Receptor Agonist Affinity Research Articles

Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology.

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

Intracellular Allosteric Antagonist of the Olfactory Receptor OR51E2.

Olfactory receptors are members of class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs). Their expression and function have been increasingly studied in nonolfactory tissues, and many have been identified as potential therapeutic targets. In this manuscript, we focus on the discovery of novel ligands for the olfactory receptor family 51 subfamily E2 (OR51E2). We performed an artificial intelligence-based virtual drug screen of a ∼2.2 million small molecule library. Cell-based functional assay identified compound 80 (C80) as an antagonist and inverse agonist, and detailed pharmacological analysis revealed C80 acts as a negative allosteric modulator by significantly decreasing the agonist efficacy, while having a minimal effect on receptor affinity for agonist. C80 binds to an allosteric binding site formed by a network of nine residues localized in the intracellular parts of transmembrane domains 3, 5, 6, 7, and H8, which also partially overlaps with a G protein binding site. Mutational experiments of residues involved in C80 binding uncovered the significance of the C2406.37 position in blocking the activation-related conformational change and keeping the receptor in the inactive form. Our study provides a mechanistic understanding of the negative allosteric action of C80 on agonist-ctivated OR51E2. We believe the identification of the antagonist of OR51E2 will enable a multitude of studies aiming to determine the functional role of this receptor in specific biologic processes. SIGNIFICANCE STATEMENT: OR51E2 has been implicated in various biological processes, and its antagonists that can effectively modulate its activity have therapeutic potential. Here we report the discovery of a negative allosteric modulator of OR51E2 and provide a mechanistic understanding of its action. We demonstrate that this modulator has an inhibitory effect on the efficacy of the agonist for the receptor and reveal a network of nine residues that constitute its binding pocket, which also partially overlaps with the G protein binding site.

Adenosine A2A and histamine H3 receptors interact at the cAMP/PKA pathway to modulate depolarization-evoked [3H]-GABA release from rat striato-pallidal terminals.

We previously reported that the activation of histamine H3 receptors (H3Rs) selectively counteracts the facilitatory action of adenosine A2A receptors (A2ARs) on GABA release from rat globus pallidus (GP) isolated nerve terminals (synaptosomes). In this work, we examined the mechanisms likely to underlie this functional interaction. Three possibilities were explored: (a) changes in receptor affinity for agonists induced by physical A2AR/H3R interaction, (b) opposite actions of A2ARs and H3Rs on depolarization-induced Ca2+ entry, and (c) an A2AR/H3R interaction at the level of adenosine 3',5'-cyclic monophosphate (cAMP) formation. In GP synaptosomal membranes, H3R activation with immepip reduced A2AR affinity for the agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS-21680) (Ki control 4.53nM; + immepip 9.32nM), whereas A2AR activation increased H3R affinity for immepip (Ki control 0.63nM; + CGS-21680 0.26nM). Neither A2AR activation nor H3R stimulation modified calcium entry through voltage-gated calcium channels in GP synaptosomes, as evaluated by microfluorometry. A2AR-mediated facilitation of depolarization-evoked [2,3-3H]-γ-aminobutyric acid ([3H]-GABA) release from GP synaptosomes (130.4 ± 3.6% of control values) was prevented by the PKA inhibitor H-89 and mimicked by the adenylyl cyclase activator forskolin or by 8-Bromo-cAMP, a membrane permeant cAMP analogue (169.5 ± 17.3 and 149.5 ± 14.5% of controls). H3R activation failed to reduce the facilitation of [3H]-GABA release induced by 8-Bromo-cAMP. In GP slices, A2AR activation stimulated cAMP accumulation (290% of basal) and this effect was reduced (- 75%) by H3R activation. These results indicate that in striato-pallidal nerve terminals, A2ARs and H3Rs interact at the level of cAMP formation to modulate PKA activity and thus GABA release.

Development of potent and proteolytically stable human neuromedin U receptor agonists

Neuromedin U (NMU) is a highly conserved endogenous peptide that is involved in a wide range of physiological processes such as regulation of feeding behavior, the stress response and nociception. The major limitation to use NMU as a therapeutic is its short half-life. Here, we describe the development of a set of novel NMU-analogs based on NMU-8, by introducing unnatural amino acids into the native sequence. This approach shows that it is possible to generate molecules with increased potency and improved plasma stability without major changes of the peptidic nature or the introduction of large conjugates. When compared to the native NMU-8 peptide, compounds 16, 18 and 20 have potent agonist activity and affinity for both NMU receptors. Selectivity towards NMUR1 was observed when the Phe residue in position 4 was modified, whereas higher potencies at NMUR2 were found when substitutions of the Pro residue in position 6 were executed. To study the effect of the modifications on the proteolytic stability of the molecules, an in vitro stability assay in human plasma at 37 °C was performed. All analyzed analogs possessed an increased resistance against enzymatic degradation in human plasma resulting in half-lifes from 4 min for NMU-8, up to more than 23 h for compound 42.

Tolerance and cross-tolerance to head twitch behavior elicited by phenethylamine- and tryptamine-derived hallucinogens in mice.

The serotonin 5-hydroxytryptamine 2A (5-HT2A) receptor is a potential therapeutic target to a host of neuropsychiatric conditions, but agonist actions at this site are linked to abuse-related hallucinogenic effects that may limit therapeutic efficacy of chronic drug administration. Tolerance to some effects of hallucinogens has been observed in humans and laboratory animals, but the understanding of tolerance and cross-tolerance between distinct structural classes of hallucinogens is limited. Here, we used the drug-elicited head twitch response (HTR) in mice to assess the development of tolerance and cross-tolerance with two phenethylamine-derived [DOI (2,5-dimethoxy-4-iodoamphetamine) and 2C-T-7 (2,5-dimethoxy-4-propylthiophenethylamine)] and two tryptamine-derived [DPT (N,N-dipropyltryptamine) and DIPT (N,N-diisopropyltryptamine)] drugs with agonist affinity for 5-HT2A receptors. Tolerance developed to HTR elicited by daily DOI or 2C-T-7, but not to HTR elicited by DPT or DIPT. DOI-elicited tolerance was not surmountable with dose, and a similar insurmountable cross-tolerance was evident when DOI-tolerant mice were tested with various doses of 2C-T-7 or DPT. These studies suggest that the use of phenethylamine-derived hallucinogens as therapeutic agents may be limited not only by their abuse potential, but also by the rapid development of tolerance that would likely be maintained even if a patient were switched to a different 5-HT2A agonist medication from a distinct structural class. However, these experiments also imply that tryptamine-derived hallucinogens might have a reduced potential for tolerance development, compared with phenethylamine-derived 5-HT2A agonists, and might therefore be more suitable for chronic administration in a therapeutic context.

Changes in A<sub>2A</sub> adenosine receptor parameters in patients affected by bipolar disorders: Correlation with antipsychotic dosage and severity of illness

Typical antipsychotics, potent D2 dopamine receptor antagonists, are the most commonly used drugs in the treatment of bipolar disorders. In the central nervous system, the discovery of antagonistic interactions between A2A adenosine receptors and D2 dopamine receptors suggests that the adenosine system may be involved in the pathogenesis of different psychiatric disorders and in the therapeutic effectiveness of antipsychotic drugs. Previously, we have demonstrated an increase in A2A receptor expression and agonist affinity in platelets from psychotic patients treated with haloperidol. This result suggests that there is also a structural and functional interaction between A2A and D2 receptors in peripheral cells. In this work, we investigated the effect of different doses of typical drugs on A2A adenosine receptor binding and correlated these parameters with the severity of symptoms. We demonstrated, for the first time, that there was a strong correlation between A2A receptor affinity constant values (Kd) and drug doses in psychotic patients with a moderate severity of illness and moderate psychotic symptoms. The correlation was completely lost in patients with severe illness and severe psychotic symptoms. These results demonstrated that in platelets of patients affected by psychosis, typical antipsychotics modulated A2A receptor binding parameters; this regulation is dependent on the degree of D2 receptor occupancy in relation to the severity of psychotic symptoms, suggesting A2A receptors are a peripheral marker for individual therapy effectiveness.

An Ampa Receptor Unique Asp-Lys Salt-Bridge has a Role in Channel Function

Several of the many steps in the mechanism of activation of tetrameric GluA receptor-channels have been explored in a variety of published structural and functional studies. The main findings from them are that 1) Agonist binding induces closure of the L1/L2 of the isolated ligand-binding domain (LBD) that is less for partial agonists than for glutamate. 2) Crystal structures of GluA LBDs bound to full agonists manifest a 180o rotation around an Asp-Ser transpeptide bond in a flexible part of L2 that leads to the formation of cross-interface H-bonds also hypothesized to play a role in receptor activation. 3) Channel conductance increases with increasing agonist concentration, consistent with multiple LBDs in the tetramer contributing to full channel activation. Here we show that the Asp of the Asp-Ser pair also makes an electrostatic contact with a Lys residue in helix-F, and that disrupting this salt-bridge in GluA3flip receptors with point mutations at D655 and K660 decreased agonist potency by 2-10 fold. In D655A where it was examined, this occurred without altering intrinsic receptor affinity for agonists in ligand binding assays or CNQX by Schild analysis. The D655A mutation also increased the efficacy of kainate relative to glutamate, and the isolated GluA-D655A LDB bound to kainate was 3° more closed than in wildtype. Pharmacological studies of the wildtype and mutant receptors expressed in human embryonic kidney cells employed whole cell patch recordings and structure data was from crystals of isolated wildtype and mutant LBDs bound to full agonists and kainate. Taken together, these studies showed that breaking the D655-K660 contact altered channel gating, indicating that tethering a flexible part of L2 to helix-F may contribute to the rapid activation of GluA receptors.

Lipid rafts constrain basal α 1A-adrenergic receptor signaling by maintaining receptor in an inactive conformation

We have reported that the α 1A-adrenergic receptor (α 1AAR) in rat-1 fibroblasts is a lipid raft protein. Here we examined whether disrupting lipid rafts by methyl-β-cyclodextrin (MCD) sequestration of cholesterol affects α 1AAR signaling. Unexpectedly, MCD increased α 1AAR-dependent basal inositol phosphate formation and p38 mitogen-activated protein kinase activation in a cholesterol-dependent manner. It also initiated internalization of surface α 1AAR, which was partially blocked by receptor inhibition. Binding assays revealed MCD-mediated increases in receptor agonist affinity as well as reciprocal decreases in inverse agonist affinity, a behavior that is usually interpreted as a shift toward the active receptor conformation. In untreated cells a fraction of the receptor was found to be present in preassociated receptor/G protein complexes, which rapidly dissociate upon receptor stimulation. Consistent with MCD-induced signaling, raft disruption resulted in an increase in receptor/G protein complexes. These results strongly suggest that lipid rafts constrain basal α 1AAR activity; however, preassembled receptor/G protein complexes could still provide a mechanism for accelerating α 1AAR signaling following stimulation.

Identification of Dopamine D1–D3 Receptor Heteromers: INDICATIONS FOR A ROLE OF SYNERGISTIC D1–D3 RECEPTOR INTERACTIONS IN THE STRIATUM

The function of dopamine D(3) receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D(1)-D(3) receptor heteromers and for an intramembrane D(1)-D(3) receptor cross-talk in living cells and in the striatum. The formation of D(1)-D(3) receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D(1)-D(3) intramembrane receptor-receptor interaction was observed, by which D(3) receptor stimulation enhances D(1) receptor agonist affinity, indicating that the D(1)-D(3) intramembrane receptor-receptor interaction is a biochemical characteristic of the D(1)-D(3) receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D(1) and D(3) receptors. According to the synergistic D(1)-D(3) intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D(3) receptor stimulation potentiates D(1) receptor-mediated behavioral effects by a different mechanism than D(2) receptor stimulation. The present study shows that a main functional significance of the D(3) receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.

Antagonistic cannabinoid CB 1/dopamine D 2 receptor interactions in striatal CB 1/D 2 heteromers. A combined neurochemical and behavioral analysis

In vitro results show the ability of the CB 1 receptor agonist CP 55,940 to reduce the affinity of D 2 receptor agonist binding sites in both the dorsal and ventral striatum including the nucleus accumbens shell. This antagonistic modulation of D 2 receptor agonist affinity was found to remain and even be enhanced after G-protein activation by Gpp(NH)p. Using the FRET technique in living HEK-293T cells, the formation of CB 1–D 2 receptor heteromers, independent of receptor occupancy, was demonstrated. These data thereby indicate that the antagonistic intramembrane CB 1/D 2 receptor–receptor interactions may occur in CB 1/D 2 formed heteromers. Antagonistic CB 1/D 2 interactions were also discovered at the behavioral level through an analysis of quinpirole-induced locomotor hyperactivity in rats. The CB 1 receptor agonist CP 55,940 at a dose that did not change basal locomotion was able to block quinpirole-induced increases in locomotor activity. In addition, not only the CB 1 receptor antagonist rimonobant but also the specific A 2A receptor antagonist MSX-3 blocked the inhibitory effect of CB 1 receptor agonist on D 2-like receptor agonist-induced hyperlocomotion. Taken together, these results give evidence for the existence of antagonistic CB 1/D 2 receptor–receptor interactions within CB 1/D 2 heteromers in which A 2A receptors may also participate.

Extracellular proton modulates GABAergic synaptic transmission in rat hippocampal CA3 neurons

Acidification, which occurs in some pathological conditions, such as ischemia and hypoxia often induces neurotoxicity. The activation of acid-sensing ion channels (ASICs), which are highly permeable to calcium, has been considered the main target responsible for calcium overload in ischemic/hypoxic brain. However, the influence of extracellular proton on GABAergic synaptic transmission is not well understood. In the rat (aged 6–12 postnatal days) hippocampal CA3 neurons dissociated with an enzyme-free, mechanical method, we show that raising the extracellular pH (to 8.5) or lowering it (to 6.0) significantly increased or decreased, respectively, the frequency and the amplitude of spontaneous inhibitory postsynaptic currents mediated by γ-aminobutyric acid A (GABA A) receptors. Interestingly, these modifications were not altered by amiloride (100 μM, an antagonist for ASICs), tetrodotoxin (0.5 μM, a sodium channel blocker), cadmium (100 μM, a nonselective blocker for voltage-gated calcium channels), or a medium containing low calcium (0.5 mM). Significantly, changes in extracellular pH biphasically altered the peak amplitude of the currents elicited by exogenous GABA in CA3 neurons dissociated with enzyme. Raising the extracellular pH (to 8.5) or lowering (to 6.5) shifted the concentration–response curves of GABA to the left or right, respectively, without altering the maximal responses. These data suggest that proton alters the apparent affinity of GABA receptors for agonist. Thus, extracellular proton modifies GABAergic synaptic transmission both presynaptically and postsynaptically, and this could be independent of ASICs and voltage-gated calcium channels. Our finding may constitute a new mechanism underlying acidification-induced neurotoxicity.

Allosteric Modulation of Dopamine D2 Receptors by Homocysteine

It has been suggested that L-DOPA-induced hyperhomocysteinemia can increase the risk of stroke, heart disease, and dementia and is an additional pathogenetic factor involved in the progression of Parkinson's disease. In Chinese hamster ovary (CHO) cells stably cotransfected with adenosine A(2A) and dopamine D2 receptors, homocysteine selectively decreased the ability of D2 receptor stimulation to internalize adenosine A(2A)-dopamine D2 receptor complexes. Radioligand-binding experiments in the same cell line demonstrated that homocysteine acts as an allosteric D2 receptor antagonist, by selectively reducing the affinity of D2 receptors for agonists but not for antagonists. Mass spectrometric analysis showed that, by means of an arginine (Arg)-thiol electrostatic interaction, homocysteine forms noncovalent complexes with the two Arg-rich epitopes of the third intracellular loop of the D2 receptor, one of them involved in A(2A)-D2 receptor heteromerization. However, homocysteine was unable to prevent or disrupt A(2A)-D2 receptor heteromerization, as demonstrated with Fluorescence Resonance Energy Transfer (FRET) experiments in stably cotransfected HEK cells. The present results could have implications for Parkinson's disease.

Characterization of specific δ-opioid binding sites in the distal small intestine of swine

Delta-opioid receptors modulate neurogenic smooth muscle contractions and mucosal ion transport in the porcine distal small intestine. We compared specific δ-opioid binding sites in neuronal membranes isolated from the inner submucosal plexus and myenteric plexus of this gut region. In both preparations, the δ-opioid receptor antagonist naltrindole displaced [ 3H]diprenorphine from two binding sites. [ 3H]Naltrindole bound specifically to a single high-affinity site that was displaced by δ-opioid receptor ligands with the rank order of affinity: naltriben >7-benzylidenenaltrexone>deltorphin II>[ d-Pen 2, d-Pen 5] enkephalin. Relative decreases in δ-opioid receptor agonist affinity in the presence of Na + and other ions differed in submucosal and myenteric membranes. The κ-opioid receptor antagonist norbinaltorphimine displaced [ 3H]naltrindole binding in myenteric membranes ( K i=7.2 nM). Delta-opioid receptors in submucosal and myenteric plexuses of porcine ileum appear to differ in some respects.

Glutamate but not glycine agonist affinity for NMDA receptors is influenced by small cations.

NMDA receptor currents desensitize in an agonist-dependent manner when either the glutamate or glycine agonist is subsaturating. This may result from a conformational change in the NMDA receptor protein that lowers glutamate and glycine binding site affinity induced by co-agonist binding, channel opening, or ion permeation. We have used whole-cell voltage clamp of cultured hippocampal neurons with agonist paired-pulse protocols to demonstrate that glutamate and glycine dissociate 7.9- and 6.8-fold slower in the absence of their respective co-agonists than when their co-agonists are present. Paired-pulse and desensitization protocols were used to show that co-agonist binding and channel opening are sufficient to cause a reduction in glycine affinity, but extracellular sodium or magnesium binding was required in addition to conformational changes leading to channel opening to reduce glutamate binding-site affinity. Use of cesium or potassium as the major extracellular cation prevented the reduction of glutamate affinity. In addition, the use of choline-, sodium-, or cesium-based intracellular solutions did not alter desensitization characteristics, indicating that the site responsible for reduction of glutamate affinity is not in the intracellular domain. The fact that the reduction of glutamate affinity is dependent on certain small extracellular cations whereas the reduction of glycine affinity is insensitive to such cations indicates that conformational changes induced by the binding of glutamate are not completely paralleled by the conformational changes induced by glycine. Although glutamate and glycine are essential co-agonists, these data suggest that they have differential roles in the process of NMDA receptor activation.

Glutamate But Not Glycine Agonist Affinity for NMDA Receptors Is Influenced by Small Cations

NMDA receptor currents desensitize in an agonist-dependent manner when either the glutamate or glycine agonist is subsaturating. This may result from a conformational change in the NMDA receptor protein that lowers glutamate and glycine binding site affinity induced by co-agonist binding, channel opening, or ion permeation. We have used whole-cell voltage clamp of cultured hippocampal neurons with agonist paired-pulse protocols to demonstrate that glutamate and glycine dissociate 7.9- and 6.8-fold slower in the absence of their respective co-agonists than when their co-agonists are present. Paired-pulse and desensitization protocols were used to show that co-agonist binding and channel opening are sufficient to cause a reduction in glycine affinity, but extracellular sodium or magnesium binding was required in addition to conformational changes leading to channel opening to reduce glutamate binding-site affinity. Use of cesium or potassium as the major extracellular cation prevented the reduction of glutamate affinity. In addition, the use of choline-, sodium-, or cesium-based intracellular solutions did not alter desensitization characteristics, indicating that the site responsible for reduction of glutamate affinity is not in the intracellular domain. The fact that the reduction of glutamate affinity is dependent on certain small extracellular cations whereas the reduction of glycine affinity is insensitive to such cations indicates that conformational changes induced by the binding of glutamate are not completely paralleled by the conformational changes induced by glycine. Although glutamate and glycine are essential co-agonists, these data suggest that they have differential roles in the process of NMDA receptor activation.

Modulation of GABA(A) receptor channel gating by pentobarbital.

1. We have studied the kinetic properties of channel gating of recombinant alpha 1 beta 2 gamma 2L GABA(A) receptors transiently expressed in human embryonic kidney 293 cells, using the cell-attached, single-channel patch-clamp technique. The receptors were activated by GABA, beta-alanine or piperidine-4-sulfonic acid (P4S), and the effects of pentobarbital (PB) on single-channel activity were examined. 2. At relatively high concentrations of agonist, single-channel activity occurred in well-defined clusters. In global terms, PB increased the mean open time for events in clusters, without changing the mean closed time. The addition of PB shifted the curve relating the probability of being open in a cluster (P(o)) to lower agonist concentrations, and that shift could be accounted for by the changes in mean open time. 3. The intracluster closed-time histograms contained four components. The durations and relative frequencies of these closed-dwell components were not affected by the presence of 40 microM PB, at any agonist concentration. The duration of one component was dependent upon the concentration of agonist used to activate the receptor. Accordingly, the inverse of the mean duration of this component will be called the effective opening rate. 4. The channel-opening rate constant (beta) was determined from the value of the effective opening rate at a saturating agonist concentration. beta was about 1900 s(-1) when the receptors were activated by GABA, 1500 s(-1) when activated by beta-alanine, and too low to be determined when P4S was administered. In the presence of 40 microM PB, beta was about 1500 s(-1) when the receptors were activated by GABA, 1400 s(-1) when activated by beta-alanine, and 50 s(-1) when activated by P4S. Hence, the potentiating effect of PB is not mediated by a change in beta. The concentration of agonist producing a half-maximal effective opening rate also remained unaffected in the presence of PB, indicating that receptor affinity for agonists is not influenced by PB. 5. The distributions of the intracluster open durations elicited by GABA could be described by the sum of three exponentials, with mean durations of about 0.4, 2.4 and 6.3 ms. The duration and relative frequency of the components did not change with GABA concentration (20 microM to 1 mM). In the presence of 40 microM PB, however, the mean duration of the longest of the open times increased (mean durations of about 0.4, 2.0 and 13 ms). The intracluster open durations elicited by beta-alanine could be described by the sum of two exponential components (1.1 and 3.5 ms). However, in the presence of 40 microM PB the open-time distribution contained three exponential components (0.2, 2 and 10 ms). Finally, openings elicited by P4S exhibited two components (0.3 and 0.9 ms). In the presence of 40 microM PB, three components could be distinguished (0.5, 2.5 and 13 ms). 6. These observations indicate that the potentiating effect of PB on GABA type A (GABA(A)) receptors reflects effects on the open state(s) of the receptors. In the case of receptors activated by GABA, the observations are consistent with the idea that the action is the result of PB stabilizing one of the open states. The actions on receptors activated by P4S or beta-alanine are also broadly consistent with this idea. However, the changes in open-time distributions caused by PB appear to be more complex. Possible explanations of the effects of PB on gating by different agonists are considered.

Regulation of Formyl Peptide Receptor Agonist Affinity by Reconstitution with Arrestins and Heterotrimeric G Proteins

Although heptahelical chemoattractant and chemokine receptors are known to play a significant role in the host immune response and the pathophysiology of disease, the molecular mechanisms and transient macroassemblies underlying their activation and regulation remain largely uncharacterized. We report herein real time analyses of molecular assemblies involving the formyl peptide receptor (FPR), a well described member of the chemoattractant subfamily of G protein-coupled receptors (GPCRs), with both arrestins and heterotrimeric G proteins. In our system, the ability to define and discriminate distinct, in vitro receptor complexes relies on quantitative differences in the dissociation rate of a fluorescent agonist as well as the guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) sensitivity of the complex, as recently described for FPR-G protein interactions. In the current study, we demonstrate a concentration- and time-dependent reconstitution of liganded, phosphorylated FPR with exogenous arrestin-2 and -3 to form a high agonist affinity, nucleotide-insensitive complex with EC(50) values of 0.5 and 0.9 microm, respectively. In contrast, neither arrestin-2 nor arrestin-3 altered the ligand dissociation kinetics of activated, nonphosphorylated FPR. Moreover, we demonstrated that the addition of G proteins was unable to alter the ligand dissociation kinetics or induce a GTP gamma S-sensitive state of the phosphorylated FPR. The properties of the phosphorylated FPR were entirely reversible upon treatment of the receptor preparation with phosphatase. These results represent to our knowledge the first report of the reconstitution of a detergent-solubilized, phosphorylated GPCR with arrestins and, furthermore, the first demonstration that phosphorylation of a nonvisual GPCR is capable of efficiently blocking G protein binding in the absence of arrestin. The significance of these results with respect to receptor desensitization and internalization are discussed.

Mechanism and impact of allosteric AMPA receptor modulation by the Ampakine TM CX546

Glutamate release at central synapses is transduced into a characteristic fast postsynaptic response by AMPA receptor gating and agonist affinity. The effect of two classes of modulators of AMPA receptor desensitization, the benzothiadiazides (cyclothiazide and IDRA 21) and the benzoylpiperidines (CX516 and CX546), were studied on gating kinetics of recombinant, native AMPA receptors and on synaptic currents. CX546 reduced the degree of desensitization more potently than CX516 or IDRA 21, but not as efficiently as cyclothiazide. In presence of CX516/CX546, desensitization of GluR2 flip receptors was inhibited more than of GluR1 flip, whereas they had no effect upon response shape or conductance. CX546 increased agonist affinity threefold on nondesensitizing AMPA receptors by slowing agonist unbinding. Analysis of modulatory action suggests that, in contrast to cyclothiazide or IDRA 21, the Ampakine TM CX546 binds specifically to the agonist bound nondesensitized receptor, most likely acting by destabilizing the desensitized receptor conformation. All modulators tested showed higher efficiency on native receptors as compared to homomeric receptors. At the glutamatergic synapse, evoked synaptic amplitudes were weakly potentiated, while EPSC decay was slowed by nearly a factor of three in the presence of CX546 or cyclothiazide. In the presence of CX546, the current induced by short pulses of glutamate from recombinant GluR2 receptors decayed with a time course that was approximately twentyfold faster than EPSCs. The unique properties of CX546 may be beneficial for therapeutical use.

Effects of maturation and acute hypoxia on receptor-IP(3) coupling in ovine common carotid arteries.

Whereas previous studies have established that many mechanisms mediating pharmacomechanical coupling are subject to regulation, evidence of physiological regulation of the coupling efficiency between receptor activation and second-messenger production is scarce. The present studies address the hypothesis that acute hypoxia and maturation can influence the mass of second-messenger production for each activated agonist-bound receptor ("receptor gain"). For this assessment, receptor density and agonist affinity values were used to calculate 5-hydroxytryptamine (5-HT) concentrations that would produce standardized numbers of bound receptors (8.5 fmol/mg protein) in each experimental group and thus minimize effects of age or hypoxia on receptor density or agonist affinity. After 3 min of exposure to these 5-HT concentrations, normoxic magnitudes of contraction were similar (as %potassium maxima) in fetal (50 +/- 14%) and adult (40 +/- 9%) arteries, but hypoxia (PO(2) approximately 9--12 Torr for 30 min) depressed contractile tensions with a significantly different time course and magnitude in fetal (30 +/- 10%) and adult (17 +/- 11%) arteries (P < 0.05). Basal inositol 1,4,5-trisphosphate (IP(3)) values (in pmol/mg protein) were significantly greater in fetal (94 +/- 16) than in adult (44 +/- 6) arteries, and integrated areas above baseline for the IP(3) time courses (in nmol-s/mg protein) were significantly greater in fetal than in adult arteries both in normoxic (14.3 +/- 1.8 vs. 9.1 +/- 1.6) and hypoxic (15.0 +/- 2.1 vs. 8.6 +/- 1.2) conditions (P < 0.05). Hypoxia altered the IP(3) time courses both in the fetus and the adult but had no significant effect on IP(3 )mobilization or receptor gain. These data demonstrate that for the 5-HT(2a) receptor predominant in this preparation, receptor gain can be experimentally determined, is not influenced by acute hypoxia, but is greater in fetal than in adult ovine carotid arteries.

Acute hypoxia modulates 5-HT receptor density and agonist affinity in fetal and adult ovine carotid arteries.

In light of recent observations that receptor-ligand binding and coupling are physiologically regulated, the present study examined the hypothesis that the direct effects of hypoxia on vascular contractility involve modulation of pharmacomechanical coupling via changes in agonist affinity and/or receptor density. Because the direct effects of hypoxia on vascular smooth muscle contractility can vary with age, we carried out these experiments using both fetal and adult arteries. In common carotid arteries from near-term fetal and adult sheep, hypoxia (PO(2) = 9-12 Torr for 30 min) reduced the maximum responses to potassium by 17.8 +/- 3.5% (fetus) and 20.5 +/- 2.2% (adult), significantly reduced the pD(2) for 5-HT in the fetus (7.01 +/- 0.1 to 6.3 +/- 0.2) but not the adult (6.1 +/- 0.1 to 6.0 +/- 0.1), and significantly reduced 5-HT-induced maximum contractions (as % maximum response to 120 mM K(+)) not in the fetus (from 114 +/- 7 to 70 +/- 10%, not significant) but only in the adult (from 83 +/- 15 to 25 +/- 7%, P < 0.05) arteries. Hypoxia significantly attenuated 5-HT binding affinity (pK(A), determined by partial irreversible blockade with phenoxybenzamine) in both fetal (from 6.5 +/- 0.2 to 6.0 +/- 0.2) and adult arteries (from 6.2 +/- 0. 2 to 5.7 +/- 0.1) and also decreased receptor density (fmol/mg protein, determined by competitive binding with ketanserin and mesulergine) in adult (from 18.3 +/- 1.1 to 10.9 +/- 1.0) but not in fetal (21.0 +/- 1.0 to 23.2 +/- 1.4) arteries. These results suggest that acute hypoxia modulates receptor-ligand binding via age-dependent modulation of agonist affinity and receptor density. These effects may contribute to hypoxic vasodilatation and help explain why the effects of hypoxia on vascular contractility differ between fetuses and adults.