Ring substituents on substituted benzamide ligands indirectly mediate interactions with position 7.39 of transmembrane helix 7 of the D4 dopamine receptor

In an effort to delineate how specific molecular interactions of dopamine receptor ligand classes vary between D2-like dopamine receptor subtypes, a conserved threonine in transmembrane (TM) helix 7 (Thr7.39), implicated as a key ligand interaction site with biogenic amine G protein-coupled receptors, was substituted with alanine in D2 and D4 receptors. Interrogation of different ligand chemotypes for sensitivity to this substitution revealed enhanced affinity in the D4, but not the D2 receptor, specifically for substituted benzamides (SBAs) having polar 4- (para) and/or 5- (meta) benzamide ring substituents. D4-T7.39A was fully functional, and the mutation did not alter the sodium-mediated positive and negative allostery observed with SBAs and agonists, respectively. With the exception of the non-SBA ligand (+)-butaclamol, which, in contrast to certain SBAs, had decreased affinity for the D4-T7.39A mutant, the interactions of numerous other ligands were unaffected by this mutation. SBAs were docked into D4 models in the same mode as observed for eticlopride in the D3 crystal structure. In this mode, interactions with TM5 and TM6 residues constrain the SBA ring position that produces distal steric crowding between pyrrolidinyl/diethylamine moieties and D4-Thr7.39. Ligand-residue interaction energy profiles suggest this crowding is mitigated by substitution with a smaller alanine. The profiles indicate sites that contribute to the SBA binding interaction and site-specific energy changes imparted by the D4-T7.39A mutation. Substantial interaction energy changes are observed at only a few positions, some of which are not conserved among the dopamine receptor subtypes and thus seem to account for this D4 subtype-specific structure-activity relationship.     

Selective modulation by membrane potential of desmethoxyverapamil binding to calcium channels in rat portal vein

(-)-[3H]Desmethoxyverapamil (D888) binds saturably to intact strips from rat portal vein bathed in physiological solution with a Kd value of 363 pM and a maximal binding capacity value of 15.6 fmol.mg-1 wet weight. Unlabeled dihydropyridines, phenylalkylamines and benzothiazepines inhibited (-)-[3H]D888 specific binding in a concentration-dependent manner. Scatchard analyses and dissociation kinetics of (-)-[3H]D888 binding revealed the existence of mutual allosteric interactions between (+)-isradipine, (+)-cis diltiazem and (-)-D888 binding sites in portal vein strips. When voltage-dependent Ca++ channels transported Ca++, Ba++, Sr++ or Na+ the binding capacity of (-)-[3H]D888 remained unchanged. In contrast, both depolarization (induced by elevation of external K+) and hyperpolarization (in the presence of cromakalim) induced a gradual decrease in (-)-D888 binding capacity. These observations suggest that membrane potential variation would change the conformational state of Ca++ channels, in such a way that it would be less favorable for access of (-)-[3H]D888 to the binding site. This would provide an experimental argument in favor of the "guarded receptor hypothesis" according to which membrane potential modulates ligand affinity by alteration of the amount of time during which the receptor binding site is available to (-)-[3H]D888.     

Binding of the dihydropyridine calcium channel blocker (+)-[3H] isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-5-methoxycarbonyl-2, 6-dimethyl-3-pyridinecarboxylate (PN200-110) to RINm5F membranes and cells: characterization and functional significance.

This report provides direct evidence for a dihydropyridine receptor/calcium channel in the insulin-secreting beta-cell line RINm5F. The receptor/channel can modulate the intracellular Ca++ concentration and the resultant insulin secretion by regulating the influx of extracellular Ca++ through dihydropyridine-sensitive voltage-dependent L-type Ca++ channels. Elevated extracellular K+ or the dihydropyridine Ca++ channel agonist, BAY k 8644 [methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethyl- phenyl)pyridine-5-carboxylate], stimulated the uptake of 45Ca++, raised [Ca++]i, and increased insulin secretion in a concentration-dependent manner. These actions were inhibited by L-type Ca++ channel blockers including nitrendipine, verapamil and diltiazem. (+)-[3H]PN200-110 bound specifically with high affinity to RINm5F cell membranes (Kd approximately 200 pM). Specific binding was inhibited competitively by dihydropyridines whereas phenylalkylamines inhibited incompletely (+)-[3H]PN200-110 binding, consistent with an allosteric interaction. The benzothiazepine diltiazem had no effect on (+)-[3H]PN200-110 binding in the presence of Ca++, but increased binding allosterically in the absence of Ca++ (in the presence of EGTA). Maximal (+)-[3H]PN200-110 binding required divalent cations, with Mg++, Mn++ and Ba++ essentially as effective as Ca++ in reversing the effects of EGTA, whereas binding was not supported by Cd++ or La . Specific high affinity (+)-[3H]PN200-110 binding was also demonstrated in intact RINm5F cells and shown to be modulated by membrane potential. Depolarization of the cells by raising extracellular K+ from 5 to 80 mM increased the affinity of (+)-[3H]PN200-110 4- to 5-fold (decreased Kd) with no significant effect on the maximum number of binding sites.     

Modulation of agonist binding to human dopamine receptor subtypes by L-prolyl-L-leucyl-glycinamide and a peptidomimetic analog

The present study was undertaken to investigate the role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) and its conformationally constrained analog 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) in modulating agonist binding to human dopamine (DA) receptor subtypes using human neuroblastoma SH-SY5Y cells stably transfected with respective cDNAs. Both PLG and PAOPA enhanced agonist [3H]N-propylnorapomorphine (NPA) and [3H]quinpirole binding in a dose-dependent manner to the DA D2L,D2S, and D4 receptors. However, agonist binding to the D1 and D3 receptors and antagonist binding to the D2L receptors by PLG were not significantly affected. Scatchard analysis of [3H]NPA binding to membranes in the presence of PLG revealed a significant increase in affinity of the agonist binding sites for the D2L, D2S, and D4 receptors. Analysis of agonist/antagonist competition curves revealed that PLG and PAOPA increased the population and affinity of the high-affinity form of the D2L receptor and attenuated guanosine 5'-(beta,gamma-imido)-triphosphate-induced inhibition of high-affinity agonist binding sites for the DA D2L receptor. Furthermore, direct NPA binding with D2L cell membranes pretreated with suramin, a compound that can uncouple receptor/G protein complexes, and incubated with and without DA showed that both PLG and PAOPA had only increased agonist binding in membranes pretreated with both suramin and DA, suggesting that PLG requires the D2L receptor/G protein complex to increase agonist binding. These results suggest that PLG possibly modulates DA D2S, D2L, and D4 receptors in an allosteric manner and that the coupling of D2 receptors to the G protein is essential for this modulation to occur.     

The binding-site crevice of the D4 dopamine receptor is coupled to three distinct sites of allosteric modulation

Most biogenic amine G protein-coupled receptors contain a conserved aspartic acid residue positioned near the intracellular side of the second transmembrane-spanning (TMS) domain that is the primary site of allosteric modulation by sodium ions and pH. Recently, zinc ions and amiloride derivatives were found to allosterically modulate antagonist binding to dopamine receptors. In the current study, the wild-type D4 dopamine receptor showed an 8-fold decrease in zinc affinity in the presence of 120 mM NaCl, but the binding of zinc to the neutral TMS2 D4-D77N mutant was completely sodium-insensitive. In contrast to zinc, methylisobutylamiloride (MIA) binding to the wild-type D4 receptor was virtually unaffected by sodium. In addition, the binding affinity for MIA was essentially unchanged in the presence of an IC(50) concentration of zinc and vice versa. Furthermore, MIA binding affinity was decreased 4-fold for the D4-D77N mutant and increased 30-fold for the TMS3 mutant D4-M107V, even though the binding affinity for zinc was similar to the wild-type D4 background for both mutants. These findings demonstrate for the first time the existence of three distinct sites of allosteric modulation within a G protein-coupled receptor.     

Classification of bovine aortic alpha-1 adrenoceptors by the criteria of ligand affinity and molecular mass

[3H]Prazosin bound to a single class of alpha-1 adrenoceptors in bovine aortic membranes with a Kd of 25 pM. Digitonin solubilized 30% of the receptors as assayed by specific [3H] prazosin binding. The rank order potency of displacing ligands was the same for both membrane-bound and soluble alpha-1 adrenoceptors [prazosin greater than phentolamine greater than yohimbine and (-)-epinephrine = (-)-norepinephrine much greater than (+)-norepinephrine]. Prazosin had a significantly lower affinity for the soluble receptor, whereas the other adrenergic ligands had the same affinity for both forms of the receptor. The alpha-1 adrenoceptor was affinity-labeled with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7- dimethoxyquinazoline and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the receptor binding subunit in both bovine aorta and rat liver was found to be 86,000 MW. It is concluded from this study that bovine aortic alpha-1 adrenoceptors have pharmacologic and biochemical characteristics similar to those in other tissues.     

Influence of ligand choice on the apparent binding profile of gallamine to cardiac muscarinic receptors. Identification of three main types of gallamine-muscarinic receptor interactions

The binding profile of the positively charged muscarinic antagonist, gallamine, was studied in rat heart homogenates. A proportion of the binding sites labeled by the tertiary muscarinic ligands [( 3H]quinuclidinyl benzilate (QNB) and [3H]atropine) were inaccessible to their quaternary analogs [( 3H]N-methyl-QNB (NMeQNB) and [3H]-N-methylscopolamine (NMS)] or gallamine. Whereas gallamine displaced the binding of [3H]NMeQNB with high affinity, biphasic competition curves were observed using [3H]NMS only at higher ligand concentrations. The rank order of potency of gallamine in allosterically decelerating ligand dissociation kinetics was: [3H]NMS greater than [3H]atropine greater than [3H]NMeQNB greater than [3H]QNB. Our calculations demonstrate that the displayed heterogeneity of gallamine binding sites detected using [3H]NMS, but not the tertiary ligands, might be accounted for by the allosteric modification of the binding of this ligand by gallamine. Based on these findings, the exhibited binding profile of gallamine to muscarinic receptors is influenced strongly by ligand choice, and also by the ligand concentration used in the binding experiment. Furthermore, it is concluded that gallamine binds to three major sites on the muscarinic receptor, thereby revealing an apparent heterogeneity of its binding sites, even in a tissue which presumably possesses one major muscarinic receptor subtype such as the heart. According to several lines of evidence, gallamine binds competitively and with high affinity to NMS-accessible sites on the receptor. Under certain experimental conditions, it also appears to identify another low-affinity site, either due to its binding to NMS-inaccessible sites or through its differential ability to alter the binding of ligands to the main binding domain on the receptor in an allosteric fashion.     

Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of [3H]prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate [Gpp(NH)p]. A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. [3H]Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with [3H]prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of critical residues in the amino terminal domain of the human NR2B subunit involved in the RO 25-6981 binding pocket

N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-alpha-(4-Hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.     

Allosteric modulation of muscarinic receptor signaling: alcuronium-induced conversion of pilocarpine from an agonist into an antagonist

Previous studies on allosteric interactions at muscarinic receptors have often focused on ligand-receptor binding interactions, because ligand binding seemed to reflect functional consequences. The prototypal allosteric agent alcuronium is known to bind with similar affinity to the M(2) subtype of muscarinic acetylcholine receptors whether or not the receptors are occupied by the agonist pilocarpine. To determine allosteric modulation of receptor signaling by alcuronium, the effects of pilocarpine were measured in contracting guinea pig left atria and on G-protein coupling in M(2)-transfected Chinese hamster ovary (CHO) cell membranes. Alcuronium dose-dependently suppressed pilocarpine-induced reduction of isometric contraction force in atria (pIC(50, Alc) = 5.63) without any effect on the EC(50) of pilocarpine, consistent with an allosteric mechanism. In contrast, alcuronium shifted the concentration-effect curve of the agonist oxotremorine M to the right without affecting the maximal effect, in a formally competitive manner (pK(A, Alc) = 5.54). If pilocarpine remained receptor bound in the presence of alcuronium, this indicates that pilocarpine can no longer act as an agonist. In support of this hypothesis, pilocarpine acted as a competitive antagonist against oxotremorine M in the presence of 10 microM alcuronium. Measuring guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in CHO-M(2) membranes yielded similar results. Alcuronium suppressed pilocarpine-induced stimulation of [(35)S]GTPgammaS binding (pIC(50, Alc) = 5.47) without shift in EC(50), whereas it competitively shifted the response to oxotremorine M (pK(A, Alc) = 5.97). [(3)H]Oxotremorine M binding data corresponded with the functional findings. In conclusion, alcuronium converted the agonist pilocarpine into an antagonist-a novel type of functional allosteric interaction.     

Mapping the structural requirements in the CB1 cannabinoid receptor transmembrane helix II for signal transduction

Amino acid residues in the transmembrane domains of the CB(1) receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB(1)) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(-)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl) hexahydrocannabinol (AM4056), and (-)-11-hydroxyldimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) for the stimulation of GTPgammaS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by approximately 50-fold) in the EC(50) for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.     

Zinc modulates antagonist interactions with D2-like dopamine receptors through distinct molecular mechanisms

Recently, zinc has been shown to modulate antagonist drug interactions with the D1 dopamine receptor (Schetz and Sibley, 1997) and the dopamine transporter (Norregaard et al., 1998). We now demonstrate that zinc also reversibly and dose-dependently modulates the specific binding of the butyrophenone antagonist [3H]methylspiperone to all D2-like dopamine receptors: D2L, D3, and D4. The molecular mechanisms of zinc regulation of these D2-like receptor subtypes are distinct because zinc inhibition of [3H]methylspiperone binding to the D4 receptor is noncompetitive by both equilibrium and kinetic measures (lower Bmax and essentially no change in koff), whereas the corresponding inhibition of zinc at D2L and D3 receptors is primarily characterized by competitive allosterism (increases in KD and koff). Interestingly, thermodynamic measurements reveal that the macroscopic properties of zinc binding are entropy-driven for all receptor subtypes, despite their having distinct molecular mechanisms. Zinc also reduces the binding affinity of the D2L receptor for [3H]raclopride, a structurally different antagonist of the substituted benzamide class. Sodium ions negatively modulate zinc inhibition of both sodium-insensitive [3H]methylspiperone binding and sodium-sensitive [3H]raclopride binding. In addition to its demonstrated effects on antagonist binding in membrane preparations, zinc also retards the functional effects of antagonist at the D2L receptor in intact cells. These findings suggest that synaptic zinc may be a factor influencing the effectiveness of therapies that rely on dopamine receptor antagonists.     

Non‐competitive interaction between raclopride and spiperone on human D2L‐receptors in intact Chinese hamster ovary cells

We recently investigated the binding properties of the antagonists [³H]‐raclopride and [³H]‐spiperone to intact Chinese hamster ovary cells expressing recombinant human D_2long‐dopamine receptors (CHO‐D_2L cells). Compared with saturation binding with [³H]‐raclopride, raclopride reduced [³H]‐spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [³H]‐raclopride binding non‐competitively. While raclopride only decreases the apparent K_D of [³H]‐raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [³H]‐raclopride binds with high affinity. Also, while the IC₅₀ of raclopride depends on the concentration of [³H]‐raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [³H]‐spiperone nor the rebinding of freshly dissociated [³H]‐spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [³H]‐raclopride and raclopride does not affect the (genuine) dissociation rate of [³H]‐spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D_2L‐receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [³H]‐raclopride and the other one with high affinity/accessibility for [³H]‐spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non‐reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D₂‐receptor antagonists.     

Characterization of the interaction of indiplon, a novel pyrazolopyrimidine sedative-hypnotic, with the GABAA receptor

Clinically used benzodiazepine and nonbenzodiazepine sedative-hypnotic agents for the treatment of insomnia produce their therapeutic effects through allosteric enhancement of the effects of the inhibitory neurotransmitter GABA at the GABA(A) receptor. Indiplon is a novel pyrazolopyrimidine sedative-hypnotic agent, currently in development for insomnia. Using radioligand binding studies, indiplon inhibited the binding of [(3)H]Ro 15-1788 (flumazenil) to rat cerebellar and cerebral cortex membranes with high affinity (K(i) values of 0.55 and 0.45 nM, respectively). [(3)H]Indiplon binding to rat cerebellar and cerebral cortex membranes was reversible and of high affinity, with K(D) values of 1.01 and 0.45 nM, respectively, with a pharmacological specificity consistent with preferential labeling of GABA(A) receptors containing alpha1 subunits. In "GABA shift" experiments and in measurements of GABA-induced chloride conductance in rat cortical neurons in culture, indiplon behaved as an efficacious potentiator of GABA(A) receptor function. In both the radioligand binding and electrophysiological experiments, indiplon had a higher affinity than zolpidem or zaleplon. These in vitro properties are consistent with the in vivo properties of indiplon as an effective sedative-hypnotic acting through allosteric potentiation of the GABA(A) receptor.     

Modulation of bovine aortic alpha-2 receptors by Na+, 5'-guanylylimidodiphosphate, amiloride and ethylisopropylamiloride: evidence for receptor G-protein precoupling

The influence of Na+ and 5'-Guanylylimidodiphosphate [Gpp(NH)p] on [3H]rauwolscine binding to alpha-2 adrenergic receptors was studied in plasma membranes prepared from bovine aorta. Both Na+ and Gpp(NH)p increased [3H]rauwolscine affinity while maximal binding capacity (Bmax) was significantly increased only with Gpp(NH)p. The increase in affinity was solely due to an increase in the association rate, while dissociation rate was not altered. In contrast, Na+ and Gpp(NH)p each lowered the affinity of the agonist epinephrine for both high- and low-affinity binding sites. The effects of Na+ and Gpp(NH)p on agonist binding were additive, such that only in their combined presence was a homogeneous class of low-affinity sites observed. This indicates that alpha-2 receptor-guanine nucleotide-binding protein (G-protein) interactions are modulated by both Na+ and Gpp(NH)p but via different mechanisms. Amiloride (100 or 300 microM) and ethylisopropylamiloride (10 microM) produced dose-dependent reductions in [3H]rauwolscine affinity, and, in the case of amiloride, also reduced Bmax. Competition at the [3H]rauwolscine binding site as well as noncompetitive, allosteric effects were present. The presence of Na+ augmented the ability of amiloride to reduce Bmax, indicating a shared locus of action. These findings illustrate that vascular alpha-2 receptors can interact with G-proteins even in the absence of agonists (i.e., receptor/G-protein precoupling) and that Na+ ion concentration regulates this interaction. Amiloride can occupy a Na(+)-shared binding site, causing an allosterically induced loss of receptor binding, suggesting that ligand binding and G-protein binding depend upon common receptor features.     

Characterization of [3H]quinpirole binding to D2-like dopamine receptors in rat brain.

The putative D2 dopamine receptor agonist quinpirole (LY 171,555) is the most widely used D2 agonist in in vivo and in vitro studies of D2 receptor-mediated effects. In addition, quinpirole may have even higher affinity for the recently described D3 dopamine receptor. The present study describes the in vitro binding properties of newly developed [3H]quinpirole in rat brain. [3H]Quinpirole binding was characterized in striatal membrane homogenate preparations using a filtration assay. Nonspecific binding was defined by 1 microM (+)-butaclamol. Specific [3H]quinpirole binding was saturable, and dependent on temperature, membrane concentration, sodium concentration and guanine nucleotides. Saturation analysis revealed high affinity binding characteristics (KD = 2.3 +/- 0.3 nM) which were confirmed by association-dissociation kinetics. The pharmacological profile of [3H]quinpirole binding in striatum was: (-)-N-n-propylnorapomorphine (+/-)-2-amino-6,7-dihydroxyl-1,2,3,4-tetrahydronaphthalene greater than or equal to quinpirole greater than apomorphine greater than bromocriptine greater than dopamine greater than SKF 38393 much greater than 5-hydroxytryptamine for putative dopamine agonists; spiperone greater than (+)-butaclamol greater than haloperidol greater than (-)-sulpiride greater than clozapine greater than SCH 23390 much greater than cinanserin for antagonists. [3H]Quinpirole binding exhibited stereoselectivity: (-)-sulpiride greater than (+)-sulpiride and (+)-butaclamol greater than (-)-butaclamol. This pharmacological profile is similar, though-not identical, to that observed for [3H] spiperone-labeled D2 receptors. The regional distribution of [3H]quinpirole binding sites roughly paralleled the distribution of [3H]spiperone binding sites, with greatest densities present in the striatum, nucleus accumbens and olfactory tubercles.(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain

Characterization of the adenosine A2 receptor has been limited due to the lack of available ligands which have high affinity and selectivity for this adenosine receptor subtype. In the present study, the binding of a highly A2-selective agonist radioligand, [3H]CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) is described. [3H]CGS 21680 specific binding to rat striatal membranes was saturable, reversible and dependent upon protein concentration. Saturation studies revealed that [3H]CGS 21680 bound with high affinity (Kd = 15.5 nM) and limited capacity (apparent Bmax = 375 fmol/mg of protein) to a single class of recognition sites. Estimates of ligand affinity (16 nM) determined from association and dissociation kinetic experiments were in close agreement with the results from the saturation studies. [3H]CGS 21680 binding was greatest in striatal membranes with negligible specific binding obtained in rat cortical membranes. Adenosine agonists ligands competed for the binding of 5 nM [3H]CGS 21680 to striatal membranes with the following order of activity; CGS 21680 = 5'-N-ethylcarboxamidoadenosine greater than 2-phenylaminoadenosine (CV-1808) = 5'-N-methylcarboxamidoadenosine = 2-chloroadenosine greater than R-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6cyclopentyltheophylline greater than S-phenylisopropyladenosine. The nonxanthine adenosine antagonist, CGS 15943A, was the most active compound in inhibiting the binding of [3H]CGS 21680. Other adenosine antagonists inhibited binding in the following order; xanthine amine congener = (1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 1,3-dipropyl-8-cyclopentylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than 8-phenyltheophylline greater than 8-cyclopentyltheophylline = xanthine carboxylic acid congener greater than 8-parasulfophenyltheophylline greater than theophylline greater than caffeine. The pharmacological profile of both adenosine agonist and antagonist compounds to compete for the binding of [3H]CGS 21680 was consistent with a selective interaction at the high affinity adenosine A2 receptor. A high positive correlation (r = 0.98, P less than .01) was observed between the pharmacological profile of adenosine ligands to inhibit the binding of [3H]CGS 21680 and the selective binding of [3H]NECA (+50 nM CPA) to high affinity A2 receptors. However, some differences between these assays were found for compounds which have moderate affinity and nonselective actions at both the A1 and A2 adenosine receptor subtypes. Unlike data obtained with nonselective adenosine ligands, the present results indicate that [3H]CGS 21680 directly labels the high affinity A2 receptor in rat brain without the need to block binding activity at the A1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

High-affinity dextromethorphan and (+)-3-(-3-hydroxyphenyl)-N-(1-propyl)piperidine binding sites in rat brain. Allosteric effects of ropizine.

Dextromethorphan (DM) binds to high- and low-affinity sites in the rat brain. The high-affinity DM binding is inhibited by nonnarcotic antitussives, opipramol and sigma ligands with nanomolar affinities. Computer-assisted modeling of homologous and heterologous competition studies between DM and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] were performed at pH 8.4. These experiments confirmed the existence of the common high-affinity DM1/sigma 1 site (R1) for which DM and (+)-3-PPP have Kd values of 20 and 10 nM, respectively. DM also binds to a second high-affinity site (R2, Kd, 20 nM) for which (+)-3-PPP has only micromolar affinity. Similarly, (+)-3-PPP binds to another high-affinity site (R3, Kd, 60 nM) for which DM has micromolar affinity. The common high-affinity DM1/sigma 1 site is allosterically modulated by the anticonvulsant ropizine, and is (+)-pentazocine sensitive, as is the homologous site in the guinea pig. However, in the rat the common DM1/sigma 1 site is 10 times smaller than in the guinea pig. This explains the apparently different effects of the allosteric modifiers in both species. The multiplicity of binding sites for DM and (+)-3-PPP resolved in this investigation will help to establish the physiological role and the pharmacological potential of the different sites. Meanwhile, the pharmacological effects of DM and sigma ligands cannot be summarily attributed to any particular binding site or receptor. This investigation also demonstrates that the use of multiple labeled and unlabeled ligands, combined with computer-assisted modeling, is essential to resolve multiple binding sites with similar affinities and to characterize the complex effects of allosteric modifiers.     

Sodium-dependent isomerization of dopamine D-2 receptors characterized using [125I]epidepride, a high-affinity substituted benzamide ligand

We have characterized the in vitro binding of a new ligand, [125I]epidepride, and used this substituted benzamide to assess the sensitivity of dopamine D-2 receptors to sodium. Both direct and indirect binding studies with [125I]epidepride and unlabeled epidepride, respectively, demonstrated that the affinity of D-2 receptors for the ligand was decreased from 20 to 30 pM in the presence of sodium to 350 to 500 pM in the absence of sodium. The density of binding sites for [125I]epidepride was identical in the presence and absence of NaCl. The time courses for association of [125I]epidepride to and dissociation from D-2 receptors in the presence of sodium were not consistent with simple bimolecular reactions, suggesting the possibility of a sodium-dependent ligand-induced receptor isomerization. Thus, dissociation of [125I]epidepride was biphasic in the presence of sodium, but monophasic in the absence of sodium. The rank order of potency for inhibition of [125I]epidepride binding by drugs was identical in rat striatum and cells expressing a D-2 receptor cDNA, and similar to the previously described pharmacological profile of D-2 receptors labeled by [3H]spiperone. [125I]Epidepride bound to two classes of binding sites in rat medial prefrontal cortex. One class, present at a density of 10 fmol/mg of protein and with a Kd value of approximately 40 pM, was pharmacologically indistinguishable from D-2 receptors in striatum and transfected cells. The pharmacological profile of the second class of sites was similar to that of alpha-2 adrenergic receptors. [125I]Epidepride had 50- to 100-fold lower affinity (approximately 2 nM) for alpha-2 receptors than for D-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of agonist and antagonist ligand-binding parameters at the human parathyroid hormone type 1 receptor: evaluation of receptor states and modulation by guanine nucleotide.

Determination of ligand-binding constants for parathyroid hormone (PTH) receptors has been hampered by a lack of suitable experimental systems and mechanistic models for data analysis. In this study, ligand binding to the cloned human PTH-1 receptor was measured using membrane-based radioligand-binding assays. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) (10 microM) reduced binding of agonist radioligands [125I]rPTH(1-34) and [125I]PTHrP(1-36) but only to a limited extent (by 29 +/- 5 and 42 +/- 3%, respectively). Radiolabeled agonist dissociation was described by three and two phases in the absence and presence of GTPgammaS, respectively. GTPgammaS treatment removed a pseudoirreversible binding phase. Inhibition of radiolabeled antagonist ([125I]bPTH(3-34)) binding was measured using a 90-min incubation, which allowed binding of ligands to closely approach the asymptotic maximum. Agonist/[125I]bPTH(3-34) displacement curves were fitted best by assuming two independent affinity states, both in the presence and absence of GTPgammaS. After a 3-h incubation, binding of PTH agonists in the presence of GTPgammaS was described by a single affinity state, indicating the presence of slow components in the binding reaction. Antagonist binding was described by a single affinity state and was not significantly affected by GTPgammaS. The data were used to evaluate potential receptor-binding models. Although other models could not be excluded, all of the observations could be explained by assuming two binding sites on the receptor that recognize two corresponding sites on agonist ligands. Using the model, it was possible to estimate receptor-ligand-binding constants and to propose a direct method for identifying ligands that interact with a putative antagonist binding region of the receptor.