Plasmon resonance studies of agonist/antagonist binding to the human delta-opioid receptor: new structural insights into receptor-ligand interactions

Structural changes accompanying the binding of ligands to the cloned human delta-opioid receptor immobilized in a solid-supported lipid bilayer have been investigated using coupled plasmon-waveguide resonance spectroscopy. This highly sensitive technique directly monitors mass density, conformation, and molecular orientation changes occurring in anisotropic thin films and allows direct determination of binding constants. Although both agonist binding and antagonist binding to the receptor cause increases in molecular ordering within the proteolipid membrane, only agonist binding induces an increase in thickness and molecular packing density of the membrane. This is a consequence of mass movements perpendicular to the plane of the bilayer occurring within the lipid and receptor components. These results are consistent with models of receptor function that involve changes in the orientation of transmembrane helices.     

Plasmon-waveguide resonance and impedance spectroscopy studies of the interaction between penetratin and supported lipid bilayer membranes

The interaction between the cell-penetrating peptide, penetratin, and solid-supported lipid bilayer membranes consisting of either egg phosphatidylcholine (PC) or a 75/25 mol% mixture of egg PC and palmitoyloleylphosphatidylglycerol has been studied by simultaneously measuring plasmon-waveguide resonance (PWR) spectra and impedance spectra of lipid-peptide mixtures. When penetratin was incorporated into an egg PC + palmitoyloleylphosphatidylglycerol bilayer, PWR measurements showed a hyperbolic increase in the average refractive index and the refractive index anisotropy, with no change in membrane thickness, over a concentration range between 0 and 2 micro M peptide. In the case of an egg PC bilayer, a biphasic dependence was observed, with a decrease in average refractive index and anisotropy and no thickness change occurring between 0 and 5 micro M peptide, and an increase in membrane thickness occurring between 5 and 15 micro M peptide with no further change in the refractive index parameters. For both membranes, the impedance spectroscopy measurements demonstrated that the electrical resistance was not altered by peptide incorporation, whereas a decrease in membrane capacitance occurred with the same concentration dependence as observed in the PWR experiments, although for the PC membrane no further changes in electrical properties were observed in the higher concentration range. A structural interpretation of these results is described, in which the peptide binds electrostatically within the headgroup region of the bilayer and influences the headgroup conformation, amount of bound water, and the lipid-packing density, without perturbing the hydrocarbon core of the bilayer.     

Plasmon-waveguide resonance studies of ligand binding to the human beta 2-adrenergic receptor

Plasmon-waveguide resonance (PWR) spectroscopy is an optical technique that can be used to probe the molecular interactions occurring within anisotropic proteolipid membranes in real time without requiring molecular labeling. This method directly monitors mass density, conformation, and molecular orientation changes occurring in such systems and allows determination of protein-ligand binding constants and binding kinetics. In the present study, PWR has been used to monitor the incorporation of the human beta(2)-adrenergic receptor into a solid-supported egg phosphatidylcholine lipid bilayer and to follow the binding of full agonists (isoproterenol, epinephrine), a partial agonist (dobutamine), an antagonist (alprenolol), and an inverse agonist (ICI-118,551) to the receptor. The combination of differences in binding kinetics and the PWR spectral changes point to the occurrence of multiple conformations that are characteristic of the type of ligand, reflecting differences in the receptor structural states produced by the binding process. These results provide new evidence for the conformational heterogeneity of the liganded states formed by the beta(2)-adrenergic receptor.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

Spontaneous human B2 bradykinin receptor activity determines the action of partial agonists as agonists or inverse agonists. Effect of basal desensitization.

In this report, we show that desensitization regulates ligand-independent, spontaneous activity of the human B2 bradykinin (BK) receptor, and the level of spontaneous receptor activity determines the action of the BK antagonists and partial receptor agonists NPC17731 and HOE140 as agonists or inverse agonists. Spontaneous receptor activity was monitored by measuring basal cellular phosphoinositide (PI) hydrolysis as a function of the density of the receptor in transiently transfected HEK293 cells. Minimal spontaneous activity of the wild-type B2 receptor was detected in these cells. Mutating a cluster of serines and threonines within the fourth intracellular domain of the receptor, which is critical for agonist-promoted desensitization, significantly increased the spontaneous receptor activity. BK, the natural B2 receptor ligand and, consequently, a full agonist, stimulated PI hydrolysis at high and low levels of spontaneous receptor activity. On the other hand, the partial agonists NPC17731 and HOE140 were stimulatory, or agonists, at the lower level of receptor activity but inhibitory, or inverse agonists, at the higher level of activity. These results show that receptors are desensitized in response to their spontaneous activity. Furthermore, these results, which refute traditional theories, show that the capacity of a drug to modulate a receptor response is not intrinsic to the drug but is also dependent on the cellular environment in which the drug acts.     

Influence of agonists and antagonists on the segmental motion of residues near the agonist binding pocket of the acetylcholine-binding protein

Using the Lymnaea acetylcholine-binding protein as a surrogate of the extracellular domain of the nicotinic receptor, we combined site-directed labeling with fluorescence spectroscopy to assess possible linkages between ligand binding and conformational dynamics. Specifically, 2-[(5-fluoresceinyl)aminocarbonyl]ethyl methanethiosulfonate was conjugated to a free cysteine on loop C and to five substituted cysteines at strategic locations in the subunit sequence, and the backbone flexibility around each site of conjugation was measured with time-resolved fluorescence anisotropy. The sites examined were in loop C (Cys-188 using a C187S mutant), in the beta9 strand (T177C), in the beta10 strand (D194C), in the beta8-beta9 loop (N158C and Y164C), and in the beta7 strand (K139C). Conjugated fluorophores at these locations show distinctive anisotropy decay patterns indicating different degrees of segmental fluctuations near the agonist binding pocket. Ligand occupation and decay of anisotropy were assessed for one agonist (epibatidine) and two antagonists (alpha-bungarotoxin and d-tubocurarine). The Y164C and Cys-188 conjugates were also investigated with additional agonists (nicotine and carbamylcholine), partial agonists (lobeline and 4-hydroxy,2-methoxy-benzylidene anabaseine), and an antagonist (methyllycaconitine). With the exception of the T177C conjugate, both agonists and antagonists perturbed the backbone flexibility of each site; however, agonist-selective changes were only observed at Y164C in loop F where the agonists and partial agonists increased the range and/or rate of the fast anisotropy decay processes. The results reveal that agonists and antagonists produced distinctive changes in the flexibility of a portion of loop F.     

Coupled plasmon-waveguide resonance spectroscopy studies of the cytochrome b6f/plastocyanin system in supported lipid bilayer membranes.

The incorporation of cytochrome (cyt) b6f into a solid-supported planar egg phosphatidylcholine (PC) bilayer membrane and complex formation with plastocyanin have been studied by a variant of surface plasmon resonance called coupled plasmon-waveguide resonance (CPWR) spectroscopy, developed in our laboratory. CPWR combines greatly enhanced sensitivity and spectral resolution with direct measurement of anisotropies in refractive index and optical extinction coefficient, and can therefore probe structural properties of lipid-protein and protein-protein interactions. Cyt b6f incorporation into the membrane proceeds in two stages. The first occurs at low protein concentration and is characterized by an increase in total proteolipid mass without significant changes in the molecular order of the system, as demonstrated by shifts of the resonance position to larger incident angles without changing the refractive index anisotropy. The second stage, occurring at higher protein concentrations, results in a decrease in both the mass density and the molecular order of the system, evidenced by shifts of the resonance position to smaller incident angles and a large decrease in the membrane refractive index anisotropy. Plastocyanin can bind to such a proteolipid system in three different ways. First, the addition of plastocyanin before the second stage of b6f incorporation begins results in complex formation between the two proteins with a KD of approximately 10 microM and induces structural changes in the membrane that are similar to those occurring during the second stage of complex incorporation. The addition of larger amounts of plastocyanin under these conditions leads to nonspecific binding to the lipid phase with a KD of approximately 180 microM. Finally, the addition of plastocyanin after the completion of the second phase of b6f incorporation results in tighter binding between the two proteins (KD approximately 1 microM). Quantitation of the binding stoichiometry indicates that two plastocyanin molecules bind tightly to the dimeric form of the cyt b6f complex, assuming random insertion of the cytochrome into the bilayer. The structural basis for these results and formation of the proteolipid membrane are discussed.     

Structures of Aplysia AChBP complexes with nicotinic agonists and antagonists reveal distinctive binding interfaces and conformations

Upon ligand binding at the subunit interfaces, the extracellular domain of the nicotinic acetylcholine receptor undergoes conformational changes, and agonist binding allosterically triggers opening of the ion channel. The soluble acetylcholine-binding protein (AChBP) from snail has been shown to be a structural and functional surrogate of the ligand-binding domain (LBD) of the receptor. Yet, individual AChBP species display disparate affinities for nicotinic ligands. The crystal structure of AChBP from Aplysia californica in the apo form reveals a more open loop C and distinctive positions for other surface loops, compared with previous structures. Analysis of Aplysia AChBP complexes with nicotinic ligands shows that loop C, which does not significantly change conformation upon binding of the antagonist, methyllycaconitine, further opens to accommodate the peptidic antagonist, alpha-conotoxin ImI, but wraps around the agonists lobeline and epibatidine. The structures also reveal extended and nonoverlapping interaction surfaces for the two antagonists, outside the binding loci for agonists. This comprehensive set of structures reflects a dynamic template for delineating further conformational changes of the LBD of the nicotinic receptor.     

Interactions of agonists and antagonists with beta-adrenergic receptors on intact L6 muscle cells

A binding assay has been developed to characterize beta-adrenergic receptors on intact L6 muscle cells. The affinity of beta-adrenergic receptors for the radioligand iodohydroxybenzylpindolol (IHYP) was the same in membrane preparations and in intact cells when determined by either equilibrium binding or kinetic analysis. The number of specific IHYP binding sites per cell was approximately the same on intact cells as on membranes. The pharmacological properties of antagonists indicated that the receptors on intact cells were identical to those on membranes. However, the beta-adrenergic receptors on intact cells had a 100-400 fold lower affinity at equilibrium for the agonist isoproterenol than did beta-adrenergic receptors on membranes. This low affinity of the receptor for agonists as measured by inhibition of radioligand binding in intact cells has also been observed in C6 (2) and S49 (3) cells. Our results suggest that beta receptors on intact cells after a 1 minute incubation was similar to the KD value for isoproterenol measured in membranes at equilibrium in the presence of GTP. After 1-2 minutes of exposure to a low concentration of agonist, binding of IHYP was no longer inhibited. These results suggest that agonists rapidly convert the beta receptors on intact cells to a state which has a low affinity for agonists. The affinity of the receptor for antagonists did not change during the incubation.     

Discrimination between agonists and antagonists by the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-selective glutamate receptor. A mutation analysis of the ligand-binding domain of GluR-D subunit

The crystal structures of the ligand-binding core of the agonist complexes of the glutamate receptor-B (GluR-B) subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-selective glutamate receptor indicate that the distal anionic group of agonist molecules are stabilized by interactions with an N-terminal region of an alpha-helix (helix F) in the lobe 2 ("domain 2," Armstrong, N., and Gouaux, E. (2000) Neuron 28, 165-181) of the two-lobed ligand-binding domain. We used site-directed mutagenesis to further analyze the role of this region in the recognition of both agonists and antagonists by the AMPA receptor. Wild-type and mutated versions of the ligand-binding domain of GluR-D were expressed in insect cells as secreted soluble polypeptides and subjected to binding assays using [(3)H]AMPA, an agonist, and [(3)H]Ro 48-8587 (9-imidazol-1-yl-8-nitro-2,3,5,6-tetrahydro[1,2,4]triazolo[1,5-c] quinazoline-2,5-dione), a high affinity AMPA receptor antagonist, as radioligands. Single alanine substitutions at residues Leu-672 and Thr-677 severely affected the affinities for all agonists, as seen in ligand competition assays, whereas similar mutations at residues Asp-673, Ser-674, Gly-675, Ser-676, and Lys-678 selectively affected the binding affinities of one or two of the agonists. In striking contrast, the binding affinities of [(3)H]Ro 48-8587 and of another competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione, were not affected by any of these alanine mutations, suggesting the absence of critical side-chain interactions. Together with ligand docking experiments, our results indicate a selective engagement of the side chains of the helix F region in agonist binding, and suggest that conformational changes involving this region may play a critical role in receptor activation.     

Effect of phosphatidylcholine bilayer thickness and molecular order on the binding of the antimicrobial peptide maculatin 1.1

Antimicrobial peptides (AMPs) interact directly with bacterial membrane lipids. Thus, changes in the lipid composition of bacterial membranes can have profound effects on the activity of AMPs. In order to understand the effect of bilayer thickness and molecular order on the activity of AMPs, the interaction of maculatin 1.1 (Mac1.1) with phosphatidylcholine (PC) model membranes composed of different monounsaturated acyl chain lengths between 14 and 22 carbons was characterised by dual polarisation interferometry (DPI) and 31P and 1H solid-state NMR techniques. The thickness and bilayer order of each PC bilayer showed a linear dependence on the acyl chain length. The binding of Mac1.1 exhibited a biphasic dependency between the amount of bound Mac1.1 and bilayer thickness, whereby the mass of bound peptide increased from C14 to C16 and then decreased from C16 to C22. Significant perturbation of 31P chemical shift anisotropy (CSA) values was only observed for DOPC (C18) and DEPC (C22), respectively. In the case of DEPC, the greater range in CSA indicated different headgroup conformations or environments in the presence of Mac1.1. Overall, the results indicated that there is a significant change in the bilayer order upon binding of Mac1.1 and this change occurred in a co-operative manner at higher concentrations of Mac1.1 with increasing bilayer thickness and order. Overall, an optimum bilayer thickness and lipid order may be required for effective membrane perturbation by Mac1.1 and increasing the bilayer thickness and order may counteract the activity of Mac1.1 and play a role in antimicrobial resistance to AMPs.     

Data-Driven Analysis in Drug Discovery

In the process of drug discovery for new chemical entities, application of appropriate pharmacological models often is not possible because the molecular mechanism of the compound is not yet elucidated. Therefore, a data-driven approach using generic tools designed to quantify characteristic patterns of concentration-response curves is required. This article outlines the options available for quantifying agonist and antagonist activity. Specifically, for agonists, the use of the Operational model for the determination of functional effects (equimolar potency ratios for full agonists, calculation of relative efficacy) is described. For antagonists, the measurement of pK_B (-log of the equilibrium dissociation constant of the antagonist-receptor complex) for orthosteric antagonists that do not alter basal response (simple competitive antagonists), increase basal response (partial agonists), and decrease basal response (in constitutively active systems; inverse agonists) is discussed. In addition, this article considers methods to discern orthosteric receptor antagonism from allosteric antagonism whereby the agonist and antagonist bind to separate sites and interact through a conformational change in the receptor. Methods for the measurement of the pK_B for allosteric modulators as well as co-operativity constants for these modulators is described.     

alpha 2B-Adrenoceptor levels govern agonist and inverse agonist responses in PC12 cells

Receptor density is an important determinant of cellular effector responses to receptor activation. We analysed cytosolic Ca(2+) responses to alpha(2)-adrenergic agents in PC12 cells expressing human alpha(2B)-adrenergic receptors (AR) at two densities (3.8 and 1.3 pmol/mg protein). The efficacy (E(max)) of agonists was greater in cells with higher receptor expression; while the potency (EC(50)) of norepinephrine and oxymetazoline was independent of alpha(2B)-AR levels. Several classical alpha(2)-AR antagonists behaved as either partial or inverse agonists in a receptor density-dependent fashion. No apparent structural similarities were found among the inverse agonists, precluding simple predictions of inverse agonist activity. Transfected PC12 cells expressing alpha(2B)-AR at relatively high density would be a useful approach to screen inverse agonists for this class of receptors. Our results further indicate that receptor density significantly influences the properties of ligands, not only of partial agonists as predicted by classical receptor theory, but also of antagonists and full agonists.     

Hydrophobic Residues of the D2 Dopamine Receptor Are Important for Binding and Signal Transduction

Abstract: Dopamine receptors belong to the seven transmembrane helix-containing, G protein-coupled receptor superfamily. Mutagenesis studies suggest that dopamine and its analogues interact with aspartate-114 in helix 3 and two helix 5 serines (194 and 197) of the D₂ receptor. In addition to these amino acids, hydrophobic residues within the receptor core may be important not only for binding but also for receptor activation. Described is a site-directed mutagenesis investigation into the roles of these hydrophobic residues in the long isoform of the human D₂ receptor. Replacement of helix 6 phenylalanines (389 or 390) with alanines resulted in disrupted binding to several agonists and antagonists and impaired inhibition of adenylyl cyclase activity. Replacement of the helix 5 phenylalanine-198 with an alanine selectively disrupted [³H]N-0437 binding, whereas the affinities for other agonists and antagonists remained unchanged. This mutant remained functionally intact when stimulated with dopamine or bromocriptine. Replacement of the helix 7 phenylalanine-411 or the helix 6 leucine-387 with alanines produced receptors that bound agonists well but were unable to inhibit adenylyl cyclase. Based on these data, two conserved helix 6 phenylalanines (389 and 390) appear to be crucial for ligand binding, and phenylalanine-411 in helix 7 and leucine-387 in helix 6 may be important for propagating conformational changes from the agonist binding site(s) to G protein coupling domain(s) of the D₂ receptor.     

Regulation of Serotonin2A Receptors in Heterologous Expression Systems

Abstract: The serotonin_2A and serotonin_2C receptors are unique among receptors coupled to guanine nucleotide binding proteins in that chronic treatment in vivo with agonists as well as antagonists decreases receptor density. In an attempt to uncover molecular events involved in down-regulation of the serotonin_2A receptor, the ability of agonists and antagonists to alter receptor density was examined in three heterologous expression systems, i.e., transfected NIH 3T3, transfected Madin-Darby canine kidney, and transfected AtT-20 cells. All three transfected cell lines exhibited pharmacological properties consistent with that predicted for cells expressing the serotonin_2A receptor. However, the three cell lines displayed different receptor regulation properties in response to drugs acting at the serotonin_2A receptor. In transfected NIH 3T3 cells, neither agonist nor antagonist treatment altered receptor density. Treatment with agonist as well as antagonist led to up-regulation of the serotonin_2A receptor in transfected Madin-Darby canine kidney cells. In transfected AtT-20 cells, treatment with agonist led to receptor down-regulation, whereas antagonist treatment increased receptor density. Thus, the cellular background in which the serotonin_2A receptor is expressed appears to determine the regulation properties of the receptor.     

High-and Low-Affinity States of Striatal D2 Receptors Are Not Affected by 6-Hydroxydopamine or Chronic Haloperidol Treatment

Specific D2 binding in rat striatum was characterized and then the effects of chronic disruption of dopaminergic activity on antagonist and agonist binding to these sites were studied. D2 receptors were defined as those sites capable of binding [3H]spiperone in the presence of cinanserin, a 5-HT2 antagonist, but not in the presence of (+)-butaclamol, a D2 and 5-HT2 blocker. Saturation, competition, and kinetic analyses suggested that D2 receptors are a homogeneous population exhibiting more complex interactions with agonists than antagonists. Antagonist binding was monophasic and guanine nucleotide-insensitive whereas agonist binding was biphasic and guanine nucleotide-sensitive. D2 receptor density was elevated by more than 40% following dopamine depletion by 6-hydroxydopamine or chronic receptor blockade by haloperidol. However neither treatment altered the affinities or magnitudes of the high- and low-affinity components associated with agonist binding to the D2 receptor.     

Discovery and SAR study of novel dihydroquinoline-containing glucocorticoid receptor agonists

We have recently reported the discovery of a novel class of glucocorticoid receptor (GR) antagonists, exemplified by 3, containing a 1,2-dihydroquinoline molecular scaffold. Further SAR studies of these antagonists uncovered chemical modifications conveying agonist functional activity to this series. These agonists exhibit good GR binding affinity and are selective against other nuclear hormone receptors.     

A 5-HT4 receptor transmembrane network implicated in the activity of inverse agonists but not agonists

Activation of G protein-coupled receptors is thought to involve disruption of intramolecular interactions that stabilize their inactive conformation. Such disruptions are induced by agonists or by constitutively active mutations. In the present study, novel potent inverse agonists are described to inhibit the constitutive activity of 5-HT(4) receptors. Using these compounds and specific receptor mutations, we investigated the mechanisms by which inverse agonists may reverse the disruption of intramolecular interactions that causes constitutive activation. Two mutations (D100(3.32)A in transmembrane domain (TMD)-III and F275(6.51)A in TMD-VI) were found to completely block inverse agonist effects without impairing their binding properties nor the molecular activation switches induced by agonists. Based on the rhodopsin model, we propose that these mutated receptors are in equilibrium between two states R and R* but are unable to reach a third "silent" state stabilized by inverse agonists. We also found another mutation in TMD-VI (W272(6.48)A) that stabilized this silent state. This mutant remained fully activated by agonists. Molecular modeling indicated that Asp-100, Phe-275, and Trp-272 might constitute a network required for stabilization of the silent state by the described inverse agonists. However, this network is not necessary for agonist activity.