High Affinity State For Agonists Research Articles

Different Alterations of Agonist and Antagonist Binding to 5-HT1A Receptor in a Rat Model of Parkinson's Disease and Levodopa-Induced Dyskinesia: A MicroPET Study.

The gold-standard treatment for Parkinson's disease is L-DOPA, which in the long term often leads to levodopa-induced dyskinesia. Serotonergic neurons are partially responsible for this, by converting L-DOPA into dopamine leading to its uncontrolled release as a "false neurotransmitter". The stimulation of 5-HT1A receptors can reduce involuntary movements but this mechanism is poorly understood. This study aimed to investigate the functionality of 5-HT1A receptors using positron emission tomography in hemiparkinsonian rats with or without dyskinesia induced by 3-weeks daily treatment with L-DOPA. Imaging sessions were performed "off" L-DOPA. Each rat underwent a positron emission tomography scan with [18F]F13640, a 5-HT1AR agonist which labels receptors in a high affinity state for agonists, or with [18F]MPPF, a 5-HT1AR antagonist which labels all the receptors. There were decreases of [18F]MPPF binding in hemiparkinsonian rats in cortical areas. In dyskinetic animals, changes were slighter but also found in other regions. In hemiparkinsonian rats, [18F]F13640 uptake was decreased bilaterally in the globus pallidus and thalamus. On the non-lesioned side, binding was increased in the insula, the hippocampus and the amygdala. In dyskinetic animals, [18F]F13640 binding was strongly increased in cortical and limbic areas, especially in the non-lesioned side. These data suggest that agonist and antagonist 5-HT1A receptor-binding sites are differently modified in Parkinson's disease and levodopa-induced dyskinesia. In particular, these observations suggest a substantial involvement of the functional state of 5-HT1AR in levodopa-induced dyskinesia and emphasize the need to characterize this state using agonist radiotracers in physiological and pathological conditions.

Cocaine self-administration differentially affects allosteric A2A-D2 receptor-receptor interactions in the striatum. Relevance for cocaine use disorder

In the current study behavioral and biochemical experiments were performed to study changes in the allosteric A2AR-D2R interactions in the ventral and dorsal striatum after cocaine self-administration versus corresponding yoked saline control. By using ex vivo [3H]-raclopride/quinpirole competition experiments, the effects of the A2AR agonist CGS 21680 (100nM) on the KiH and KiL values of the D2-like receptor (D2-likeR) were determined. One major result was a significant reduction in the D2-likeR agonist high affinity state observed with CGS 21680 after cocaine self-administration in the ventral striatum compared with the yoked saline group. The results therefore support the hypothesis that A2AR agonists can at least in part counteract the motivational actions of cocaine. This action is mediated via the D2-likeR by targeting the A2AR protomer of A2AR-D2-likeR heteroreceptor complexes in the ventral striatum, which leads to the reduction of D2-likeR protomer recognition through the allosteric receptor-receptor interaction. In contrast, in the dorsal striatum the CGS 21680-induced antagonistic modulation in the D2-likeR agonist high affinity state was abolished after cocaine self-administration versus the yoked saline group probably due to a local dysfunction/disruption of the A2AR-D2-likeR heteroreceptor complexes. Such a change in the dorsal striatum in cocaine self-administration can contribute to the development of either locomotor sensitization, habit-forming learning and/or the compulsive drug seeking by enhanced D2-likeR protomer signaling. Potential differences in the composition and stoichiometry of the A2AR-D2R heteroreceptor complexes, including differential recruitment of sigma 1 receptor, in the ventral and dorsal striatum may explain the differential regional changes observed in the A2A-D2-likeR interactions after cocaine self-administration.

Absolute abundances and affinity states of dopamine receptors in mammalian brain: A review

The abundances of dopamine (DA) D(1) and D(2) receptors have been assayed with radioligands in membrane preparations and by autoradiography in vitro, and also in living brain using positron emission tomography (PET). This review compares the saturation binding parameters (B(max) and K(D) ) obtained in striatum by these several methods, and in different species. Some uncertainty in quantitation is derived from the incomplete specificities of commonly used ligands, especially Sch 23,390 for D(1) sites and spiperone for D(2) -like sites. In striatal membrane preparations, the D(1) B(max) ranges from 10 to 139 pmol g(-1) tissue, whereas the D(2) B(max) ranges from 8 to 42 pmol g(-1) tissue. Receptor concentrations in human material, despite the more extended post mortem interval, are roughly similar to those reported in rodent and nonhuman primate. Estimates of B(max) by quantitative autoradiography are generally five times higher than corresponding results for similar ligands in membrane preparations. The saturation binding parameters in living striatum have been estimated by serial PET studies with ligands over a range of specific activities. The few PET estimates of D(1) B(max) , (40-80 pmol g(-1) ) and numerous PET estimates of D(2) B(max) (20-40 pmol g(-1) ) are in general agreement with membrane estimates, but fall far short of the mean of autoradiographic results in vitro. Apparent affinities for D(1) and D(2) ligands in vivo are typically 10 times lower than for corresponding in vitro studies, presumably because the unbound ligand concentration is not corrected for the free fraction in living brain tissue. The disparate B(max) results by method suggest the presence of a large reservoir or reserve of D(1) and D(2) receptors in intact brain sections, which are unavailable to PET ligands in vivo, and which may be lost during the preparation of washed membranes. A subset of receptors existing in a high affinity state for agonists is detected in washed membrane preparations, in which the coupling to intracellular G-proteins may have become artificially limiting. However, in most PET and autoradiographic studies in vitro, agonist and antagonist ligands have similar B(max) . Discrepancies in the literature highlight the need for a better understanding of affinity states in vivo and trafficking of G-protein coupled receptors between plasma membrane and intracellular compartments.

A thermodynamic model of the action–perception cycle in brain dynamics

We previously demonstrated that in vivo estradiol treatment markedly attenuatesα2-adrenoceptor function and coupling to G-proteins in the hypothalamus of female rats. Ligand binding studies indicated that 48 h exposure to estradiol decreases the number ofα2-adrenergic receptors in the agonist high affinity state. In the present studies, when [3H]RX821002 was used to label brainα2-adrenoceptors, the density of binding sites significantly increased in the hypothalamus and preoptic area 48 h after estrogen treatment. Moreover, the thermodynamics of ligand binding toα2-adrenergic receptors in membranes of female rat hypothalamus were modified by the same estradiol treatments that reduceα2-adrenoceptor function. In hypothalamic membranes from ovariectomized control rats, antagonist (RX821002)-receptor binding was primarily entropy-driven while agonist (oxymetazoline) binding had a higher enthalpy component. In membranes from estradiol-exposed animals, the entropic contribution to both agonist and antagonist binding was markedly increased, and the enthalpy component was reduced. Since the thermodynamic characteristics of ligand-receptor binding are strongly correlated with efficacy in activating signal transduction [36], these data raise the intriguing possibility that steroids regulate transmembrane signaling by stabilization of a receptor conformation with reduced intrinsic efficacy.

Differential regulation of serotonin-1A receptor-stimulated [ 35S]GTPγS binding in the dorsal raphe nucleus by citalopram and escitalopram

The effect of chronic citalopram or escitalopram administration on 5-HT 1A receptor function in the dorsal raphe nucleus was determined by measuring [ 35S]GTPγS binding stimulated by the 5-HT 1A receptor agonist ( R)-(+)-8-OH-DPAT (1nM–10 μM). Although chronic administration of citalopram or escitalopram has been shown to desensitize somatodendritic 5-HT 1A autoreceptors, we found that escitalopram treatment decreased the efficacy of 5-HT 1A receptors to activate G proteins, whereas citalopram treatment did not. The binding of [ 3H]8-OH-DPAT to the coupled, high affinity agonist state of the receptor was not altered by either treatment. Interestingly, escitalopram administration resulted in greater occupancy of serotonin transporter sites as measured by the inhibition of [ 3H]cyanoimipramine binding. As the binding and action of escitalopram is limited by the inactive enantiomer R-citalopram present in racemic citalopram, we propose that the regulation of 5-HT 1A receptor function in the dorsal raphe nucleus at the level of receptor-G protein interaction may be a result of greater inhibition of the serotonin transporter by escitalopram.

Serotonin‐1A receptor function in the dorsal raphe nucleus following chronic administration of the selective serotonin reuptake inhibitor sertraline

Serotonin-1A (5-HT(1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT(1A) receptors to activate G proteins was a general mechanism by which 5-HT(1A) receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT(1A) receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT(1A) autoreceptor function was not accompanied by a decrease in 5-HT(1A) receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT(1A) somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT(1A) receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT(1A) autoreceptors is regulated.

Analysis of brain adrenergic receptors in dopamine-β-hydroxylase knockout mice

The biosynthesis of norepinephrine occurs through a multi-enzymatic pathway that includes the enzyme dopamine-β-hydroxylase (DBH). Mice with a homozygous deletion of DBH ( Dbh−/−) have a selective and complete absence of norepinephrine. The purpose of this study was to assess the expression of alpha-1, alpha-2 and beta adrenergic receptors (α 1-AR, α 2-AR and β-AR) in the postnatal absence of norepinephrine by comparing noradrenergic receptors in Dbh−/− mice with those in Dbh heterozygotes ( Dbh+/−), which have normal levels of norepinephrine throughout life. The densities of α 1-AR, α 2-AR and β-AR were assayed with [ 3H]prazosin, [ 3H]RX21002 and [ 125I]-iodo-pindolol autoradiography, respectively. The α 2-AR agonist high affinity state was examined with [ 125I]- para-iodoclonidine autoradiography and α 2-AR functionality by α 2-AR agonist-stimulated [ 35S]GTPγS autoradiography. The density of α 1-AR in Dbh−/− mice was similar to Dbh+/− mice in most brain regions, with an up-regulation in the hippocampus. Modest decreases in α 2-AR were found in septum, hippocampus and amygdala, but these were not reflected in α 2-AR functionality. The density of β-AR was up-regulated to varying degrees in many brain regions of Dbh−/− mice compared to the heterozygotes. These findings indicate that regulation of noradrenergic receptors by endogenous norepinephrine depends on receptor type and neuroanatomical region.

Characterization of the Residues in Helix 8 of the Human β1-Adrenergic Receptor That Are Involved in Coupling the Receptor to G Proteins

Several key amino acids within amphipathic helix 8 of the human beta1-adrenergic receptor (beta1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type beta1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type beta1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.

Probing the Reorganization of the Nicotinic Acetylcholine Receptor during Desensitization by Time-Resolved Covalent Labeling Using [3H]AC5, a Photoactivatable Agonist

The structural reorganizations occurring on the nicotinic acetylcholine receptor (nAChR) during activation and subsequent desensitization have been investigated through time-resolved photoaffinity labeling using a photoactivatable nicotinic agonist. [(3)H]AC5 is a photosensitive nicotinic probe with high affinity for the desensitized state of the Torpedo marmorata receptor (K(D) = 5 nM) that displays full agonist activity on the Torpedo californica receptor expressed in oocytes (EC(50) = 1.2 microM). Photoaffinity labeling of this receptor in the desensitized state showed a predominant specific labeling of gamma and delta subunits, whereas the alpha subunit was barely labeled. Using a stopped-flow device combined with a flash photolysis quenching system, we investigated the covalent mapping of the subunits as a function of incubation time of the receptor with [(3)H]AC5 (17 ms-1.25 h). During agonist-induced desensitization, specific labeling increased substantially, with similar time constants for gamma and delta subunits (0.016 s(-1)), whereas labeling of the alpha subunit remained relatively low. Therefore, the repartition of radioactivity shifted during desensitization from a weak but predominant labeling of the alpha and gamma subunits toward a substantial labeling of gamma and delta subunits. The observed time-dependent labeling pattern together with AC5 docking into a homology model of the T. californica nAChR suggest a subunit reorganization during agonist-induced desensitization, leading to a tightly packed arrangement that corresponds to a stable high affinity state for agonists.

The Interaction of a Constitutively Active Arrestin with the Arrestin-Insensitive 5-HT2AReceptor Induces Agonist-Independent Internalization

5-HT(2A) serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT(2A) receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT(2A) receptor phosphorylation. Because the arrestin-insensitivity of 5-HT(2A) receptors is cell-type selective, we used a "constitutively active" arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this "constitutively active" arrestin mutant (Arr2-R169E) can force 5-HT(2A) receptors to be regulated by arrestins. Cotransfection of 5-HT(2A) receptors with Arr2-R169E induced agonist-independent 5-HT(2A) receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT(2A) receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT(2A) receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT(2A) receptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor.

Pharmacokinetics and brain distribution in non human primate of R(-)[ 123I]DOI, A 5HT 2A/2C serotonin agonist

Our goal was to synthesize with high specific activity R(-)-1-(2,5-Dimethoxy-4-[ 123I]iodophenyl)-2-aminopropane [R(-)[ 123I]DOI], an in vitro potent and selective 5-HT 2A/2C serotonin agonist, and study in vivo its plasma pharmacokinetics and brain distribution in baboon by SPECT. The purpose was to evaluate this radiotracer as a potential tool in discerning the role of the agonist high affinity state of 5-HT 2 receptors in depression and other neurological disorders. The radiotracer was prepared by electrophilic radioiodination of the N-trifluoroacetyl precursor of R(-)-1-(2,5-Dimethoxyphenyl)-2-aminopropane [R(-)DMA-TFA] with high-purity sodium [ 123I]iodide in the presence of chloramine-T, followed by amino deprotection with KOH in isopropanol (labeling yield: 73%, radiochemical yield: 62%, radiochemical purity: 99%). In vivo studies in baboon showed high accumulation of radioactivity in thalamus, the frontoparietal cortex, temporal, occipital and the striatum regions, with slightly lower accumulation in the midbrain and cerebellum. Ketanserin did not displaced the radioactivity in any of these brain regions. Plasma metabolite analysis was performed using methanol protein precipitation, the methanol fractions contained from 68% to 92% of the mixture of a labeled metabolite and parent compound. The recovery coefficient of unmetabolized R(-)[ 123I]DOI was 68%. The percent parent compound present in the extracted fraction, measured by HPLC, decreased gradually with time from 99.8% to 0.3% still present after 4.7 hours post injection whereas the percentage of the only one detected metabolite increased conversely. Free fraction determination (f 1), was 31 ± 0.9% (n = 3). For comparison purposes, ex-vivo brain distribution, displacement and metabolite analysis was also carried out in rodents. Although R(-)[ 123I]DOI displayed good brain uptake and localized in serotonergic areas of the brain, its target to non target ratio and its insensitivity to ketanserin displacement suggest high nonspecific uptake, therefore non potentially useful as brain imaging radiotracer for visualization of the agonist high-affinity state of 5-HT 2A receptors and for visualizing 5-HT 2C receptors by SPECT.

Chapter 4 Cholesterol and steroid hormones: modulators of oxytocin receptor function

The function and physiological regulation of the oxytocin-receptor system is strongly steroid-dependent. This is, unexpectedly, only partially reflected by the promoter sequences in the oxytocin receptor and favors the idea that posttranscriptional mechanisms may also play a significant role for the physiological regulation of the oxytocin-receptor system. Our data indicate that cholesterol acts as an allosteric modulator of the oxytocin receptor and stabilizes both membrane-associated and solubilized OT receptors in a high-affinity state for agonists and antagonists. Moreover, high-affinity OT receptors are 2-fold enriched in cholesterol-rich plasma membrane domains in HEK293 fibroblasts stably expressing the human OT receptor. Biochemical data suggest a direct and cooperative molecular interaction of cholesterol molecules with OT receptors. To localize the cholesterol interacting domain of the oxytocin receptor the C-terminal part including the last two transmembrane domains have been exchanged by the corresponding sequences of the cholecystokinin type B receptor, which is functionally not dependent on cholesterol. Concerning its ligand-binding behavior this chimeric receptor protein showed the same dependence on cholesterol and its analogues as the wild type oxytocin receptor. From mutagenesis experiments and studies with receptor chimera between the OTR and cholecystokinin type B receptor, we conclude that a major part of the cholesterol interacting domain may be localized in the first part of the oxytocin receptor, possibly in a domain nearby the agonist binding site. Progesterone is considered to be essential to maintain the uterine quiescence. High concentrations of progesterone (> 10 microM) attenuate or block the signaling of several GPCRs, including the OT receptor via a fast, reversible and non-genomic pathway. Progesterone is known to inhibit both cholesterol biosynthesis and the intracellular trafficking of cholesterol. We therefore test the hypothesis that progesterone affects the signal transduction and subdomain localization of receptors via its influence on cholesterol trafficking. Since cholesterol-rich subdomains (rafts) are considered to be organization centers for cellular signal transduction, changes of the level or distribution of cholesterol may have profound effects on receptor-mediated signaling in general. Using fluorescence recovery after photobleaching (FRAP) measurements with GFP-tagged oxytocin receptors the influence of steroids on the mobility and distribution of the oxytocin receptor in the plasma membrane was analyzed. Progesterone had no effect on the lateral mobility of the oxytocin receptor, but it led to marked inhibition of cellular motility such as vesicle trafficking and movements of filopodia. Non-genomic effects of progesterone and estradiol with respect to receptor signaling as well as the influence of cholesterol on signal transduction will be discussed in more detail.

Receptor crosstalk protein, calcyon, regulates affinity state of dopamine D1 receptors

The recently cloned protein, calcyon, potentiates crosstalk between G s-coupled dopamine D1 receptors and heterologous G q/11-coupled receptors allowing dopamine D1 receptors to stimulate intracellular Ca 2+ release, in addition to cAMP production. This crosstalk also requires the participating G q/11-coupled receptors to be primed by their agonists. We examined the ability of calcyon and priming to regulate the affinity of dopamine D1 receptors for its ligands. Receptor binding assays were performed on HEK293 cell membrane preparations expressing dopamine D1 receptors either alone or in combination with calcyon. Co-expression of dopamine D1 receptor and calcyon affected neither the affinity of this receptor for antagonists nor the affinity of agonist binding to this receptor high and low-affinity states. However, the presence of calcyon dramatically decreased the proportion of the high-affinity dopamine D1 receptor agonist binding sites. This decrease was reversed by carbachol, which primes the receptor crosstalk by stimulating endogenous G q/11-coupled muscarinic receptors. Our findings suggest that calcyon regulates the ability of dopamine D1 receptors to achieve the high-affinity state for agonists, in a manner that depends on priming of receptor crosstalk.

Functional coupling with Gα q and Gα i1 protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli

To analyze the coupling of Gα subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Gα subunits covalently linked to the receptor C-terminus. The presence of Gα q or Gα i/q, in which the six C-terminal residues of Gα i1 were replaced with those from Gα q, increased the percentage of receptors in the agonist high-affinity state. This effect was less pronounced for wild-type Gα i1 and not observed for Gα i/s. Functional coupling of neurotensin receptor to Gα was demonstrated by neurotensin-induced [ 35S]GTPγS binding for the Gα q, Gα i/q and Gα i1 subunits, but not for Gα i/s. Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.

5-HT(1A) receptor agonist-antagonist binding affinity difference as a measure of intrinsic activity in recombinant and native tissue systems.

1. It has been reported that radiolabelled agonist : antagonist binding affinity ratios can predict functional efficacy at several different receptors. This study investigates whether this prediction is true for recombinant and native tissue 5-HT(1A) receptors. 2. Saturation studies using [(3)H]-8-OH-DPAT and [(3)H]-MPPF revealed a single, high affinity site (K(D)approximately 1 nM) in HEK293 cells expressing human 5-HT(1A) receptors and rat cortex. In recombinant cells, [(3)H]-MPPF labelled 3 - 4 fold more sites than [(3)H]-8-OH-DPAT suggesting the presence of more than one affinity state of the receptor. [(3)H]-Spiperone labelled a single, lower affinity site in HEK293 cells expressing h5-HT(1A) receptors but did not bind to native tissue 5-HT(1A) receptors. These data suggest that, in transfected HEK293 cells, human 5-HT(1A) receptors exist in different affinity states but in native rat cortical tissue the majority of receptors appear to exist in the high agonist affinity state. 3. Receptor agonists inhibited [(3)H]-MPPF binding from recombinant 5-HT(1A) receptors in a biphasic manner, whereas antagonists and partial agonists gave monophasic inhibition curves. All compounds displaced [(3)H]-8-OH-DPAT and [(3)H]-spiperone binding in a monophasic manner. In rat cortex, all compounds displaced [(3)H]-MPPF and [(3)H]-8-OH-DPAT in a monophasic manner. 4. Functional evaluation of compounds, using [(35)S]-GTPgammaS binding, produced a range of intrinsic activities from full agonism, displayed by 5-HT and 5-CT to inverse agonism displayed by spiperone. 5. [(3)H]-8-OH-DPAT : [(3)H]-MPPF pK(i) difference correlated well with functional intrinsic activity (r=0.86) as did [(3)H]-8-OH-DPAT : [(3)H]-spiperone pK(i) difference with functional intrinsic activity (r=0.96). 6. Thus agonist : antagonist binding affinity differences may be used to predict functional efficacy at human 5-HT(1A) receptors expressed in HEK293 cells where both high and low agonist affinity states are present but not at native rat cortical 5-HT(1A) receptors in which only the high agonist affinity state was detectable.

(3)H]-SB-269970--A selective antagonist radioligand for 5-HT(7) receptors.

Binding of the 5-HT(7) receptor antagonist radioligand [(3)H]-SB-269970 to human 5-HT(7(a)) receptors expressed in HEK293 cell membranes (h5-HT(7(a))/293) and to guinea-pig cerebral cortex membranes, was characterized and compared with [(3)H]-5-CT binding. [(3)H]-SB-269970 (1 nM) showed full association with h5-HT(7(a))/293 membranes after 40 min. Specific binding at equilibrium represented >90% of total binding and was fully reversible by methiothepin (10 microM), full dissociation occurring by 100 min. The association (k(+1)) and dissociation (k(-1)) rate constants were 0.05 nM(-1)min(-1) and 0.05 min(-1) respectively, giving a K(D) (k(-1)/k(+1)) of 1.0 nM. [(3)H]-SB-269970 bound saturably and apparently monophasically to both h5-HT(7(a))/293 and guinea-pig cortex membranes, with K(D) values of 1.25+/-0.05 and 1.7+/-0.3 nM respectively. The B(max) for [(3)H]-SB-269970 to both h5-HT(7(a))/293 and guinea-pig cortex membranes (5780+/-380 and 125+/-8.2 fmoles mg protein(-1) respectively) was similar to that for [(3)H]-5-CT (6190+/-940 and 143+/-19 fmoles mg protein(-1) respectively). These data suggest that, in each tissue, both radioligands labelled the same population of receptors, which appear to be present in an agonist high affinity state. The profile of compound inhibition of [(3)H]-SB-269970 binding to h5-HT(7(a))/293 and guineapig cortex membranes correlated well (corr. coeff. 0.98) with those for [(3)H]-5-CT binding and were consistent with the profiles reported previously for the human 5-HT(7(a)) and guinea-pig cortex 5-HT(7) receptors using [(3)H]-5-CT. Hill slopes for inhibition of [(3)H]-SB-269970 and [(3)H]-5-CT binding were close to 1, consistent with binding to a single receptor population in both tissues. [(3)H]-SB-269970 represents the first selective 5-HT(7) antagonist radioligand, which should aid further characterization of 5-HT(7) receptors in recombinant and native tissues and help establish their role in brain function.

Agonist high and low affinity state ratios predict drug intrinsic activity and a revised Ternary complex mechanism at serotonin 5‐HT2A and 5‐HT2C receptors

Agonist high and low affinity state ratios predict drug intrinsic activity and a revised Ternary complex mechanism at serotonin 5‐HT2A and 5‐HT2C receptors

Nonselective coupling of the human mu-opioid receptor to multiple inhibitory G-protein isoforms.

The human mu-opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2-1 pmol x mg-1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (mu, delta and kappa) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10-fold to 100-fold lower. Addition of heterotrimeric Gi,o-proteins purified from bovine brain shifted the mu-opioid receptor into a high-affinity state for agonists. Using individually purified Galpha-subunits re-associated with betagamma-dimers, we showed that alphao1, alphao2, alphai1, alphai2 and alphai3 reconstituted high-affinity agonist binding with equal efficiency. This suggests that the structural determinants of the mu-opioid receptor responsible for G-protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G-protein activity by cell-specific factors which should conveniently be analysed using the reconstitution assay described here.

Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.

Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies.

Activating mutations of the serotonin 5-HT2C receptor.

Site-directed mutagenesis was performed to create a mutant serotonin 5-HT2C receptor that would mimic the active conformation of the native receptor. Structural alteration of receptor conformation was achieved by changing amino acid no. 312 from serine to phenylalanine (S312F) or lysine (S312K). After expression in COS-7 cells, the binding affinity of 5-HT for [3H]mesulergine-labeled 5-HT2C receptors increased from 203 nM (native) to 76 nM for S312F and 6.6 nM for S312K mutant receptors. 5-HT potency for stimulation of phosphatidylinositol (PI) hydrolysis increased from 70 nM (native) to 28 nM for S312F and 2.7 nM for S312K mutant receptors. The mutant receptors were constitutively active, stimulating PI hydrolysis in the absence of agonist. S312F and S312K mutations resulted in twofold and five-fold increases, respectively, in basal levels of PI hydrolysis. Mianserin and mesulergine displayed inverse agonist activity by decreasing basal levels of PI hydrolysis stimulated by S312K mutant receptors. [3H]5-HT and [3H]mesulergine labeled the same number of S312K mutant receptors and 5'-guanylylimidodiphosphate had no effect on [3H]5-HT binding. These results indicate that serine --> lysine mutation at amino acid no. 312 produces an agonist high-affinity state of the 5-HT2C receptor that spontaneously couples to G proteins and stimulates PI hydrolysis in the absence of agonist.