Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 2. Stopped-flow studies with agonists and antagonists.

The kinetics of cholinergic ligand binding to membrane-bound acetylcholine receptor from Torpedo californica have been followed in a stopped-flow photometer, by using the fluorescent probe ethidium. The overall reaction amplitude, as a function of ligand concentration, can be fit to the law of mass action for both agonist and antagonists. All agonists show at least biphasic kinetics, and the concentration dependence of the kinetic parameters is fit by a common mechanism involving sequential binding of ligands with increasingly lower affinity. The receptor-ligand precomplexes isomerize to different noninterconvertible final complexes depending on the number of ligands bound. In contrast, the kinetics observed with antagonists cannot be fit to a common model. These kinetics are always much slower than those observed with agonists, and the relaxation rates depend only weakly on antagonist concentration.     

X-ray structures of progesterone receptor ligand binding domain in its agonist state reveal differing mechanisms for mixed profiles of 11β-substituted steroids

We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu(723). This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp(755), resulting in a decreased steric clash with Met(909) but inducing a new internal clash with Val(912) in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11β substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.     

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Model for the Effect of Estrogen Antagonists on Cooperative Estradiol Binding

Abstract

Partial agonists such as estriol and estrone have been reported to diminish or even eliminate the upward convexity of the Scatchard plot of the binding of labeled estradiol to estrogen receptor. This has been interpreted as agonist interference with the receptor dimerization induced by estradiol. In order to investigate how a partial agonist or antagonist might interfere with dimerization we have developed a theoretical mass-action law model, where soluble receptors can dimerize and bind to two different ligands. Special attention was devoted to manifestations of positive cooperativity to determine whether they could be modified by competition with a second ligand. This was done using a computer program that evaluated a large set of combinations of affinity constants in an effort to explore all possible situations. The model could reproduce the effect of a second ligand on the cooperative binding of estradiol to the estrogen receptor but only if the second ligand was anticooperative, which is not the case of estriol, estrone and tamoxifen. Furthermore, even when the Scatchard plot was linear, the model still required dimerization of the receptor in most of the cases, showing that the addition of an antagonist may eliminate the upward curvature of the Scatchard without truly eliminating dimerization or cooperativity. We conclude that the effect of a second ligand on the binding of labeled estradiol to estrogen receptor is not necessarily due to interference with dimerization and/or cooperativity. The inability of this model to fully explain the published data for estriol, estrone, clomiphene, and tamoxifen suggests that a more complex mechanism is involved.     

Mechanism of agonist and antagonist binding to alpha 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex

The alpha 2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the alpha 2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the alpha 2 AR in human platelet membranes on the second time scale. Binding of the alpha 2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 X 10(5) M-1 s-1 and a dissociation rate constant of 1.11 X 10(-3) s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the alpha 2 adrenergic full agonist [3H]UK 14,304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on [3H]UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant kappa + of 5 X 10(6) M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the alpha 2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the alpha 2 AR with the N protein or a slow, ligand-independent conformational change of the alpha 2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.     

Control and oscillation in ligand receptor interactions according to the law of mass action

The law of mass action is almost universally applied to interactions of both endogenous ligands and drugs with their specific receptors and results in the familiar hyperbolic equilibrium binding curve of bound (y) vs free (z) concentrations. Whereas the concentration of a drug molecule is governed by its pharmacokinetic properties and, possibly, by intrinsic control mechanisms, natural ligands are certainly controlled since their concentrations normally remain within specific limits. This paper represents a further study of control of this kinetic process in a model based on ligand production (rate F), first-order elimination (rate constant E) and a feedback function of occupancy, phi(y), that modulates these. In the controlled situation the system equilibrium occurs at states called critical points (yc,zc) at which both dy/dt and dz/dt are simultaneously zero. There are only a finite number of such points along the hyperbolic binding curve and these may be either stable or unstable. The basal state is the normal operating point of the system and is necessarily stable; that is, perturbations producing states away from it will return in time to this point. We have previously shown that phi'(y) less than or equal to 0 is a sufficient condition for stability. Accordingly, for a continuous control curve, an adjacent critical point will be unstable, and have phi'(y) greater than 0. If the system coordinates get sufficiently close to such an unstable point there is propulsion to extreme states and loss of control. The distance between the stable and unstable points determines whether a dose (or release) of the ligand will be controlled or not. The current paper focuses on the geometrical properties of the binding and control curves and how these relate to the stability of critical points and the overall control of ligand doses. In particular we show how the magnitude of the (negative) slope of the control curve at the basal point affects the frequency of oscillation about the basal state. It is further shown that high frequency control results in lower receptor occupancy, a result that may explain desensitization.     

Identification of two serine residues involved in agonist activation of the beta-adrenergic receptor

Pharmacophore mapping of the ligand binding domain of the beta-adrenergic receptor has revealed specific molecular interactions which are important for agonist and antagonist binding to the receptor. Previous site-directed mutagenesis experiments have demonstrated that the binding of amine agonists and antagonists to the receptor involves an interaction between the amine group of the ligand and the carboxylate side chain of Asp113 in the third hydrophobic domain of the receptor (Strader, C. D., Sigal, I. S., Candelore, M. R., Rands, E., Hill, W. S., and Dixon, R. A. F. (1988) J. Biol. Chem. 263, 10267-10271). We have now identified 2 serine residues, at positions 204 and 207 in the fifth hydrophobic domain of the beta-adrenergic receptor, which are critical for agonist binding and activation of the receptor. These serine residues are conserved with G-protein-coupled receptors which bind catecholamine agonists, but not with receptors whose endogenous ligands do not have the catechol moiety. Removal of the hydroxyl side chain from either Ser204 or Ser207 by substitution of the serine residue with an alanine attenuates the activity of catecholamine agonists at the receptor. The effects of these mutations on agonist activity are mimicked selectively by the removal of the catechol hydroxyl moieties from the aromatic ring of the agonist. The data suggest that the interaction of catecholamine agonists with the beta-adrenergic receptor involves two hydrogen bonds, one between the hydroxyl side chain of Ser204 and the meta-hydroxyl group of the ligand and a second between the hydroxyl side chain of Ser207 and the para-hydroxyl group of the ligand.     

The third cytoplasmic loop of a yeast G-protein-coupled receptor controls pathway activation, ligand discrimination, and receptor internalization.

To identify functional domains of G-protein-coupled receptors that control pathway activation, ligand discrimination, and receptor regulation, we have used as a model the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. From a collection of random mutations introduced in the region coding for the third cytoplasmic loop of Ste2p, six ste2sst alleles were identified by genetic screening methods that increased alpha-factor sensitivity 2.5- to 15-fold. The phenotypic effects of ste2sst and sst2 mutations were not additive, consistent with models in which the third cytoplasmic loop of the alpha-factor receptor and the regulatory protein Sst2p control related aspects of pheromone response and/or desensitization. Four ste2sst mutations did not dramatically alter cell surface expression or agonist binding affinity of the receptor; however, they did permit detectable responses to an alpha-factor antagonist. One ste2sst allele increased receptor binding affinity for alpha-factor and elicited stronger responses to antagonist. Results of competition binding experiments indicated that wild-type and representative mutant receptors bound antagonist with similar affinities. The antagonist-responsive phenotypes caused by ste2sst alleles were therefore due to defects in the ability of receptors to discriminate between agonist and antagonist peptides. One ste2sst mutation caused rapid, ligand-independent internalization of the receptor. These results demonstrate that the third cytoplasmic loop of the alpha-factor receptor is a multifunctional regulatory domain that controls pathway activation and/or desensitization and influences the processes of receptor activation, ligand discrimination, and internalization.     

Simulation of the different biological activities of diethylstilbestrol (DES) on estrogen receptor alpha and estrogen-related receptor gamma

We describe the application of the molecular dynamics (MD) and molecular mechanics-generalized Born/surface area (MM-GB/SA) approaches to the simulation of the different biological activity of diethylstilbestrol (DES) on two highly homologous nuclear receptors-estrogen receptor alpha (ER-alpha) and estrogen-related receptor gamma (ERR-gamma). DES exerts an agonistic effect against ER-alpha and an antagonistic effect against ERR-gamma. Using the x-ray crystal structures of ER-alpha in the canonical agonist bound form (PDB code: 3ERD) and antagonist bound form (PDB code: 3ERT), ERR-gamma homology models have been constructed for the receptor in two different conformations. MM-GB/SA binding free energy calculations of DES in the ER-alpha and ERR-gamma structures suggest that DES exhibits a greater free energy of binding in the agonist bound conformation of ER-alpha, while the antagonist bound conformation is preferred for ERR-gamma. Further dissection of the free energy contributions coupled with calculation of the ligand binding pocket volume suggests that the van der Waals interactions for DES within the smaller binding pocket volume of ERR-gamma are less favorable and this is the main factor for DES antagonism in ERR-gamma. This approach has potential general applicability to the prediction of the biological activity of nuclear receptor ligands.     

Study of a structurally similar kappa opioid receptor agonist and antagonist pair by molecular dynamics simulations

Among the structurally similar guanidinonaltrindole (GNTI) compounds, 5′-GNTI is an antagonist while 6′-GNTI is an agonist of the κOR opioid receptor. To explore how a subtle alteration of the ligand structure influences the receptor activity, we investigated two concurrent processes: the final steps of ligand binding at the receptor binding site and the initial steps of receptor activation. To trace these early activation steps, the membranous part of the receptor was built on an inactive receptor template while the extracellular loops were built using the ab initio CABS method. We used the simulated annealing procedure for ligand docking and all-atom molecular dynamics simulations to determine the immediate changes in the structure of the ligand–receptor complex. The binding of an agonist, in contrast to an antagonist, induced the breakage of the “3–7 lock” between helices TM3 and TM7. We also observed an action of the extended rotamer toggle switch which suggests that those two switches are interdependent.     

Interaction of Serotonin1A Receptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin1A (5-HT1A) receptor, an important member of the G-protein-coupled receptor superfamily. 2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics. 3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane. 4. The local anesthetics showed inhibition of agonist binding to the 5-HT1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT1A receptor brought about by local anesthetics is independent of the membrane cholesterol content. 5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.     

Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.     

In vitro binding and functional studies of Ac-RYYRIK-ol and its derivatives, novel partial agonists of the nociceptin/orphanin F/Q receptor

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.     

Direct observation of G-protein binding to the human delta-opioid receptor using plasmon-waveguide resonance spectroscopy

Using a recently developed method (Salamon, Z., Macleod, H. A., and Tollin, G. (1997) Biophys. J. 73, 2791-2797), plasmon-waveguide resonance spectroscopy, we have been able, for the first time, to directly measure the binding between the human brain delta-opioid receptor (hDOR) and its G-protein effectors in real-time. We have found that the affinity of the G-proteins toward the receptor is highly dependent on the nature of the ligand pre-bound to the receptor. The highest affinity was observed when the receptor was bound to an agonist ( approximately 10 nm); the lowest when receptor was bound to an antagonist ( approximately 500 nm); and no binding at all was observed when the receptor was bound to an inverse agonist. We also have found direct evidence for the existence of an additional G-protein binding conformational state that corresponds to the unliganded receptor, which has a G-protein binding affinity of approximately 60 nm. Furthermore, GTP binding to the receptor.G-protein complex was only observed when the agonist was pre-bound. Similar studies were carried out using the individual G-protein subtypes for both the agonist and the unliganded receptor. Significant selectivity toward the different G-protein subtypes was observed. Thus, the unliganded receptor had highest affinity toward the Galphao (Kd approximately 20 nm) and lowest affinity toward the Galphai2 ( approximately 590 nm) subtypes, whereas the agonist-bound state had highest affinity for the Galphao and Galphai2 subtypes (Kd approximately 9 nm and approximately 7 nm, respectively). GTP binding was also highly selective, both with respect to ligand and G-protein subtype. We believe that this methodology provides a powerful new way of investigating transmembrane signaling.     

Mutagenesis of the Human 5-HT1B Receptor: Differences from the Closely Related 5-HT1A Receptor and the Role of Residue F331 in Signal Transduction

Abstract

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT_1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT_1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.     

Novel ligands for the opioid receptors: synthesis and structure-activity relationships among 5'-aryl and 5'-heteroaryl 17-cyclopropylmethyl-4,5 alpha-epoxypyrido[2',3':6,7]morphinans

A series of pyridomorphinans possessing an aryl (10a-s) or heteroaryl (11a-h) substituent at the 5'-position of the pyridine ring of 17-cyclopropylmethyl-4,5 alpha-epoxypyrido[2',3':6,7]morphinan was synthesized and evaluated for binding and functional activity at the opioid delta, mu, and kappa receptors. All of these pyridomorphinans bound with higher affinity at the delta site than at mu or kappa sites. The binding data on isomeric compounds revealed that there exists greater bulk tolerance for substituents placed at the o-position of the phenyl ring than at m- or p-positions. Among the ligands examined, the 2-chlorophenyl (10l), 2-nitrophenyl (10n), 2-pyridyl (11a), and 4-quinolinyl (11g) compounds bound to the delta receptor with subnanomolar affinity. Compound 10c with the p-tolyl substituent displayed the highest mu/delta selectivity (ratio=42) whereas compound 10l with the 2-chlorophenyl substituent displayed the highest kappa/delta selectivity (ratio=23). At 10 microM concentration, the in vitro functional activity determined using [(35)S]GTP-gamma-S binding assays showed that all of the compounds were antagonists devoid of any significant agonist activity at the delta, mu, and kappa receptors. Antagonist potency determinations of three selected ligands revealed that the p-tolyl compound 10c is a potent delta selective antagonist. In the [(35)S]GTP-gamma-S assays this compound had a functional antagonist K(i) value of 0.2, 4.52, and 7.62 nM at the delta, mu, and kappa receptors, respectively. In the smooth muscle assays 10c displayed delta antagonist potency with a K(e) value of 0.88 nM. As an antagonist, it was 70-fold more potent at the delta receptors in the MVD than at the mu receptors in the GPI. The in vitro delta antagonist profile of this pyridomorphinan 10c resembles that of the widely used delta selective antagonist ligand naltrindole.     

Synthesis and In vitro binding affinities of 1-azabicyclic compounds as muscarinic ligands

Two series of compounds, 2 and 3, were synthesized and their binding affinities were evaluated for the human recombinant muscarinic M(1) receptor subtype expressed in CHO cells. Comparing their binding affinities for the NMS binding sites and the Oxo-M binding sites, they were assumed as agonists. In particular, compound 2e was a good ligand for the agonist binding sites with an IC(50) of 23 nM, which represents over 1585 times stronger binding than for the antagonist binding sites.