the human cannabinoid receptor subtype 1 (CB1) and cannabinoid receptor subtype 2 (CB2) cannabinoid receptors was made to test two hypotheses: (1) D6.30 may be crucial for the functions of CB1 and CB2 receptors. (2) D6.30 may participate in an ionic lock with R3.50 that keeps the receptors in an inactive conformation. Specific ligand binding and ligand-induced inhibition of forskolin-stimulated cAMP accumulation were observed with human embryonic kidney epithelial cell line (HEK293) cells expressing wild-type CB1 and CB2, as well as CB1D6.30N and CB2D6.30N mutant receptors. There was however a decrease in maximum response of the mutant receptors compared to their wild-type counterparts, suggesting that D6.30 is essential for full activation of both CB1 and CB2 receptors. Both CB1D6.30N and CB2D6.30N demonstrated a level of constitutive activity no greater than that of their wild-type counterparts, indicating that either D6.30 does not participate in a salt bridge with R3.50, or the salt bridge is not critical for keeping cannabinoid receptors in the inactive conformation.     

Complementary roles of the DRY motif and C-terminus tail of GPCRS for G protein coupling and beta-arrestin interaction

β-arrestin mediates the desensitization of GPCRs and acts as an adaptor molecule to recruit the receptor complex to clathrin-rich regions. Class-A GPCRs subsequently dissociate from β-arrestin but class-B GPCRs internalize with β-arrestin in the endocytic vesicles. Here the dopamine D₂ and D₃ receptors, which have similar structural features but different intracellular trafficking properties, were used in an attempt to better understand the structural requirements for the classification of GPCRs. The C-terminus tail of the vasopressin type-2 receptor was added to the ends of D₂R and D₃R to increase their affinity to β-arrestin. A point mutation was introduced into the DRY motif to change their basal activation levels. Among a battery of constructs in which the C-terminus tail and/or DRY motif was altered, class-B behavior was observed with the constructs whose affinities for β-arrestin were increased complementarily and their signaling was either maintained or regained. In conclusion, the DRY motif and C-terminal tail of the GPCRs determine complementarily their intracellular trafficking behavior by regulating the affinity to β-arrestin and G protein coupling.     

Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

Chemokine receptors play a major role in immune system regulation and have consequently been targets for drug development leading to the discovery of several small molecule antagonists. Given the large size and predominantly extracellular receptor interaction of endogenous chemokines, small molecules often act more deeply in an allosteric mode. However, opposed to the well described molecular interaction of allosteric modulators in class C 7-transmembrane helix (7TM) receptors, the interaction in class A, to which the chemokine receptors belong, is more sparsely described. Using the CCR5 chemokine receptor as a model system, we studied the molecular interaction and conformational interchange required for proper action of various orthosteric chemokines and allosteric small molecules, including the well known CCR5 antagonists TAK-779, SCH-C, and aplaviroc, and four novel CCR5 ago-allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5 chemokines (CCL3 and CCL5), with CCR2-like high affinities and potencies throughout the CCR5 signaling unit. Concomitantly, high affinity binding of small molecule CCR5 agonists and antagonists was retained in the transmembrane region. Importantly, whereas the agonistic and antagonistic properties were preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units.     

The role of the DRY motif of human MC4R for receptor activation

We constructed several mutant human MC4R cDNAs by site directed mutagenesis and expressed these receptors in COS-1 cells. The conserved DRY motif among GPCRs was mutated to generate eight mutants. While no MC4R ligand binding was detected in any of the mutants, one mutant, D146A, resulted in higher cAMP production in cells than the wild-type receptor without ligand stimulation.     

Functional role of the "ionic lock"--an interhelical hydrogen-bond network in family A heptahelical receptors

Activation of family A G-protein-coupled receptors involves a rearrangement of a conserved interhelical cytoplasmic hydrogen bond network between the E(D)RY motif on transmembrane helix 3 (H3) and residues on H6, which is commonly termed the cytoplasmic “ionic lock.” Glu134^3.49 of the E(D)RY motif also forms an intrahelical salt bridge with neighboring Arg135^3.50 in the dark-state crystal structure of rhodopsin. We examined the roles of Glu134^3.49 and Arg135^3.50 on H3 and Glu247^6.30 and Glu249^6.32 on H6 on the activation of rhodopsin using Fourier transform infrared spectroscopy of wild-type and mutant pigments reconstituted into lipid membranes. Activation of rhodopsin is pH-dependent with proton uptake during the transition from the inactive Meta I to the active Meta II state. Glu134^3.49 of the ERY motif is identified as the proton-accepting group, using the Fourier transform infrared protonation signature and the absence of a pH dependence of activation in the E134Q mutant. Neutralization of Arg135^3.50 similarly leads to pH-independent receptor activation, but with structural alterations in the Meta II state. Neutralization of Glu247^6.30 and Glu249^6.32 on H6, which are involved in interhelical interactions with H3 and H7, respectively, led to a shift toward Meta II in the E247Q and E249Q mutants while retaining the pH sensitivity of the equilibrium. Disruption of the interhelical interaction of Glu247^6.30 and Glu249^6.32 on H6 with H3 and H7 plays its role during receptor activation, but neutralization of the intrahelical salt bridge between Glu134^3.49 and Arg135^3.50 is considerably more critical for shifting the photoproduct equilibrium to the active conformation. These conclusions are discussed in the context of recent structural data of the β₂-adrenergic receptor.     

Full and Partial Agonists of Thromboxane Prostanoid Receptor Unveil Fine Tuning of Receptor Superactive Conformation and G Protein Activation

The intrahelical salt bridge between E/D^3.49 and R^3.50 within the E/DRY motif on helix 3 (H3) and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E^3.49/6.30 in the thromboxane prostanoid (TP) receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA) with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET) indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow “resistant” to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.     

Computationally‐predicted CB1 cannabinoid receptor mutants show distinct patterns of salt‐bridges that correlate with their level of constitutive activity reflected in G protein coupling levels,thermal stability,and ligand binding

The cannabinoid receptor 1 (CB1), a member of the class A G‐protein‐coupled receptor (GPCR) family, possesses an observable level of constitutive activity. Its activation mechanism, however, has yet to be elucidated. Previously we discovered dramatic changes in CB1 activity due to single mutations; T3.46A, which made the receptor inactive, and T3.46I and L3.43A, which made it essentially fully constitutively active. Our subsequent prediction of the structures of these mutant receptors indicated that these changes in activity are explained in terms of the pattern of salt‐bridges in the receptor region involving transmembrane domains 2, 3, 5, and 6. Here we identified key salt‐bridges, R2.37 + D6.30 and D2.63 + K3.28, critical for CB1 inactive and active states, respectively, and generated new mutant receptors that we predicted would change CB1 activity by either precluding or promoting these interactions. We find that breaking the R2.37 + D6.30 salt‐bridge resulted in substantial increase in G‐protein coupling activity and reduced thermal stability relative to the wild‐type reflecting the changes in constitutive activity from inactive to active. In contrast, breaking the D2.63 + K3.28 salt‐bridge produced the opposite profile suggesting this interaction is critical for the receptor activation. Thus, we demonstrate an excellent correlation with the predicted pattern of key salt‐bridges and experimental levels of activity and conformational flexibility. These results are also consistent with the extended ternary complex model with respect to shifts in agonist and inverse agonist affinity and provide a powerful framework for understanding the molecular basis for the multiple stages of CB1 activation and that of other GPCRs in general. Proteins 2013; 81:1304–1317. © 2013 Wiley Periodicals, Inc.