Low affinity of beta-adrenergic receptors for agonists on intact cells is not due to receptor sequestration

The low affinity of beta-adrenergic receptors for agonists described on intact cells at 37 degrees C has usually been interpreted in terms of reduced accessibility of agonists (which are usually hydrophilic) for sequestered receptors. We challenged this hypothesis by eliminating the plasma membrane barrier with low doses of the detergent digitonin. In human mononuclear leukocytes (MNL) permeabilized with digitonin, sequestered receptors became accessible to hydrophilic ligands such as agonists, but the affinity was still low. Then we investigated the relationship between low affinity agonist binding and sequestration using concanavalin A, which blocks sequestration. Even when sequestration was blocked, the affinity of the beta-adrenergic receptors for agonists was low. We conclude that: (a) low affinity agonist binding is independent of receptor sequestration; (b) the receptors which undergo conformational change are those that are sequestered; (c) the low affinity appears before sequestration occurs. This receptor conformational change could be the first step in agonist-induced desensitization.     

A Fluorescence Resonance Energy Transfer-based M2 Muscarinic Receptor Sensor Reveals Rapid Kinetics of Allosteric Modulation

Allosteric modulators have been identified for several G protein-coupled receptors, most notably muscarinic receptors. To study their mechanism of action, we made use of a recently developed technique to generate fluorescence resonance energy transfer (FRET)-based sensors to monitor G protein-coupled receptor activation. Cyan fluorescent protein was fused to the C terminus of the M₂ muscarinic receptor, and a specific binding sequence for the small fluorescent compound fluorescein arsenical hairpin binder, FlAsH, was inserted into the third intracellular loop; the latter site was labeled in intact cells by incubation with FlAsH. We then measured FRET between the donor cyan fluorescent protein and the acceptor FlAsH in intact cells and monitored its changes in real time. Agonists such as acetylcholine and carbachol induced rapid changes in FRET, indicative of agonist-induced conformational changes. Removal of the agonists or addition of an antagonist caused a reversal of this signal with rate constants between 400 and 1100 ms. The allosteric ligands gallamine and dimethyl-W84 caused no changes in FRET when given alone, but increased FRET when given in the presence of an agonist, compatible with an inactivation of the receptors. The kinetics of these effects were very rapid, with rate constants of 80–100 ms and ≈200 ms for saturating concentrations of gallamine and dimethyl-W84, respectively. Because these speeds are significantly faster than the responses to antagonists, these data indicate that gallamine and dimethyl-W84 are allosteric ligands and actively induce a conformation of the M₂ receptor with a reduced affinity for its agonists.     

Conformational changes at the agonist binding domain of the N-methyl-D-aspartic acid receptor

The conformational changes in the agonist binding domain of the glycine-binding GluN1 and glutamate-binding GluN2A subunits of the N-methyl D-aspartic acid receptor upon binding agonists of varying efficacy have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances indicate a cleft closure conformational change at the GluN1 subunit upon binding agonists; however, no significant changes in the cleft closure are observed between partial and full agonists. This is consistent with the previously reported crystal structures for the isolated agonist binding domain of this receptor. Additionally, the LRET-based distances show that the agonist binding domain of the glutamate-binding GluN2A subunit exhibits a graded cleft closure with the extent of cleft closure being proportional to the extent of activation, indicating that the mechanism of activation in this subunit is similar to that of the glutamate binding α-amino-5-methyl-3-hydroxy-4-isoxazole propionate and kainate subtypes of the ionotropic glutamate receptors.     

Molecular basis of partial agonism at the neurotransmitter alpha2A-adrenergic receptor and Gi-protein heterotrimer

To characterize the mechanism by which heterotrimeric G-proteins interpret the signals coming from various neurotransmitters of diverse efficacies (agonists and partial agonists) acting on alpha(2A)-adrenergic receptors, we used a fluorescent resonance energy transfer-based approach to study the effects of these partial agonists on the activation process of both the alpha(2A)-adrenergic receptor and its cognate G(i)-protein. We show that ligands of different efficacies switch the receptor into distinct conformational states, which in turn set the speed and extent of the G(i)-protein signaling. Thus, in cells the efficacy by which a receptor responds to diverse ligands is caused by the ability of the G-protein to differentiate between distinct receptor conformations. The data provide a new key characteristic underlying the mechanism of partial agonism at G-protein-coupled receptors.     

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

The concept of “functional selectivity” or “biased signaling” suggests that a ligand can have distinct efficacies with regard to different signaling pathways. We have investigated the question of whether biased signaling may be related to distinct agonist-induced conformational changes in receptors using the β₂-adrenergic receptor (β₂AR) and its two endogenous ligands epinephrine and norepinephrine as a model system. Agonist-induced conformational changes were determined in a fluorescently tagged β₂AR FRET sensor. In this β₂AR sensor, norepinephrine caused signals that amounted to only ≈50% of those induced by epinephrine and the standard “full” agonist isoproterenol. Furthermore, norepinephrine-induced changes in the β₂AR FRET sensor were slower than those induced by epinephrine (rate constants, 47 versus 128 ms). A similar partial β₂AR activation signal was revealed for the synthetic agonists fenoterol and terbutaline. However, norepinephrine was almost as efficient as epinephrine (and isoproterenol) in causing activation of G_s and adenylyl cyclase. In contrast, fenoterol was quite efficient in triggering β-arrestin2 recruitment to the cell surface and its interaction with β₂AR, as well as internalization of the receptors, whereas norepinephrine caused partial and slow changes in these assays. We conclude that partial agonism of norepinephrine at the β₂AR is related to the induction of a different active conformation and that this conformation is efficient in signaling to G_s and less efficient in signaling to β-arrestin2. These observations extend the concept of biased signaling to the endogenous agonists of the β₂AR and link it to distinct conformational changes in the receptor.     

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.     

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists

Background and Objective

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M₁ mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).

Methods

Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M₁ mAChR, respectively. The optimized FRET receptor construct (M₁-cam5) was expressed stably in HEK293 cells.

Results

The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα_q/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M₁ FRET (F_EYFP/F_ECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.

Conclusion

The M₁ FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M₁ mAChR activation.     

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.     

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.     

Cooperative conformational changes in a G-protein-coupled receptor dimer, the leukotriene B(4) receptor BLT1

We have used an isolated receptor, the leukotriene B(4) receptor BLT1, to analyze the mechanism of receptor activation in a G-protein-coupled receptor dimer. The isolated receptor is essentially a dimer whether the agonist is present or not, provided the detergent used stabilizes the inactive dimeric assembly. We have produced a receptor mutant where Cys(97) in the third transmembrane domain has been replaced by a serine. This mutation leads to an approximately 100-fold decrease in the affinity for the agonist. 5-Hydroxytryptophan has then been introduced at position 234 in the C97A mutant sixth transmembrane domain. Agonist binding to the labeled receptor is associated with variations in the fluorescence properties of 5-hydroxytryptophan due to specific agonist-induced conformational changes. The C97A mutant labeled with 5-hydroxytryptophan has then been associated with a wild-type receptor in a dimeric complex that has been subsequently purified. The purified complex activates its G-protein partner in a similar manner as the wild-type homodimer. Due to the difference in the affinity for the agonist between the wild-type and mutant protomers in this dimer, we have been able to reach a state where one of the protomers, the mutant, is in its unliganded state, whereas the other, the wild type, is loaded with the agonist. We show that agonist binding to the wild-type receptor induces specific changes in the conformation of the unliganded protomer, as evidenced by the variations in the emission of the 5-hydroxytryptophan residue in the mutant receptor. These data provide a direct demonstration for agonist-induced cooperative conformational changes in a GPCR dimer.     

A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines

Insect cells are an underexplored resource for functional G-protein-coupled receptor (GPCR) assays, despite a strong record in biochemical (binding) assays. Here we describe the use of vectors capable of creating stably transformed insect cell lines to generate a cell-based functional GPCR assay. This assay employs the luminescent photoprotein aequorin and the promiscuous G-protein subunit Galpha16 and is broadly applicable to human GPCRs. We demonstrate that the assay can quantitate ligand concentration-activity relationships for seven different human GPCRs, can differentiate between partial and full agonists, and can determine rank order potencies for both agonists and antagonists that match those seen with other assay systems. Human Galpha16 improves signal strength but is not required for activity with some receptors. The coexpression of human and bovine betagamma subunits and/or phospholipase Cbeta makes no difference to agonist efficacy or potency. Two different receptors expressed in the same cell line respond to their specific agonists, and two different cell lines (Sf9 and High 5) are able to functionally detect the same expressed GPCR. Sf9 cells have the capability to produce fully functional human receptors, allied to a low background of endogenous receptors, and so are a valuable system for investigating orphan GPCRs and receptor dimerization.     

Conformation state-sensitive antibodies to G-protein-coupled receptors

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.     

Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.     

Measurement of changes in fluorescence resonance energy transfer between gonadotropin-releasing hormone receptors in response to agonists

Oligomerization of membrane-bound G-protein-coupled receptors has recently emerged as an important step in cellular signaling. Fluorescence resonance energy transfer (FRET) has undergone a revival as the method of choice for demonstrating in vivo protein-protein interactions and receptor dimerization. We have used chimeras of gonadotropin-releasing harmone (GnRH) receptors and various fluorescent proteins to investigate receptor dimerization in relation to receptor activation. Two pairs of FRET-compatible fluorescent proteins were used: sapphire with topaz, and enhanced green fluorescent protein (eGFP) with dsRed. Changes in the ratio between acceptor and donor fluorescence were measured after addition of buserelin, a GnRH agonist, and antide, a GnRH antagonist. For both pairs of fluorescent proteins, an increase in the ratio of acceptor to donor intensities was observed immediately after addition of buserelin as would be predicted if FRET occurred due to the microaggregation of receptors conjugated with different fluorescent proteins. No change in FRET was observed in time for cells in medium or after addition of antide. The increase in FRET signal was not uniform throughout a cell.     

Agonist-bound structures of G protein-coupled receptors

G protein-coupled receptors (GPCRs) play a major role in intercellular communication by binding small diffusible ligands (agonists) at the extracellular surface. Agonist-binding induces a conformational change in the receptor, which results in the binding and activation of heterotrimeric G proteins within the cell. Ten agonist-bound structures of non-rhodopsin GPCRs published last year defined for the first time the molecular details of receptor activated states and how inverse agonists, partial agonists and full agonists bind to produce different effects on the receptor. In addition, the structure of the β(2)-adrenoceptor coupled to a heterotrimeric G protein showed how the opening of a cleft in the cytoplasmic face of the receptor as a consequence of agonist binding results in G protein coupling and activation of the G protein.     

Measurement of the millisecond activation switch of G protein-coupled receptors in living cells

Hormones and neurotransmitters transduce signals through G protein-coupled receptors (GPCR). Despite their common signaling pathways, however, the responses they elicit have different temporal patterns. To reveal the molecular basis for these differences we have developed a generally applicable fluorescence-based technique for real-time monitoring of the activation switch of GPCRs in living cells. We used such direct measurements to investigate the activation of the alpha(2A)-adrenergic receptor (alpha(2A)AR; neurotransmitter) and the parathyroid hormone receptor (PTHR; hormone) and observed much faster kinetics than expected: approximately 40 ms for the alpha(2A)AR and approximately 1 s for the PTHR. The different switch times are in agreement with the different receptors' biological functions. Agonists and antagonists could rapidly switch the receptors on or off, whereas a partial agonist caused only a partial signal. This approach allows the comparison of agonist and partial agonist intrinsic activities at the receptor level and provides evidence for millisecond activation times of GPCRs.     

Inverse, protean, and ligand-selective agonism: matters of receptor conformation.

Concepts regarding the mechanisms by which drugs activate receptors to produce physiological response have progressed beyond considering the receptor as a simple on-off switch. Current evidence suggests that the idea that agonists produce only varying degrees of receptor activation is obsolete and must be reconciled with data to show that agonist efficacy has texture as well as magnitude. Thus, agonists can block system constitutive response (inverse agonists), behave as positive and inverse agonists on the same receptor (protean agonists), and differ in the stimulus pattern they produce in physiological systems (ligand-selective agonists). The molecular mechanism for this seemingly diverse array of activities is the same, namely, the selective microaffinity of ligands for different conformational states of the receptor. This paper reviews evidence for the existence of the various types of agonism and the potential therapeutic utility of different agonist types.-Kenakin, T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation.     

Regulation of heptaspanning-membrane-receptor function by dimerization and clustering

G-protein-coupled receptors form homomers and heteromers; agonist-induced conformational changes within interacting receptors of the oligomer modify their pharmacology, signalling and/or trafficking. When these receptors are activated, the oligomers rearrange and cluster and a novel mechanism of receptor-operation regulation by oligomer intercommunication is possible. This intercommunication would be assisted by components of the plasma membrane and by scaffolding proteins. Receptor cross-sensitization, cross-desensitization and novel, integrated receptor responses can then develop between oligomeric receptor complexes of the cluster without direct contact between them. This concept gives a new perspective to the understanding of neurotransmission and neuronal plasticity.     

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.