The all-trans-15-syn-retinal chromophore of metarhodopsin III is a partial agonist and not an inverse agonist

Meta III is formed during the decay of rhodopsin's active receptor state at neutral to alkaline pH by thermal isomerization of the retinal Schiff base C15=N bond, converting the ligand from all-trans 15-anti to all-trans 15-syn. The thereby induced change of ligand geometry switches the receptor to an inactive conformation, such that the decay pathway to Meta III contributes to the deactivation of the signaling state at higher pH values. We have examined the conformation of Meta III over a wider pH range and found that Meta III exists in a pH-dependent conformational equilibrium between this inactive conformation at neutral to alkaline pH and an active conformation similar to that of Meta II, which, however, is assumed at very acidic pH only. The apparent pKa of this transition is around 5.1 and thus several units lower than that of the Meta I/Meta II photoproduct equilibrium with its all-trans 15-anti ligand, but still about 1 unit higher than that of the opsin conformational equilibrium in the absence of ligand. The all-trans-15-syn-retinal chromophore is therefore not an inverse agonist like 11-cis- or 9-cis-retinal, which lock the receptor in an inactive conformation, but a classical partial agonist, which is capable of activating the receptor, yet with an efficiency considerably lower than the full agonist all-trans 15-anti. As the Meta III chromophore differs structurally from this full agonist only in the isomeric state of the C15=N bond, this ligand represents an excellent model system to study principal mechanisms of partial agonism which are helpful to understand the partial agonist behavior of other ligands.     

Changes in conformation at the cytoplasmic ends of the fifth and sixth transmembrane helices of a yeast G protein-coupled receptor in response to ligand binding

The third intracellular loop (IL3) of G protein-coupled receptors (GPCRs) is an important contact domain between GPCRs and their G proteins. Previously, the IL3 of Ste2p, a Saccharomyces cerevisiae GPCR, was suggested to undergo a conformational change upon activation as detected by differential protease susceptibility in the presence and absence of ligand. In this study using disulfide cross-linking experiments we show that the Ste2p cytoplasmic ends of helix 5 (TM5) and helix 6 (TM6) that flank the amino and carboxyl sides of IL3 undergo conformational changes upon ligand binding, whereas the center of the IL3 loop does not. Single Cys substitution of residues in the middle of IL3 led to receptors that formed high levels of cross-linked Ste2p, whereas Cys substitution at the interface of IL3 and the contiguous cytoplasmic ends of TM5 and TM6 resulted in minimal disulfide-mediated cross-linked receptor. The alternating pattern of residues involved in cross-linking suggested the presence of a 3(10) helix in the middle of IL3. Agonist (WHWLQLKPGQPNleY) induced Ste2p activation reduced cross-linking mediated by Cys substitutions at the cytoplasmic ends of TM5 and TM6 but not by residues in the middle of IL3. Thus, the cytoplasmic ends of TM5 and TM6 undergo conformational change upon ligand binding. An α-factor antagonist (des-Trp, des-His-α-factor) did not influence disulfide-mediated Ste2p cross-linking, suggesting that the interaction of the N-terminus of α-factor with Ste2p is critical for inducing conformational changes at TM5 and TM6. We propose that the changes in conformation revealed for residues at the ends of TM5 and TM6 are affected by the presence of G protein but not G protein activation. This study provides new information about role of specific residues of a GPCR in signal transduction and how peptide ligand binding activates the receptor.     

Galpha-subunits differentially alter the conformation and agonist affinity of kappa-opioid receptors

Although ligand-induced conformational changes in G protein-coupled receptors (GPCRs) are well-documented, there is little direct evidence for G protein-induced changes in GPCR conformation. To investigate this possibility, the effects of overexpressing Galpha-subunits (Galpha16 or Galphai2) with the kappa-opioid receptor (KOR) were examined. The changes in KOR conformation were subequently examined via the substituted cysteine accessibility method (SCAM) in transmembrane domains 6 (TM6) and 7 (TM7) and extracellular loop 2 (EL2). Significant conformational changes were observed on TM7, the extracellular portion of TM6, and EL2. Seven SCAM-sensitive residues (S3107.33, F3147.37, and I3167.39 to Y3207.43) on TM7 presented a cluster pattern when the KOR was exposed to baseline amounts of G protein, and additional residues became sensitive upon overexpression of various G proteins. In TM7, S3117.34 and N3267.49 were found to be sensitive in Galpha16-overexpressed cells and Y3137.36, N3227.45, S3237.46, and L3297.52 in Galphai2-overexpressed cells. In addition, the degree of sensitivity for various TM7 residues was augmented, especially in Galphai2-overexpressed cells. A similar phenomenon was also observed for residues in TM6 and EL2. In addition to an enhanced sensitivity of certain residues, our findings also indicated that a slight rotation was predicted to occur in the upper part of TM7 upon G protein overexpression. These relatively modest conformational changes engendered by G protein overexpression had both profound and differential effects on the abilities of agonists to bind to KOR. These data are significant because they demonstrate that Galpha-subunits differentially modulate the conformation and agonist affinity of a prototypical GPCR.     

Molecular mechanism underlying inverse agonist of angiotensin II type 1 receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.     

Ligand-stabilized conformational states of human beta(2) adrenergic receptor: insight into G-protein-coupled receptor activation

G-protein-coupled receptors (GPCRs) are known to exist in dynamic equilibrium between inactive- and several active-state conformations, even in the absence of a ligand. Recent experimental studies on the β₂ adrenergic receptor (β₂AR) indicate that structurally different ligands with varying efficacies trigger distinct conformational changes and stabilize different receptor conformations. We have developed a computational method to study the ligand-induced rotational orientation changes in the transmembrane helices of GPCRs. This method involves a systematic spanning of the rotational orientation of the transmembrane helices (TMs) that are in the vicinity of the ligand for predicting the helical rotations that occur on ligand binding. The predicted ligand-stabilized receptor conformations are characterized by a simultaneous lowering of the ligand binding energy and a significant gain in interhelical and receptor-ligand hydrogen bonds. Using the β₂AR as a model, we show that the receptor conformational state depends on the structure and efficacy of the ligand for a given signaling pathway. We have studied the ligand-stabilized receptor conformations of five different ligands, a full agonist, norepinephrine; a partial agonist, salbutamol; a weak partial agonist, dopamine; a very weak agonist, catechol; and an inverse agonist, ICI-115881. The predicted ligand-stabilized receptor models correlate well with the experimentally observed conformational switches in β₂AR, namely, the breaking of the ionic lock between R131^3.50 at the intracellular end of TM3 (part of the DRY motif) and E268^6.30 on TM6, and the rotamer toggle switch on W286^6.48 on TM6. In agreement with trp-bimane quenching experiments, we found that norepinephrine and dopamine break the ionic lock and engage the rotamer toggle switch, whereas salbutamol, a noncatechol partial agonist only breaks the ionic lock, and the weak agonist catechol only engages the rotamer toggle switch. Norepinephrine and dopamine occupy the same binding region, between TM3, TM5, and TM6, whereas the binding site of salbutamol is shifted toward TM4. Catechol binds deeper into the protein cavity compared to the other ligands, making contact with TM5 and TM6. A part of the catechol binding site overlaps with those of dopamine and norepinephrine but not with that of salbutamol. Virtual ligand screening on 10,060 ligands on the norepinephrine-stabilized receptor conformation shows an enrichment of 38% compared to ligand unbound receptor conformation. These results show that ligand-induced conformational changes are important for developing functionally specific drugs that will stabilize a particular receptor conformation. These studies represent the first step toward a more universally applicable computational method for studying ligand efficacy and GPCR activation.     

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.     

Ligand-specific changes in M3 muscarinic acetylcholine receptor structure detected by a disulfide scanning strategy

G protein-coupled receptor (GPCR) function can be modulated by different classes of ligands including full and inverse agonists. At present, little is known about the conformational changes that agonist ligands induce in their target GPCRs. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced structural changes in a series of cysteine (Cys)-substituted mutant M 3 muscarinic acetylcholine receptors. One of our goals was to study whether the cytoplasmic end of transmembrane domain V (TM V), a region known to be critically involved in receptor/G protein coupling, undergoes a major conformational change, similar to the adjacent region of TM VI. Another goal was to determine and compare the disulfide cross-linking patterns observed after treatment of the different mutant receptors with full versus inverse muscarinic agonists. Specifically, we generated 20 double Cys mutant M 3 receptors harboring one Cys substitution within the cytoplasmic end of TM V (L249-I253) and a second one within the cytoplasmic end of TM VI (A489-L492). These receptors were transiently expressed in COS-7 cells and subsequently characterized in pharmacological and disulfide cross-linking studies. Our cross-linking data, in conjunction with a three-dimensional model of the M 3 muscarinic receptor, indicate that M 3 receptor activation does not trigger major structural disturbances within the cytoplasmic segment of TM V, in contrast to the pronounced structural changes predicted to occur at the cytoplasmic end of TM VI. We also demonstrated that full and inverse muscarinic agonists had distinct effects on the efficiency of disulfide bond formation in specific double Cys mutant M 3 receptors. The present study provides novel information about the dynamic changes that accompany M 3 receptor activation and how the receptor conformations induced (or stabilized) by full versus inverse muscarinic agonists differ from each other at the molecular level. Because all class I GPCRs are predicted to share a similar transmembrane topology, the conclusions drawn from the present study should be of broad general relevance.     

Role of the extracellular amino terminus and first membrane-spanning helix of dopamine D1 and D5 receptors in shaping ligand selectivity and efficacy

Transmembrane (TM) helices of human D1-like dopaminergic receptors (hD1R and hD5R) harbor the same residues implicated in ligand binding and activation of catecholamine G protein-coupled receptors (GPCRs). Yet, hD1R and hD5R naturally display the distinct functional properties shared by wild type and constitutively active mutant GPCRs, respectively. Interestingly, we show in the present study that a class of synthetic phenylbenzazepine agonists containing a methyl on the azepine ring exhibited lower affinity for the more constitutively activated hD5R. These results cannot be explained by the “allosteric ternary complex model” postulating a higher agonist affinity for constitutively active GPCRs. We have also explored the functional role of distinct extracellular amino terminus (NT) and TM1 regions of hD1R and hD5R using a chimerical approach. Of these two regions, our studies suggest that TM1 predominantly shapes D1-like ligand affinity and selectivity. Additionally, NT and TM1 of hD1R and hD5R play no role in receptor constitutive activity but differentially modulate dopamine-mediated responsiveness. The TM1 exchange mediated drastic changes in intrinsic efficacy and activity of phenylbenzazepine drugs displaying partial agonism at hD1R and hD5R. Phenylbenzazepines were converted into strong partial agonists or full agonists in cells expressing hD1R-TM1^D5 chimera while being switched from full agonists to partial agonists and partial agonists to antagonists in cells harboring hD5R-TM1^D1 chimera. TM1 exchange had no effect on antipsychotic-mediated inverse agonism. In summary, our study shows that NT and TM1 of D1-like receptors control ligand binding and agonist-induced activation, poising these regions as important structural determinants for catecholamine GPCR function.     

Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa(2),Ile(5),Trp(23),Tyr(36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-l-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked to hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu(11),d-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.     

Long Range Effect of Mutations on Specific Conformational Changes in the Extracellular Loop 2 of Angiotensin II Type 1 Receptor

The topology of the second extracellular loop (ECL2) and its interaction with ligands is unique in each G protein-coupled receptor. When the orthosteric ligand pocket located in the transmembrane (TM) domain is occupied, ligand-specific conformational changes occur in the ECL2. In more than 90% of G protein-coupled receptors, ECL2 is tethered to the third TM helix via a disulfide bond. Therefore, understanding the extent to which the TM domain and ECL2 conformations are coupled is useful. To investigate this, we examined conformational changes in ECL2 of the angiotensin II type 1 receptor (AT1R) by introducing mutations in distant sites that alter the activation state equilibrium of the AT1R. Differential accessibility of reporter cysteines introduced at four conformation-sensitive sites in ECL2 of these mutants was measured. Binding of the agonist angiotensin II (AngII) and inverse agonist losartan in wild-type AT1R changed the accessibility of reporter cysteines, and the pattern was consistent with ligand-specific “lid” conformations of ECL2. Without agonist stimulation, the ECL2 in the gain of function mutant N111G assumed a lid conformation similar to AngII-bound wild-type AT1R. In the presence of inverse agonists, the conformation of ECL2 in the N111G mutant was similar to the inactive state of wild-type AT1R. In contrast, AngII did not induce a lid conformation in ECL2 in the loss of function D281A mutant, which is consistent with the reduced AngII binding affinity in this mutant. However, a lid conformation was induced by [Sar¹,Gln²,Ile⁸] AngII, a specific analog that binds to the D281A mutant with better affinity than AngII. These results provide evidence for the emerging paradigm of domain coupling facilitated by long range interactions at distant sites on the same receptor.     

Structural insights into agonist-induced activation of G-protein-coupled receptors

Recent years have seen tremendous breakthroughs in structure determination of G-protein-coupled receptors (GPCRs). In 2011, two agonist-bound active-state structures of rhodopsin have been published. Together with structures of several rhodopsin activation intermediates and a wealth of biochemical and spectroscopic information, they provide a unique structural framework on which to understand GPCR activation. Here we use this framework to compare the recent crystal structures of the agonist-bound active states of the β(2) adrenergic receptor (β(2)AR) and the A(2A) adenosine receptor (A(2A)AR). While activation of these three GPCRs results in rearrangements of TM5 and TM6, the extent of this conformational change varies considerably. Displacements of the cytoplasmic side of TM6 ranges between 3 and 8? depending on whether selective stabilizers of the active conformation are used (i.e. a G-protein peptide in the case of rhodopsin or a conformationally selective nanobody in the case of the β(2)AR) or not (A(2A)AR). The agonist-induced conformational changes in the ligand-binding pocket are largely receptor specific due to the different chemical nature of the agonists. However, several similarities can be observed, including a relocation of conserved residues W6.48 and F6.44 towards L5.51 and P5.50, and of I/L3.40 away from P5.50. This transmission switch links agonist binding to the movement of TM5 and TM6 through the rearrangement of the TM3-TM5-TM6 interface, and possibly constitutes a common theme of GPCR activation.     

Molecular basis of melanocortin-4 receptor for AGRP inverse agonism

We have investigated receptor structural components of the melanocortin-4 receptor (MC4R) responsible for ligand-dependent inverse agonism. We utilized agouti-related protein (AGRP), an inverse agonist which reduces MC4R basal cAMP production, as a tool to determine the molecular mechanism. We tested a series of chimeric receptors and utilized MC4R and MC1R as templates, in which AGRP is an inverse agonist for MC4R but not for MC1R. Our results indicate that replacements of the extracellular loops 1, 2 and 3 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity. However, replacement of the N terminus of MC4R with the same region of MC1R decreases AGRP inverse agonism. Replacement of transmembrane domains 3, 4, 5 and 6 of MC4R with the corresponding regions of MC1R did not affect AGRP inverse agonist activity but mutation of D90A in transmembrane 2 (TM2) and D298A in TM7 abolished AGRP inverse activity. Deletion of the distal MC4R C terminus fails to maintain AGRP mediated reduction in basal cAMP production although it maintains NDP-MSH mediated cAMP production. In conclusion, our results indicate that the N terminus and the distal C terminus of MC4R do appear to play important roles in AGRP inverse agonism but not NDP-MSH mediated receptor activation. Our results also indicate that the residues D90 in TM2 and D298 in TM7 of hMC4R are involved in not only NDP-MSH mediated receptor activation but also AGRP mediated inverse agonism.     

Molecular characterization of a purified 5-HT4 receptor: a structural basis for drug efficacy

Serotonin 5-HT(4(a)) receptor, a G-protein-coupled receptor (GPCR), was produced as a functional isolated protein using Escherichia coli as an expression system. The isolated receptor was characterized at the molecular level by circular dichroism (CD) and steady-state fluorescence. A specific change in the near-UV CD band associated with the GPCR disulfide bond connecting the third transmembrane domain to the second extracellular loop (e2) was observed upon agonist binding to the purified receptor. This is a direct experimental evidence for a change in the conformation of the e2 loop upon receptor activation. Different variations were obtained depending whether the ligand was an agonist (partial or full) or an inverse agonist. In contrast, antagonist binding did not induce any variation. These observations provide a first direct evidence for the fact that free (or antagonist-occupied), active (partial- or full agonist-occupied) and silent (inverse agonist-occupied) states of the receptor involve different arrangements of the e2 loop. Finally, ligand-induced changes in the fluorescence emission profile of the purified receptor confirmed that the partial agonist stabilized a single, well-defined, conformational state and not a mixture of different states. This result is of particular interest in a pharmacological perspective since it directly demonstrates that the efficacy of a drug is likely due to the stabilization of a ligand-specific state rather than selection of a mixture of different conformational states of the receptor.     

Identification of an agonist-induced conformational change occurring adjacent to the ligand-binding pocket of the M(3) muscarinic acetylcholine receptor

To study the conformational changes that convert G protein-coupled receptors (GPCRs) from their resting to their active state, we used the M(3) muscarinic acetylcholine receptor, a prototypical class A GPCR, as a model system. Specifically, we employed a recently developed in situ disulfide cross-linking strategy that allows the formation of disulfide bonds in Cys-substituted mutant M(3) muscarinic receptors present in their native membrane environment. At present, little is known about the conformational changes that GPCR ligands induce in the immediate vicinity of the ligand-binding pocket. To address this issue, we generated 11 Cys-substituted mutant M(3) muscarinic receptors and characterized these receptors in transfected COS-7 cells. All analyzed mutant receptors contained an endogenous Cys residue (Cys-532(7.42)) located within the exofacial segment of transmembrane domain (TM) VII, close to the agonist-binding site. In addition, all mutant receptors harbored a second Cys residue that was introduced into the exofacial segment of TM III, within the sequence Leu-142(3.27)-Asn-152(3.37). Disulfide cross-linking studies showed that muscarinic agonists, but not antagonists, promoted the formation of a disulfide bond between S151(3.36)C and Cys-532. A three-dimensional model of the inactive state of the M(3) muscarinic receptor indicated that Cys-532 and Ser-151 face each other in the center of the TM receptor core. Our cross-linking data therefore support the concept that agonist activation pulls the exofacial segments of TMs VII and III closer to each other. This structural change may represent one of the early conformational events triggering the more pronounced structural reorganization of the intracellular receptor surface. To the best of our knowledge, this is the first direct demonstration of a conformational change occurring in the immediate vicinity of the binding site of a GPCR activated by a diffusible ligand.     

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights into agonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.     

Identification of an efficacy switch region in the ghrelin receptor responsible for interchange between agonism and inverse agonism

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation.     

Both ligand- and cell-specific parameters control ligand agonism in a kinetic model of g protein-coupled receptor signaling

G protein-coupled receptors (GPCRs) exist in multiple dynamic states (e.g., ligand-bound, inactive, G protein-coupled) that influence G protein activation and ultimately response generation. In quantitative models of GPCR signaling that incorporate these varied states, parameter values are often uncharacterized or varied over large ranges, making identification of important parameters and signaling outcomes difficult to intuit. Here we identify the ligand- and cell-specific parameters that are important determinants of cell-response behavior in a dynamic model of GPCR signaling using parameter variation and sensitivity analysis. The character of response (i.e., positive/neutral/inverse agonism) is, not surprisingly, significantly influenced by a ligand's ability to bias the receptor into an active conformation. We also find that several cell-specific parameters, including the ratio of active to inactive receptor species, the rate constant for G protein activation, and expression levels of receptors and G proteins also dramatically influence agonism. Expressing either receptor or G protein in numbers several fold above or below endogenous levels may result in system behavior inconsistent with that measured in endogenous systems. Finally, small variations in cell-specific parameters identified by sensitivity analysis as significant determinants of response behavior are found to change ligand-induced responses from positive to negative, a phenomenon termed protean agonism. Our findings offer an explanation for protean agonism reported in beta2--adrenergic and alpha2A-adrenergic receptor systems.     

Constitutive activation of angiotensin II type 1 receptor alters the orientation of transmembrane Helix-2

A key step in transmembrane (TM) signal transduction by G-protein-coupled receptors (GPCRs) is the ligand-induced conformational change of the receptor, which triggers the activation of a guanine nucleotide-binding protein. GPCRs contain a seven-TM helical structure essential for signal transduction in response to a large variety of sensory and hormonal signals. Primary structure comparison of GPCRs has shown that the second TM helix contains a highly conserved Asp residue, which is critical for agonist activation in these receptors. How conformational changes in TM2 relate to signal transduction by a GPCR is not known, because activation-induced conformational changes in TM2 helix have not been measured. Here we use modification of reporter cysteines to measure water accessibility at specific residues in TM2 of the type 1 receptor for the octapeptide hormone angiotensin II. Activation-dependent changes in the accessibility of Cys76 on TM2 were measured in constitutively activated mutants. These changes were directly correlated with measurement of function, establishing the link between physical changes in TM2 and function. Accessibility changes were measured at several consecutive residues on TM2, which suggest that TM2 undergoes a transmembrane movement in response to activation. This is the first report of in situ measurement of TM2 movement in a GPCR.     

Direct assessment of CXCR4 mutant conformations reveals complex link between receptor structure and G(alpha)(i) activation

Ligand binding to G protein-coupled receptors (GPCRs) is thought to induce changes in receptor conformation that translate into activation of downstream effectors. The link between receptor conformation and activity is still insufficiently understood, as current models of GPCR activation fail to take an increasing amount of experimental data into account. To elucidate structure-function relationships in GPCR activation, we used bioluminescence resonance energy transfer to directly assess the conformation of mutants of the chemokine receptor CXCR4. We analyzed substitutions in the arginine cage DRY motif and in the conserved asparagine N(3.35)119, which are pivotal molecular switches for receptor conformation and activation. G(alpha)(i) activation of the mutants was either similar to wild-type CXCR4 (D133N, Y135A, and N119D) or resulted in loss of activity (R134A and N119K). Mutant N119S was constitutively active but further activated by agonist. Bioluminescence resonance energy transfer analysis suggested no simple correlation between conformational changes in response to ligand binding and activation of G(alpha)(i) by the mutants. Different conformations of active receptors were detected (for wild-type CXCR4, D133N, and N119S), suggesting that different receptor conformations are able to trigger G(alpha)(i) activity. Several conformations were also found for inactive mutants. These data provide biophysical evidence for different receptor conformations being active with respect to a single readout. They support models of GPCR structure-activity relationships that take this conformational flexibility of active receptors into account.