Low Affinity Agonist Binding Research Articles

SR9883 is a novel small-molecule enhancer of α4β2* nicotinic acetylcholine receptor signaling that decreases intravenous nicotine self-administration in rats.

Most smokers attempting to quit will quickly relapse to tobacco use even when treated with the most efficacious smoking cessation agents currently available. This highlights the need to develop effective new smoking cessation medications. Evidence suggests that positive allosteric modulators (PAM) and other enhancers of nicotinic acetylcholine receptor (nAChR) signaling could have therapeutic utility as smoking cessation agents. 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283) was used as a starting point for medical chemistry efforts to develop novel small molecule enhancers of α4β2* nAChR stoichiometries containing a low-affinity agonist binding site at the interface of α4/α4 and α4/α5 subunits. The NS9283 derivative SR9883 enhanced the effect of nicotine on α4β2* nAChR stoichiometries containing low-affinity agonist binding sites, with EC50 values from 0.2-0.4 μM. SR9883 had no effect on α3β2* or α3β4* nAChRs. SR9883 was bioavailable after intravenous (1 mg kg-1) and oral (10-20 mg kg-1) administration and penetrated into the brain. When administered alone, SR9883 (5-10 mg kg-1) had no effect on locomotor activity or intracranial self-stimulation (ICSS) thresholds in mice. When co-administered with nicotine, SR9883 enhanced locomotor suppression and elevations of ICSS thresholds induced by nicotine. SR9883 (5 and 10 mg kg-1) decreased responding for intravenous nicotine infusions (0.03 mg kg-1 per infusion) but had no effect on responding for food rewards in rats. These data suggest that SR9883 is useful for investigating behavioral processes regulated by certain α4β2* nAChR stoichiometries. SR9883 and related compounds with favorable drug-like physiochemical and pharmacological properties hold promise as novel treatments of tobacco use disorder.

Combined treatment with Sigma1R and A2AR agonists fails to inhibit cocaine self-administration despite causing strong antagonistic accumbal A2AR-D2R complex interactions: the potential role of astrocytes.

Previous studies have indicated that acute treatment with the monoamine stabilizer OSU-6162 (5 mg/kg), which has a high affinity for Sigma1R, significantly increased the density of accumbal shell D2R-Sigma1R and A2AR-D2R heteroreceptor complexes following cocaine self-administration. Ex vivo studies using the A2AR agonist CGS21680 also suggested the existence of enhanced antagonistic accumbal A2AR-D2R allosteric interactions after treatment with OSU-6162 during cocaine self-administration. However, a 3-day treatment with OSU-6162 (5 mg/kg) failed to alter the behavioral effects of cocaine self-administration. To test these results and the relevance of OSU-6162 (2.5 mg/kg) and/or A2AR (0.05 mg/kg) agonist interactions, we administered low doses of receptor agonists during cocaine self-administration and assessed their neurochemical and behavioral effects. No effects were observed on cocaine self-administration; however, marked and highly significant increases using the proximity ligation assay (PLA) were induced by the co-treatment on the density of the A2AR-D2R heterocomplexes in the nucleus accumbens shell. Significant decreases in the affinity of the D2R high- and low-affinity agonist binding sites were also observed. Thus, in low doses, the highly significant neurochemical effects observed upon cotreatment with an A2AR agonist and a Sigma1R ligand on the A2AR-D2R heterocomplexes and their enhancement of allosteric inhibition of D2R high-affinity binding are not linked to the modulation of cocaine self-administration. The explanation may be related to an increased release of ATP and adenosine from astrocytes in the nucleus accumbens shell in cocaine self-administration. This can lead to increased activation of the A1R protomer in a putative A1R-A2AR-D2R complex that modulates glutamate release in the presynaptic glutamate synapse. We hypothesized that the integration of changes in presynaptic glutamate release and postjunctional heteroreceptor complex signaling, where D2R plays a key role, result in no changes in the firing of the GABA anti-reward neurons, resulting in no reduction in cocaine self-administration in the present experiments.

New insight into active muscarinic receptors with the novel radioagonist [3H]iperoxo

Activation of G protein-coupled receptors involves major conformational changes of the receptor protein ranging from the extracellular transmitter binding site to the intracellular G protein binding surface. GPCRs such as the muscarinic acetylcholine receptors are commonly probed with radioantagonists rather than radioagonists due to better physicochemical stability, higher affinity, and indifference towards receptor coupling states of the former. Here we introduce tritiated iperoxo, a superagonist at muscarinic M2 receptors with very high affinity. In membrane suspensions of transfected CHO-cells, [3H]iperoxo – unlike the common radioagonists [3H]acetylcholine and [3H]oxotremorine M – allowed labelling of each of the five muscarinic receptor subtypes in radioagonist displacement and saturation binding studies. [3H]iperoxo revealed considerable differences in affinity between the even- and the odd-numbered muscarinic receptor subtypes with affinities for the M2 and M4 receptor in the picomolar range. Probing ternary complex formation on the M2 receptor, [3H]iperoxo dissociation was not influenced by an archetypal allosteric inverse agonist, reflecting activation-related rearrangement of the extracellular loop region. At the inner side of M2, the preferred Gi protein acted as a positive allosteric modulator of [3H]iperoxo binding, whereas Gs and Gq were neutral in spite of their robust coupling to the activated receptor. In intact CHO-hM2 cells, endogenous guanylnucleotides promoted receptor/G protein-dissociation resulting in low-affinity agonist binding which, nevertheless, was still reported by [3H]iperoxo. Taken together, the muscarinic superagonist [3H]iperoxo is the best tool currently available for direct probing activation-related conformational transitions of muscarinic receptors.

The influence of beta-arrestin2 on cannabinoid CB1 receptor coupling to G-proteins and subcellular localization and relative levels of beta-arrestin1 and 2 in mouse brain

Context: Beta-arrestins are known to couple to some G-protein-coupled receptors (GPCRs) to regulate receptor internalization, G-protein coupling and signal transduction, but have not been investigated for most receptors, and for very few receptors in vivo. Previous studies have shown that beta-arrestin2 deletion enhances the efficacy of specific cannabinoid agonists. Objective: The present study hypothesized that brain cannabinoid CB1 receptors are regulated by beta-arrestin2. Methods: Beta-arrestin2+/+ and −/− mice were used. Western blotting was used to determine the relative levels of each beta-arrestin subtype in mouse brain. Receptor binding was measured to determine whether deletion of beta-arrestin2 influences agonist binding to brain CB1 receptors, or the subcellular localization of CB1 in brain membranes subjected to differential centrifugation. A variety of cannabinoid agonists from different chemical classes were investigated for their ability to activate G-proteins in the presence and absence of beta-arrestin2 in cerebellum, hippocampus and cortex. Results: No differences were found in the density of beta-arrestin1 or cannabinoid CB1 receptors in several brains of beta-arrestin2+/+ versus −/− mice. Differences between genotypes were found in the proportion of high- and low-affinity agonist binding sites in brain areas that naturally express higher levels of beta-arrestin2. Cortex from beta-arrestin2−/− mice contained less CB1 in the P1 fraction and more CB1 in the P2 fraction compared to beta-arrestin2+/+. Of the agonists assayed for activity, only Δ9-tetrahydrocannabinol (THC) exhibited a difference between genotypes, in that it was less efficacious in beta-arrestin2−/− than +/+ mouse membranes. Conclusion: Beta-arrestin2 regulates cannabinoid CB1 receptors in brain.

Subunit-specific desensitization of heteromeric kainate receptors

Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two-electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration-response curves that were bell-shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration-response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration-response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high-affinity binding site leads to channel opening; and (c) the low-affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK-293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000-fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration-response curve overlapped with the wildtype curve, whereas the declining phase was right-shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high-affinity agonist binding to a non-desensitizing GluK4 subunit opens the heteromeric channel, whereas low-affinity agonist binding to GluK2 desensitizes the whole channel complex.

Physiological significance of high- and low-affinity agonist binding to neuronal and recombinant AMPA receptors

Radioligand binding studies have shown that AMPA receptors exist in two variants that differ about twenty-fold in their binding affinities, with brain receptors being mainly of the low-affinity type and recombinantly expressed receptors having almost exclusively high affinity. However, the physiological correlate of high- and low-affinity binding is not yet known. In this study we examined if physiological experiments similarly reveal evidence for two distinct receptor variants. We therefore measured equilibrium desensitization by glutamate and determined IC 50 values for neuronal receptors and for the homomeric receptors GluR1–4 expressed in HEK293 cells. Contrary to the prediction that these IC 50 values exhibit large differences commensurate with those of high- and low-affinity binding, values for homomeric receptors (1–18 μM) were on an average not different from those of neuronal receptors (3–10 μM). Moreover, simulations with kinetic receptor models suggest that the IC 50 values for neuronal and recombinant receptors correspond to the binding affinity of the low-affinity receptor variant. These findings indicate that the high-affinity binding measured in heterologous expression systems represents an immature receptor variant that does not contribute to the currents recorded from these cells, and that the functional low-affinity receptors are present in such small number that they are effectively masked in binding assays by the high-affinity receptors. Thus, in order to compare experimentally determined saturation binding profiles with those predicted by kinetic receptor models and with dose-response curves from physiological studies, it will be imperative to develop methods for isolating first the low-affinity receptors.

Effects of urea pretreatment on the binding properties of adenosine A1 receptors

The effect of denaturation and/or extraction of nonintegral membrane proteins by 7 M urea on the binding of the antagonist [3H]cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([3H]DPCPX), and the agonists adenosine, (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclohexyladenosine (CHA), was investigated at human A1 adenosine receptors stably expressed in CHO cells. Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 microM 5'-guanylylimidodiphosphate (Gpp(NH)p) or urea each caused equivalent increases in specific [3H]DPCPX binding. Neither the binding kinetics nor the affinity of [3H]DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. At 25 degrees C in ADA-pretreated membranes, the competition isotherms for R-PIA and CHA were characterized by two affinity states. Gpp(NH)p (100 microM) reduced, but did not abolish, the value of the high-affinity dissociation constant. Similar results were obtained after treatment with urea for R-PIA, whereas the high-affinity state for CHA was abolished. At 37 degrees C, urea pretreatment, but not 100 microM Gpp(NH)p, abolished high-affinity agonist competition binding. There was no significant effect of any of the treatments on the low-affinity agonist binding state. In urea-pretreated membranes, exogenously added adenosine competed according to a simple mass-action model with a pK(L) of 5.66+/-0.05 (n=3). Compared to the more common approaches of ADA treatment and/or use of guanine nucleotides, our findings suggest that urea pretreatment represents an inexpensive and useful approach for investigating the binding properties of adenosine A1 ligands (including adenosine) to the G protein-uncoupled form of the receptor.

Selective Inhibition of α1A-Adrenergic Receptor Signaling by RGS2 Association with the Receptor Third Intracellular Loop

Regulators of G-protein signaling (RGS) proteins act directly on Galpha subunits to increase the rate of GTP hydrolysis and to terminate signaling. However, the mechanisms involved in determining their specificities of action in cells remain unclear. Recent evidence has raised the possibility that RGS proteins may interact directly with G-protein-coupled receptors to modulate their activity. By using biochemical, fluorescent imaging, and functional approaches, we found that RGS2 binds directly and selectively to the third intracellular loop of the alpha1A-adrenergic receptor (AR) in vitro, and is recruited by the unstimulated alpha1A-AR to the plasma membrane in cells to inhibit receptor and Gq/11 signaling. This interaction was specific, because RGS2 did not interact with the highly homologous alpha1B- or alpha1D-ARs, and the closely related RGS16 did not interact with any alpha1-ARs. The N terminus of RGS2 was required for association with alpha1A-ARs and inhibition of signaling, and amino acids Lys219, Ser220, and Arg238 within the alpha1A-AR i3 loop were found to be essential for this interaction. These findings demonstrate that certain RGS proteins can directly interact with preferred G-protein-coupled receptors to modulate their signaling with a high degree of specificity.

Impaired G protein coupling of the neurotensin receptor 1 by mutations in extracellular loop 3

The neurotensin receptor 1, NTS1, is a G protein-coupled receptor. We have shown previously that the NTS1 receptor-binding site of the peptide agonist involved residues in extracellular loop 3 and at the extracellular junction of transmembrane domains 4 and 6. Here, we investigated by site-directed mutagenesis residues in extracellular loop 3 that might be involved in agonist-induced activation of the rat NTS1 (rNTS1) receptor. Wild type and mutated receptors were expressed in COS (African green monkey kidney fibroblasts) cells. Labeled agonist and antagonist binding as well as inositol phosphate and cAMP productions were studied. Compared to the wild type NTS1 receptor, the W339A, F344A, H348A and Y349A mutant receptors exhibited (i) decreased proportion of high over low affinity agonist binding sites, (ii) increased sensitivity of high affinity agonist binding to GTPγS, and (iii) impaired G protein coupling of high affinity agonist-receptor complexes. The data are consistent with the C-terminal part of extracellular loop 3 being essential for allowing high affinity agonist-NTS1 receptor complexes to couple to G proteins.

A Conformational Intermediate between the Resting and Desensitized States of the Nicotinic Acetylcholine Receptor

The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.

CoMFA-based prediction of agonist affinities at recombinant wild type versus serine to alanine point mutated D2 dopamine receptors.

Agonist affinity changes dramatically as a result of serine to alanine mutations (S193A, S194A, and S197A) within the fifth transmembrane region of D2 dopamine receptors and other receptors for monoamine neurotransmitters. However, agonist 2D-structure does not predict which drugs will be sensitive to which point mutations. Modeling drug-receptor interactions at the 3D level offers considerably more promise in this regard. In particular, a comparison of the same test set of agonists across receptors differing minimally (point mutations) offers promise to enhance the understanding of the structural bases for drug-receptor interactions. We have previously shown that comparative molecular field analysis (CoMFA) can be applied to comparisons of affinity at recombinant D1 and D2 dopamine receptors for the same set of agonists, a differential QSAR. Here, we predicted agonist K(L) for the same set of agonists at wild type D2 vs S193A, S194A, and S197A receptors using CoMFA. Each model used bromocriptine as the template. ln(1/K(L)) values for the low-affinity agonist binding conformation at recombinant wild type and mutant D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA of the 16 aligned agonist structures. The resulting CoMFA models yielded cross-validated R(2) (q(2)) values ranging from 0.835 to 0.864 and simple R(2) values ranging from 0.999 to 1.000. Predictions of test compound affinities at WT and each mutant receptor were close to measured affinity values. This finding confirmed the predictive ability of the models and their differences from one another. The results strongly support the idea that CoMFA models of the same training set of compounds applied to WT vs mutant receptors can accurately predict differences in drug affinity at each. Furthermore, in a "proof of principle", two different templates were used to derive the CoMFA model for the WT and S193A mutant receptors. Pergolide was chosen as an alternate template because it showed a significant increase in affinity as a result of the S193A mutation. In this instance both the bromocriptine- and pergolide-based CoMFA models were similar to one another but different from those for the WT receptor using bromocriptine- or pergolide- as templates. The pergolide-based S193A model was more strikingly different from that of the WT receptor than was the bromocriptine-based S193A model. This suggests that a "dual-template" approach to differential CoMFA may have special value in elucidating key differences across related receptor types and in determining important elements of the drug-receptor interaction.

Reduced High-Affinity Agonist Binding at the M1 Muscarinic Receptor in Alzheimer's Disease Brain: Differential Sensitivity to Agonists and Divalent Cations

M1 muscarinic acetylcholine receptor (M1AchR)–G protein coupling, as measured by high-affinity agonist binding, was examined in membranes prepared from postmortem human temporal cortex (Brodmann area 38) from individuals with Alzheimer's disease (AD, n = 8) and age-matched controls (n = 6). Binding competitions between the M1AchR-selective antagonist [3H]pirenzepine ([3H]PZ) and muscarinic agonists carbachol, acetylcholine, oxotremorine, and oxotremorine M were conducted. In the presence of 1 mM MgCl2, the inhibition of [3H]PZ binding by carbachol, acetylcholine, or oxotremorine M was best described by a two-affinity state model for control and AD cases, while oxotremorine binding affinity was best fit to a single-state model. Although both control and AD groups had similar KD values for the high- and low-affinity agonist binding sites, the proportion of M1AchRs exhibiting high affinity for carbachol and acetylcholine was reduced by 48 and 33%, respectively, in AD membranes relative to controls (P < 0.05). No changes in the binding of the oxotremorine M or oxotremorine were noted. The nonhydrolyzable guanine nucleotide GppNHp (100μM) reduced the proportion of M1AchRs with high affinity for agonists in both control and AD membranes. Substitution of 1 mM MnCl2 for MgCl2 restored high-affinity carbachol binding at the M1AchR in AD membranes similar to that seen in controls. In the presence of 1 mM MnCl2, agonist binding in controls did not differ from 1 mM MgCl2. In the absence of cations (1 mM EDTA), no differences between control and AD M1AchR carbachol binding were observed. Thus, the loss of high-affinity agonist binding at the M1AchR in AD is dependent on the agonist and cation studied.

Functional high- and low affinity agonist binding sites at native dorsal horn AMPA receptors.

Transmission and processing of nociceptive information in the superficial dorsal horn (DH) of the spinal cord involves activation of AMPA-type glutamate receptors (AMPARs). We have studied the properties of native AMPARs in freshly dissociated laminae I-II neurones from postnatal rats using a modified form of the concentration-clamp technique for fast agonist application. Analysis of kainic acid dose-response curves showed the existence of two types of functional agonist binding sites governing AMPAR activation. These sites differ by their affinity for the agonist. Depending on the neurotransmitter concentration reached in the synaptic cleft, the relative contribution of high affinity and low affinity sites might play an important role in the shaping of AMPAR-mediated postsynaptic currents.

CoMFA-based prediction of agonist affinities at recombinant D1 vs D2 dopamine receptors.

We have previously shown that using agonist affinity at recombinant receptors selectively expressed in clonal cells as the dependent variable in three-dimensional quantitative structure-activity relationship studies (3D-QSAR) presents a unique opportunity for accuracy and precision in measurement. Thus, a comparison of affinity's structural determinants for a set of compounds at two different recombinant dopamine receptors represents an attainable goal for 3D-QSAR. A molecular database of bound conformations of 16 structurally diverse agonists was established by alignment with a high-affinity template compound for the D1 receptor, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4, 5-tetrahydro-1H-benzazepin. A second molecular database of the bound conformations of the same compounds was established against a second template for the D2 receptor, bromocriptine. These aligned structures suggested three-point pharmacophore maps (one cationic nitrogen and two electronegative centers) for the two dopamine receptors, which differed primarily in the height of the nitrogen above the plane of the catechol ring and in the nature of the hydrogen-bonding region. The ln(1/KL) values for the low-affinity agonist binding conformation at recombinant D1 and D2 dopamine receptors stably expressed in C6 glioma cells were used as the target property for the CoMFA (comparative molecular field analysis) of the 16 aligned structures. The resulting CoMFA models yielded cross-validated R2 (q2) values (standard error of prediction) of 0. 879 (1.471, with five principal components) and 0.834 (1.652, with five principal components) for D1 and D2 affinity, respectively. The simple R2 values (standard error of the estimate) were 0.994 (0.323) and 0.999 (0.116), respectively, for D1 and D2 receptor. F values were 341 and 2465 for D1 and D2 models, respectively, with 5 and 10 df. The predictive utility of the CoMFA model was evaluated at both receptors using the dopamine agonists, apomorphine and 7-OH-DPAT. Predictions of KL were accurate at both receptors. Flexible 3D searches of several chemical databases (NCI, MDDR, CMC, ACD, and Maybridge) were done using basic pharmacophore models at each receptor to determine the similarity of hit lists between the two models. The D1 and D2 models yielded different lists of lead compounds. Several of the lead compounds closely resembled high-affinity training set compounds. Finally, homology modeling of agonist binding to the D2 receptor revealed some consistencies and inconsistencies with the CoMFA-derived D2 model and provided a possible rationale for features of the D2 CoMFA contour map. Together these results suggest that CoMFA-homology based models may provide useful insights concerning differential agonist-receptor interactions at related receptors. The results also suggest that comparisons of CoMFA models for two structurally related receptors may be a fruitful approach for differential QSAR.

Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing stopped-flow fluorescence spectroscopy.

We have detected the binding of the fluorescent agonist Dns-C6-Cho to both low- and high-affinity states of the nicotinic acetylcholine receptor (nAcChoR) using sequential mixing stopped-flow fluorescence spectroscopy. Our approach to resolving low- and high-affinity binding was to first preincubate receptor membranes with the fluorescent partial agonist Dns-C6-Cho for 15 ms to 1000 s and then to follow the fluorescence decay upon chemical dilution into excess acetylcholine. The fast and slow decays, reflecting Dns-C6-Cho dissociation from low- and high-affinity receptors, had rates of 140 +/- 27 s-1 and 0.1 +/- 0.02 s-1, respectively. With increasing preincubation times, the number of low-affinity receptors decreased while the number of high-affinity receptors increased in a Dns-C6-Cho concentration-dependent manner consistent with current models for agonist-induced affinity state conversion. At receptor-activating concentrations of Dns-C6-Cho, the apparent rates with which high-affinity receptors formed approximated those of ion flux desensitization, implying that the fast desensitized state has an agonist dissociation rate that is indistinguishable from the equilibrium slow desensitized state. The KD for the low-affinity binding site was determined to be 1.1 microM from the increase in the amplitude of the fast decay with Dns-C6-Cho concentration with preincubation times that were sufficiently brief to minimize affinity state conversion. Assuming a bimolecular association rate of 10(8) M-1 s-1, a second estimate of 1.4 microM was made for low-affinity binding. We also detected a fluorescence enhancement consistent with a conformational isomerization of Dns-C6-Cho-inhibited nAcChoRs.

β-adrenergic signalling in neoplastic lung type 2 cells: glucocorticoid-dependent and -independent defects

Tumorigenic mouse lung-derived type 2 cell lines have large reductions in both beta-adrenergic-stimulated cAMP production and ligand binding to beta-adrenergic receptors. These tumorigenic cells are also relatively insensitive to glucocorticoids. Because glucocorticoids regulate both beta-adrenergic receptor expression and receptor coupling to the stimulatory guanine nucleotide binding protein Gs interactions between the glucocorticoid and beta-adrenergic signalling systems were examined. This study demonstrates that beta-adrenergic ligand binding and agonist sensitivity are increased in a tumorigenic cell line stably expressing a normal glucocorticoid receptor transgene. However, although the transfected tumour cells and non-tumorigenic cells have similar amounts and affinities of beta-adrenergic agonist and antagonist binding, similar amounts of Gs subunits and similar forskolin-stimulated adenylyl cyclase activities, the former remain much less isoproterenol responsive. Competition binding studies demonstrate that tumour cell beta-adrenergic receptors have both high- and low-affinity agonist binding but are functionally uncoupled from Gs. This uncoupling may involve an alteration in Gs, as guanine nucleotides exhibit a reduced ability to stimulate adenylyl cyclase. Thus, some aspects of tumorigenic cell dysfunction in beta-adrenergic signalling can be ameliorated by interactions with the glucocorticoid pathway, but additional defects are also involved.

The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H-antagonist binding produced by GTP which has been observed in essentially all studies of A1 receptors and has been ascribed previously to precoupling of A1 receptors to G-proteins in the absence of agonists.

The function of a highly-conserved arginine residue in activation of the muscarinic M 1 receptor

Arg 123 in the rat muscarinic M 1 receptor is part of the highly conserved triad Asp-Arg-Tyr found at the junction of transmembrane helix 3 with the second intracellular loop. Mutation of Arg 123 to Lys, Ala, Leu, Glu and Gln had no effect on levels of receptor expression in COS-7 cells, or on affinities for the antagonist N-methylscopolamine. Acetylcholine stimulation of the Lys 123 receptor evoked the same maximum phosphoinositide response as the wild type, although the potency was reduced six-fold, but mutation to other residues strongly disrupted receptor function. Mutation of Arg 123 always decreased the ratio of the high affinity to the low affinity agonist binding constant, but the absolute effect on the latter varied from a 4-fold increase for the Lys 123 to a small decrease for the Leu 123 mutation. These results suggest that a positive charge at position 123 is central to the activation of G-proteins by the muscarinic M 1 receptor.

Estrogen uncouples beta-adrenergic receptor from the stimulatory guanine nucleotide-binding protein in female rat hypothalamus.

The responsiveness of adenylyl cyclase to beta-adrenergic receptor stimulation was investigated in membranes prepared from hypothalamus-preoptic area and cortex of ovariectomized female rats injected with oil vehicle or estradiol benzoate 24 or 48 h before death. Membranes from the hypothalamus-preoptic area of ovariectomized animals displayed a concentration-dependent stimulation of adenylyl cyclase when incubated with the beta-adrenergic receptor agonist, isoproterenol (10(-7)-10(-5) M). This response was suppressed in membranes from estrogen-treated animals. The effect of estrogen was observed 48 h, but not 24 h, after hormone administration. In addition, estrogen had no measurable effect on hypothalamic adenylyl cyclase activation by either GTP (10(-8)-10(-5) M) or forskolin (10(-8)-10(-6) M), on beta-adrenergic receptor density, or on antagonist binding affinity measured with the beta-adrenergic antagonist [125I]iodocyanopindolol. Analysis of isoproterenol displacement of iodocyanopindolol binding revealed that estrogen reduced agonist binding affinity in hypothalamus-preoptic area membranes. In membranes from ovariectomized controls, high affinity agonist binding to the beta-adrenergic receptor was apparent and was abolished by guanine nucleotides. However, membranes from estradiol-treated rats demonstrated only low affinity agonist binding that was unaffected by guanine nucleotides. Estradiol did not detectably alter concentrations of either cholera or pertussis toxin substrates in hypothalamus-preoptic area membranes. These data indicate that estrogen promotes a stable time-dependent desensitization of beta-adrenergic receptor activation of adenylyl cyclase in hypothalamus and preoptic area by uncoupling the receptor from the guanine nucleotide-binding protein, G8.

Forskolin-Stimulated Adenylyl Cyclase Activity Is Decreased but β2-Adrenoceptor Function Is Unchanged in Primary Hypertension

beta 2-Adrenoceptor function may be decreased in primary hypertension, resulting in increased peripheral resistance. To study the beta 2-adrenoceptor function, we used circulating mononuclear leukocytes (MNL) as a model system. Twenty untreated hypertensive subjects [(HT) 10 men and 10 women] and 20 age- and sex-matched healthy normotensive (NT) volunteers were studied. The beta 2-adrenoceptor density was not significantly different between HT and NT, but the dissociation constants for the high- and low-affinity agonist binding states, studied by isoprenaline competition assays, were significantly higher in HT. Stimulation of adenylyl cyclase with isoprenaline (10 microM, beta 2-adrenoceptor-mediated stimulation) was not significantly different between the two groups. Forskolin-mediated direct stimulation of adenylyl cyclase was significantly higher in women than in men. For both sexes, the forskolin-induced cyclic AMP production was lower in the HT group, reaching statistical significance in the men. No major abnormalities were observed in beta 2-adrenoceptor function in mononuclear leukocytes. The putative relation between the decreased forskolin-mediated adenylyl cyclase activity and primary hypertension requires further study.