An Evaluation of the Interactions of Antiestrogens with Pituitary and Striatal Dopamine Receptors

Abstract

We have examined the ability of various antiestrogens (AE's) to compete with ³H-spiroperidol for binding to membrane preparations from striatal tissue and anterior pituitary glands of immature female rats in order to determine the affinity of binding of AE's to D-2 dopamine receptors. Scatchard analyses revealed the presence of a single class of high affinity receptor sites in both the striatum and pituitary with a dissociation constant (K_d) of 0.33 nM and 0.40 nM, respectively, for the dopamine antagonist spiroperidol. The AE's tamoxifen, 4-hydroxy-tamoxifen (TAM-OH), CI-628, LY 117018, and a structurally related compound t-butyl-phenoxyethyl diethylamine (BPEA) were all able to compete with spiroperidol for binding to D-2 receptors and demonstrated relative binding affinities of 0.4-0.06%, with spiroperidol set at 100%. Dopamine displayed a lower affinity, 0.01%. Estradiol failed to compete with spiroperidol for D-2 receptor binding while the non-steroidal estrogen diethylstilbestrol (DES) showed very week competition. For the lipophilic AE's, alteration of the level of their non-specific binding greatly affected their relative affinities in these competitive binding assays. The amine side chain on an aromatic ring appears to be a critical structural requirement in allowing the AE's to bind to the dopamine receptor. The relatively low affinity of AE's for the dopamine receptor and the high degree of interaction of AE's with other proteins suggest that only limited occupancy of D-2 receptors by AE's is likely in vivo.     

Characterization of [3H]LS‐3‐134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand

LS‐3‐134 is a substituted N‐phenylpiperazine derivative that has been reported to exhibit: (i) high‐affinity binding (K_i value 0.2 nM) at human D3 dopamine receptors, (ii) > 100‐fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low‐affinity binding (K_i > 5000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin‐dependent activation of the adenylyl cyclase inhibition assay, LS‐3‐134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [³H]‐labeled LS‐3‐134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10–15 min at 37°C) and binds with high affinity to both human (K_d = 0.06 ± 0.01 nM) and rat (K_d = 0.2 ± 0.02 nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [³H]LS‐3‐134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [³H]LS‐3‐134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.     

3H-dopamine binding to rat striatal D-2 and D-3 sites: Enhancement by magnesium and inhibition by guanine nucleotides and sodium

Previous studies have demonstrated high affinity ³H-dopamine binding sites on mammalian striatal membranes. These putative dopamine receptors of unknown physiological significance have been termed D-3 sites. Such studies have failed, however, to demonstrate high affinity ³H-dopamine binding to D-2 sites, which can be labeled by ³H-butyrophenones, and which represent the putative dopamine receptors most stronly implicated in the behavioral correlates of dopaminergic CNS activity. We now report that preincubation of membrane homogenates with Mg⁺⁺ and inclusion of Mg⁺⁺ (1–10mM) or other divalent metal cations during binding allows high affinity D-2 specific ³H-dopamine binding in rat striatal membranes, and that these ions also increase the Bmax of D-3 specific ³H-dopamine binding. GTP, GDP, and GppNHp can completely abolish all D-2 specific ³H-dopamine binding, while only a magnesium-dependent portion of D-3 sites appears to be GTP sensitive. These data are consistent with the hypothesis that the striatal D-2 receptor exists in two agonist affinity states whose interconversion is effected by guanine nucleotides and divalent metal cations. The GTP sensitive/magnesium dependent nature of ³H-dopamine binding to so-called D-3 sites suggests that some such sites may in fact represent a high agonist-affinity state of the D-1 adenylate cyclase stimulating dopamine receptor also found in this tissue.     

Preparation of 123I-labeled 2′-iodospiperone and imaging of D2 dopamine receptors in the human brain using SPECT

[¹²³I]2′-ISP was readily prepared using a radioiodine exchange reaction with a radiochemical yield of approx. 50% after HPLC purification. The radiochemical purity of the product was more than 98% and the specific activity was 5.55–11.1 GBq/μmol. Biodistribution studies performed in mice indicated that injection of [¹²³I]2′-ISP with albumin produced a higher gastric uptake and a lower brain uptake than injection of the radioligand in a weakly acidic solution. In addition, toxicity tests performed in mice demonstrated that acute toxic effects would be very unlikely to be encountered if 2′-ISP was used for diagnostic purposes. A preliminary imaging study with [¹²³I]2′-ISP in a healthy human volunteer showed its specific uptake by the basal ganglia, a region of the brain known to have a high density of D₂ dopamine receptors.     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

Comparison of Neuroleptic Binding Characteristics in Rat Striatum and Renal Cortex

Abstract

The binding characteristics of the dopaminergic ligand, ³H- spiperone, were compared in renal cortical and striatal membrane homogenates of the rat. This ligand labelled a single class of high affinity binding sites in striatum with an apparent dissociation constant (Kd) of 0.13 nM and a maximal number of binding sites (Bmax) of 890 fmol/mg protein representing D-2 receptors. In the renal cortex, ³H-spiperone identified a population of binding sites with a Bmax and a Kd of 310 fmol/mg protein and 5.1 nM, respectively. The antagonist displacing profile suggests the dopaminergic nature of the renal binding site. The affinities of dopamine antagonists for the peripheral ³H-spiperone binding site were in general in the micromolar range while the affinities of D-2 or D-2/D-1 dopamine antagonists in striatum were in the nanomolar range. Moreover, these sites showed differential stereoselectivity for (+)- and (-)-isomers of sulpiride. In conclusion, the presence of a D-2/DA-2 dopamine receptor population in renal cortex could not be confirmed. The pharmacological properties of the peripheral ³H-spiperone binding site are also different from the DA-1 receptor but seem to resemble those previously reported for dopamine receptors in sympathetic ganglia and adrenal medulla.     

Affinities of methylphenidate derivatives for dopamine,norepinephrine and serotonin transporters

We have synthesized several derivatives of dl-threo-methylphenidate (Ritalin) bearing substituents on the phenyl ring. IC₅₀ values for binding of these compounds to rat brain monoamine transporters were assessed using [³H]WIN 35,428 (striatal membranes, dopamine transporters, DAT), [³H]nisoxetine (frontal cortex membranes, norepinephrine transporters, NET) and [³H]paroxetine (brain stem membranes, 5HT transporters, 5HTT). Affinities (1/Ki) decreased in the order: DAT > NET ⪢ 5HTT. Substitution at the para position of dl-threo-methylphenidate generally led to retained or increased affinity for the dopamine transporter (bromo > iodo > methoxy > hydroxy). Substitution at the meta position also increased affinity for the DAT (m-bromo > methylphenidate; m-iodo-p-hydroxy > p-hydroxy). Substitution at the ortho position with bromine considerably decreased affinity. Similar IC₅₀ values for binding of o-bromomethylphenidate to the dopamine transporter were measured at 0, 22 and 37 degrees. N-Methylation of the piperidine ring of methylphenidate also considerably reduced affinity. The dl-erythro isomer of obromomethylphenidate did not bind to the DAT (IC₅₀ > 50,000 nM). Affinities at the dopamine and norepinephrine transporters for substituted methylphenidate derivatives were well correlated (r² = 0.90). Abilities of several methylphenidate derivatives to inhibit [³H]dopamine uptake in striatal synaptosomes corresponded well with inhibition of [³H]WIN 35, 428 binding. None of the compounds examined exhibited significant affinity to dopamine D1 or D2 receptors (IC₅₀ > 500 or 5,000 nM, respectively), as assessed by inhibition of binding of [³H]SCH 23390 or [¹²³I]epidepride, respectively, to striatal membranes.     

The characteristics of adrenoceptors in homogenates of human cerebral cortex labelled by (3H)-rauwolscine

The alpha-2-adrenoceptor antagonist (³H)-rauwolscine has been used to label adrenoreptors in membranes from human cerebral cortex. The radioligand binds with high affinity (K_D 2.08 nM) to a single population of sites with a density of 135 fmoles/mg protein. Adrenoceptor antagonists displaced binding in a simple monomolecular fashion with an order of affinity rauwolscine > yohimbine > phentolamine > corynanthine > prazosin indicating binding to alpha-2-adrenoceptors. Agonists displaced with an order of affinity clonidine > (-) adrenaline > (-) noradrenaline > dopamine > (-) isoprenaline but all displayed apparent Hill coefficients less than unity indicating heterogeneity of binding. The relatively high affinity of the alpha-1 antagonist prazosin for (³H)-rauwolscine binding sites in rat cerebral cortex was not observed in the human tissue which had pharmacological properties similar to those described previously in human platelet.     

Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

An iodinated compound, [125I]-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin -7-ol, has been recently reported [Sidhu, A., & Kebabian, J.W. (1985) Eur. J. Pharmacol. 113, 437-440] to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained their ability to functionally couple to the HeLa adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chronic Antagonist Treatment Does not Alter the Mode of Interaction of Dopamine with Rat Striatal Dopamine Receptors

Abstract

Chronic treatment with the D₁ and D₂ dopamine receptor antagonists SCH 23390 (0.5 mg/kg) and haloperidol decanoate (25 mg/kg) caused an up-regulation in D₁ and D₂ receptor densities, respectively, with no change in K_D. Dopamine (20 μM) interacted with both receptor subtypes in a mixed competitive/non-competitive manner, causing a reduction in ligand binding affinity and an apparent decrease in receptor density. In the presence of dopamine, both vehicle-treated and SCH 23390-treated striatal preparations showed a significant loss in affinity for ³H-SCH 23390 binding to D₁ receptors and a decrease in D₁ receptor density of approximately 26%. Similarly, dopamine caused a substantial loss in ³H-spiperone binding affinity to D₂ receptors and a 46% decrease in B_max in both vehicle-treated and haloperidol-treated membranes. Thus, receptor up-regulation does not appear to alter the mode of interaction of dopamine with rat striatal dopamine receptors.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D2 and D3 receptors

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

[3H]Spiroxatrine labels a serotonin1A-like site in the rat hippocampus

[³H]Spiroxatrine was examined as a potential ligand for the labeling of 5-HT_1A sites in the rat hippocampus. Analysis of the binding of [³H]spiroxatrine in the absence and presence of varying concentrations of three monoamine neurotransmitters revealed that serotonin (5-HT) had high affinity (IC₅₀= 20.7 nM for the [³H]spiroxatrine binding sites, consistent with the labeling of 5-HT₁ sites, while dopamine and norepinephrine had very low affinity (IC₅₀=57600 nM and >10⁻⁴ M respectively). Saturation studies of the binding of [³H]spiroxatrine revealed a single population of sites with a K_d=2.21 nM. Further pharmacologic characterization with the 5-HT_1A ligands 8-hydroxy-2-(di-n-propylamino) tetralin, ipsapirone, and WB4101 and the butyrophenone compounds spiperone and haloperidol gave results that were consistent with [³H]spiroxatrine labeling 5-HT_1A sites. This ligand produced stable, reproducible binding with a good ratio of specific to nonspecific binding. The binding of [³H]spiroxatrine was sensitive to GTP, suggesting that this ligand may act as an agonist. This was supported by the finding that spiroxatrine inhibits forskolin-stimulated adenylate cyclase activity (a proposed 5-HT_1A receptor model) in the rat hippocampus. Since [³H]spiroxatrine is structurally distinct from other currently available radioligands for the 5-HT_1A site, it should provide new information about the properties of this putative serotonergic receptor.     

Pharmacological Characterization of Mammalian D1 and D2 Dopamine Receptors Expressed in Drosophila Schneider‐2 Cells

Abstract

Mammalian D₁ and D₂ dopamine receptors were stably expressed in Drosophila Schneider‐2 (S2) cells and screened for their pharmacological properties. Saturable, dose‐dependent, high affinity binding of the D₁‐selective antagonist [³H]SCH‐23390 was detected only in membranes from S2 cells induced to express rat dopamine D₁ receptors, while saturable, dose‐dependent, high affinity binding of the D₂‐selective antagonist [³H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D₂ receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D₁ and D₂ receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D₁ receptor bound the (+)‐enantiomer of the D₁‐selective agonist SKF38393 with higher affinity than the (?)‐enantiomer, while the dopamine D₂ receptor bound the (?)‐enantiomer of the D₂‐selective agonist norpropylapomorphine with higher affinity than the (+)‐enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPγS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D₁ and D₂ receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D₁ and D₂ dopamine receptors free of promiscuous G protein contaminants.     

Preparation and biological evaluation of 18F-labeled benzamide analogs as potential dopamine D2 receptor ligands

Three ¹⁸F-labeled benzamide derivatives were prepared and evaluated as potential ligands to study the dopamine D₂ receptor phenomenon. The compounds are analogs of iodobenzamide, eticlopride and raclopride and are labeled with an N-2-[¹⁸F]fluoroethyl functionality on the pyrrolidine ring. The compounds were tested in vitro for binding affinity and found to exhibit somewhat lower affinity than the non-fluorinated analog. In vivo distribution studies revealed that all compounds were more highly bound to plasma proteins than was raclopride. In addition, compartmentation of radioactivity demonstrated nonspecific binding to be the predominate retention in the brain as reflected by the low caudate to cerebellum ratios for these compounds. These three ¹⁸F-labeled benzamide derivatives are inferior to raclopride and iodobenzamide for studies of the D₂ receptor system using positron emission tomography.     

Design,Synthesis, Lipophilic Properties,and Binding Affinities of Potential Ligands in Positron Emission Tomography (PET) for Visualization of Brain Dopamine D4 Receptors

We report the synthesis of compounds structurally related to the high‐affinity dopamine D₄ receptor ligand N‐{2‐[4‐(3‐cyanopyridin‐2‐yl)piperazin‐1‐yl]ethyl}‐3‐methoxybenzamide ( 1e ). All compounds were specifically designed as potential PET radioligands for brain D₄ receptor visualization, having lipophilicity within a range for brain uptake and weak non‐specific binding (0.75<cLogP<3.15) and bearing a substituent for easy access to labeling with the positron emitter isotope ¹¹C or ¹⁸F. The best compound of the series, N‐{2‐[4‐(4‐chlorophenyl)piperazin‐1‐yl]ethyl}‐6‐fluoropyridine‐3‐carboxamide ( 7a ), displayed excellent selectivity over D₂ and D₃ receptors (>100‐fold), but its D₄ receptor affinity was suboptimal for imaging of brain D₄ receptors (K_i=30 nM ).     

Characterization of Muscarinic Acetylcholine Receptors in Cultured Bovine Aortic Endothelial Cells

Abstract

The binding characteristics of [³H]quinuclidinyl benzilate ([³H]QNB) to isolated crude membranes of cultured bovine aortic endothelial cells were investigated. [³H]QNB bound to endothelial cell membranes with high affinity (k_D = 0.056 nM) and limited capacity (132 fmol/mg DNA). The binding specificity, order of affinity and inhibition constants (K_i) were determined by displacement of bound [³H]QNB with unlabeled ligands. The order of affinity was QNB > atropine > 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) > p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) (M₃ antagonist) > pirenzepine (M₁ antagonist) > AFDX-116 (M₂ antagonist) > (4-hydroxy-2-butynyl) trimethylammonium chloride m-chlorocarbanilate (McN-A-343, M₁ agonist). These observations suggest that muscarinic receptors of endothelial cells in culture are likely to be of M₃ and M₁ subtype. Northern blot analysis of receptor subtypes using cDNA probes did not provide conclusive results due to the low level expression of these receptors in cultured cells. Solubilization of protein bound [³H]QNB with 1% digitonin and 0.02% cholate followed by analysis on sucrose density gradients demonstrated the presence of a specifically bound [³H]QNB-protein complex sedimenting at the 6.2S region of the gradient. These data demonstrate the presence of muscarinic acetylcholine receptor protein in cultured bovine aortic endothelial cells.     

Synthesis and biological evaluation of C-2 halogenated analogs of salvinorin A

Salvinorin A (1), the main active ingredient of Salvia divinorum, is a potent and selective κ opioid receptor (KOPR) agonist. Based on the SAR, its C-2 position is one of the key binding sites and has very little space tolerance (3–4 carbons atoms) and limited to only lipophilic groups. In our attempt to prepare PET brain imaging agent for mapping KOPR, a series of C-2 halogenated analogs have been synthesized and screened for binding affinity at κ (KOPR), μ (MOPR), and δ (DOPR). These C-2 halogenated analogs with sequential changes of atomic radius and electron density serve as excellent molecular probes for further investigating the binding pocket at C-2, particularly on the effects of α verses β configuration at C-2 position. The results of KOPR binding and functional studies reveal β isomer in general binds better than α isomer with the exception of iodinated analogs and none of the C-2 halogenated analogs shows any improvement of KOPR binding affinity. Interestingly, functional assay has characterized that 6b is a partial agonist with E_max of 46% of the kappa receptor full agonist U50,488H at 250 nM (K_i). We have also observed that the affinity to the kappa receptor increases with atomic radius (I > Br > Cl > F) which is in good agreement with halogen bonding interactions reported in the literature.     

Purification of bovine striatal dopamine D-2 receptor by affinity chromatography

Bovine striatal dopamine D-2 receptor has been purified approximately 2000-fold by affinity chromatography. The receptor, solubilized with cholic acid and sodium chloride, was adsorbed on haloperidol-linked Sepharose CL-6B and eluted with spiroperidol. The adsorption of receptor to the affinity matrix was biospecific as preincubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor. The process also displayed stereoselectivity with respect to (+)- and (-)-butaclamol. Nondopaminergic agents such as mianserin and propranolol failed to exhibit any effect on the adsorption process. Elution of the receptor was also biospecific, as dopaminergic drugs were most effective (spiroperidol greater than haloperidol greater than dopamine) in eluting the bound receptor; whereas other agents, e.g. propranolol, mianserin, and acetic acid, were only slightly effective. One-cycle affinity purification resulted in a recovery of 12% of the original membrane-bound dopamine D-2 receptor with a specific activity of 169,600 fmol/mg of protein as assayed with [3H]spiroperidol binding. The order of potency of D-2 agonists (N-propylnorapomorphine greater than NO434 greater than apomorphine greater than dopamine) and antagonists (spiroperidol greater than (+)-butaclamol greater than domperidone) with the purified preparation was found to be similar to that of the solubilized dopamine D-2 receptor.     

Identification of a hexapeptide binding region in the nociceptin (ORL1) receptor by photo-affinity labelling with Ac-Arg-Bpa-Tyr-Arg-Trp-Arg-NH2

The interaction of Ac-Arg-Tyr-Tyr-Arg-Trp-Arg-NH₂ (HP1), a high-affinity partial agonist of the opioid receptor like (ORL1) receptor, has been investigated using the photo-labile analogue [p-benzoyl-l-Phe (Bpa)²]-HP1. In recombinant CHO cells expressing the human ORL1 receptor, [Bpa²]-HP1 binds the receptor with high affinity (K; ∼3 nM) and is as potent as HP1 in stimulating GTPγS binding (50-60% of nociceptin maximal effect). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radio-iodinated [Bpa²]-HP1 results in the irreversible labelling of a glycoprotein of M_r∼66 kDa, as determined by SDS-PAGE. Cyanogen bromide (CNBr) and enzymatic footprints of the photo-labelled receptor and an engineered receptor mutant (L113M), containing an additional CNBR cleavage site, allowed the photoreactive region to be identified as ORL1[107-113] at the C-terminal of TM helix II. In addition the presence of a disulphide bridge between Cysl23 and Cys200 has been confirmed biochemically.