Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.     

CD52 is the ''major maturation-associated'' sperm membrane antigen

A thromboxane A2 receptor cDNA was isolated from a human placenta library by polymerase chain reaction (PCR) and was expressed in insect (Sf21) cells using baculovirus system. The recombinant receptor exhibited [3H]-SQ29548 and [125I]-BOP binding activities with Kd values of 1.01 +/- 0.09 nM and 1.63 +/- 0.23 nM, respectively. The receptor binding activity was inhibited by dithiothreitol in a time- and concentration-dependent manner, indicating the involvement of disulfide linkage in ligand binding. The role of the four conserved cysteinyl residues in ligand binding was further examined by site-directed mutagenesis. Each of the four cysteinyl residues was respectively mutated to a serine residue. C102S, C105S, and C183S mutants exhibited no ligand binding activity although successful expression was achieved as revealed by immunoblot analysis, whereas C257S mutant retained most of the binding activity. Homology analysis of all prostanoid receptors indicates that Cys-105 (first extracellular loop) and Cys-183 (second extracellular loop) are conserved and are presumed to form a disulfide bond for receptor stability as suggested by the inhibition of ligand binding by dithiothreitol reduction. Loss of binding activity by C102S mutant revealed that the sulfhydryl group of Cys-102 must play an essential role in ligand binding. Molecular modeling proposed that the Ser-201 is involved in interacting with TXA2 by forming hydrogen bonding. Point mutations of both Ser-201 and a conserved Ser-255 did not affect the ligand binding specificity and affinity for [3H]-SQ29548, but have significantly altered Kd values for [125I]-BOP. These results indicate that various cysteinyl and serine residues of thromboxane A2 receptor may play different roles in ligand binding.     

Identification of high-affinity binding sites for the insulin sensitizer rosiglitazone (BRL-49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferator-activated receptor gamma

A radioiodinated ligand, [125I]SB-236636 [(S)-(-)3-[4-[2-[N-(2-benzoxazolyl)-N-methylamino]ethoxy]3-[125I]i odo phenyl]2-ethoxy propanoic acid], which is specific for the gamma isoform of the peroxisomal proliferator activated receptor (PPARgamma), was developed. [125I]SB-236636 binds with high affinity to full-length human recombinant PPARgamma1 and to a GST (glutathione S-transferase) fusion protein containing the ligand binding domain of human PPARgamma1 (KD = 70 nM). Using this ligand, we characterized binding sites in adipose-derived cells from rat, mouse and humans. In competition experiments, rosiglitazone (BRL-49653), a potent antihyperglycemic agent, binds with high affinity to sites in intact adipocytes (IC50 = 12, 4 and 9 nM for rat, 3T3-L1 and human adipocytes, respectively). Binding affinities (IC50) of other thiazolidinediones for the ligand binding domain of PPARgamma1 were comparable with those determined in adipocytes and reflected the rank order of potencies of these agents as stimulants of glucose transport in 3T3-L1 adipocytes and antihyperglycemic agents in vivo: rosiglitazone > pioglitazone > troglitazone. Competition of [125I]SB-236636 binding was stereoselective in that the IC50 value of SB-219994, the (S)-enantiomer of an alpha-trifluoroethoxy propanoic acid insulin sensitizer, was 770-fold lower than that of SB-219993 [(R)-enantiomer] at recombinant human PPARgamma1. The higher binding affinity of SB-219994 also was evident in intact adipocytes and reflected its 100-fold greater potency as an antidiabetic agent. The results strongly suggest that the high-affinity binding site for [125I]SB-236636 in intact adipocytes is PPARgamma and that the pharmacology of insulin-sensitizer binding in rodent and human adipocytes is very similar and, moreover, predictive of antihyperglycemic activity in vivo.     

A detergent-solubilized nicotinic acetylcholine receptor of Caenorhabditis elegans

We have used a spin column assay to study the detergent-solubilized levamisole receptor, a nicotinic acetylcholine receptor of the nematode Caenorhabditis elegans. The receptor can be successfully solubilized in detergent solutions of Triton X-100, Lubrol PX, or sodium cholate. Centrifugal gel filtration assay using the tritiated ligand [3H]meta-aminolevamisole ([3H]MAL) provides a greater signal and a better signal-to-noise ratio for soluble levamisole receptor binding than either polyethylene glycol precipitation or DEAE filter assay with the same ligand. As for membrane-bound receptor, the detergent-solubilized levamisole receptor consists of more than one affinity state. Detergent solubilization appears to increase the affinity of all states for [3H]MAL (Kd for the highest affinity solubilized [3H]MAL binding state, 41 +/- 5 pM). Data is presented on the equilibrium binding and the association and dissociation reaction rates of the receptor. The similar relative efficacy with which various compounds inhibit specific [3H]MAL binding and deficiencies in solubilizable high affinity specific [3H]MAL binding in two receptor mutants show that the solubilized receptor is the same nicotinic acetylcholine receptor that is detected by assaying membrane-bound specific [3H]MAL binding. The detergent-solubilized levamisole receptor is stable at 0 degree to 4 degrees C, making receptor purification feasible.     

Membrane-induced secondary structures of neuropeptides: a comparison of the solution conformations adopted by agonists and antagonists of the mammalian tachykinin NK1 receptor

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.     

Importance of extracellular domains for ligand binding in the thyrotropin-releasing hormone receptor

The role of putative extracellular sequences for ligand binding in the TRH receptor was examined using deletion or substitution mutations. Each mutant receptor was transiently expressed in TRH receptor-minus GH(1)2C(1)b rat pituitary cells, and binding of 4 Nu Mu [3H]pGlu-N(tau)-MeHis-Pro-NH2 ([3H] MeTRH) was measured. When binding was not detected, signal transduction at 10 microM MeTRH was measured to assess receptor expression. Deletion of most of the N-terminal sequences (Glu(2)-Leu(22)), including two potential glycosylation sites, had no effect on the affinity of the receptor for MeTRH. Segmental deletions or simultaneous substitution of multiple amino acid residues in the first, second, or third extracellular loop (EL1, EL2, or EL3) resulted, however, in total loss of [3H]MeTRH binding, suggesting important roles for the loop sequences in either receptor expression or ligand binding. Individual substitutions were made to test further the role of the specific extracellular loop sequences in TRH binding. In EL1, conversion of Tyr93 to Ala resulted in more than 20-fold decrease in affinity for MeTRH. In EL2 and the top portion of the fifth transmembrane helix, conversion of Tyr181 to Phe, Tyr188 to Ala, and Phe199 to Ala resulted in a large ( > 100-fold) decrease in affinity for MeTRH, and conversion of Tyr 188 to Phe and Phe196 to Ala caused an agonist-specific 4- to 5-fold decrease in affinity. In EL3, conversion of Asn289 to Ala and of Ser290 to Ala caused a large ( > 100-fold) decrease in affinity for MeTRH. These results suggest important roles for the extracellular loops in high affinity TRH binding and lead us to propose a model in which TRH binds to the extra-cellular domain of its receptor.     

125I-Tyr1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes

Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-)2], both nonradioactive [I-Tyr1]biphalin and radioactive [125I-Tyr1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr1]biphalin to the delta and mu opioid receptors are not independent. [125I-Tyr1]Biphalin binds to delta receptors as shown in NG108-15 cell membranes. Nevertheless, [125I]biphalin binding to delta receptors in rat brain membranes was hardly evident and mu receptor binding predominated or at least was much more readily detectable in this preparation.     

NMR study of the transforming growth factor-alpha (TGF-alpha)-epidermal growth factor receptor complex. Visualization of human TGF-alpha binding determinants through nuclear Overhauser enhancement analysis

The study of human transforming growth factor-alpha (TGF-alpha) in complex with the epidermal growth factor (EGF) receptor extracellular domain has been undertaken in order to generate information on the interactions of these molecules. Analysis of 1H NMR transferred nuclear Overhauser enhancement data for titration of the ligand with the receptor has yielded specific data on the residues of the growth factor involved in contact with the larger protein. Significant increases and decreases in nuclear Overhauser enhancement cross-peak intensity occur upon complexation, and interpretation of these changes indicates that residues of the A- and C-loops of TGF-alpha form the major binding interface, while the B-loop provides a structural scaffold for this site. These results corroborate the conclusions from NMR relaxation studies (Hoyt, D. W., Harkins, R. N., Debanne, M. T., O'Connor-McCourt, M., and Sykes, B. D. (1994) Biochemistry 33, 15283-15292), which suggest that the C-terminal residues of the polypeptide are immobilized upon receptor binding, while the N terminus of the molecule retains considerable flexibility, and are consistent with structure-function studies of the TGF-alpha/EGF system indicating a multidomain binding model. These results give a visualization, for the first time, of native TGF-alpha in complex with the EGF receptor and generate a picture of the ligand-binding site based upon the intact molecule. This will undoubtedly be of utility in the structure-based design of TGF-alpha/EGF agonists and/or antagonists.     

Interaction of tryptamine and ergoline compounds with threonine 196 in the ligand binding site of the 5-hydroxytryptamine6 receptor

We examined the ligand-binding site of the 5-hydroxytryptamine6 (5-HT6) receptor using site-directed mutagenesis. Interactions with residues in two characteristic positions of trans-membrane region V are important for ligand binding in several bioamine receptors. In the 5-HT6 receptor, one of these residues is a threonine (Thr196), whereas in most other mammalian 5-HT receptors, the corresponding residue is alanine. After transient expression in human embryonic kidney 293 cells, we determined the effects of the mutation T196A on [3H]d-lysergic acid diethylamide (LSD) binding and adenylyl cyclase stimulation. This mutation produced a receptor with a 10-fold reduced affinity for [3H]LSD and a 6-fold reduced affinity for 5-HT. The potency of both LSD and 5-HT for stimulation of adenylyl cyclase was also reduced by 18- and 7-fold, respectively. The affinity of other N1-unsubstituted ergolines (e.g., ergotamine, lisuride) was reduced 10-30 fold, whereas the affinity of N1-methylated ergolines (e.g., metergoline, methysergide, mesulergine) and other ligands, such as methiothepine, clozapine, ritanserin, amitriptyline, and mainserin, changed very little or increased. This indicates that in wild-type 5-HT6 receptor, Thr196 interacts with the N1 of N1-unsubstituted ergolines and tryptamines, probably forming a hydrogen bond. Based on molecular modeling, a serine residue in transmembrane region IV of the 5-HT2A receptor has previously been proposed to interact with the N1-position of 5-HT. When the corresponding residue of the 5-HT6 receptor (Ala154) was converted to serine, no change in the affinity of twelve 5-HT6 receptor ligands or in the potency of 5-HT and LSD could be detected, suggesting that this position does not contribute to the ligand binding site of the 5-HT6 receptor.     

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.     

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.     

Identification of an opioid kappa receptor subtype-selective N-substituent for (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine

A three-component library of compounds was prepared in parallel using multiple simultaneous solution-phase synthetic methodology. The compounds were biased toward opioid receptor antagonist activity by incorporating (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (a potent, nonselective opioid pure antagonist) as one of the monomers. The other two monomers, which included N-substituted or unsubstituted Boc-protected amino acids and a range of substituted aryl carboxylic acids, were selected to add chemical diversity. Screening of these compounds in competitive binding experiments with the kappa opioid receptor selective ligand [3H]U69,593 led to the discovery of a novel kappa opioid receptor selective ligand, N-?(2'S)-[3-(4-hydroxyphenyl)propanamido]-3'-methylbutyl?-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (8, RTI-5989-29). Additional structure-activity relationship studies suggested that 8 possesses lipophilic and hydrogen-bonding sites that are important to its opioid receptor potency and selectivity. These sites appear to exist predominantly within the kappa receptor since the selectivity arises from a 530-fold loss of affinity of 8 for the mu receptor and an 18-fold increase in affinity for the kappa receptor relative to the mu-selective ligand, (+)-N-[trans-4-phenyl-2-butenyl]-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (5a). The degree of selectivity observed in the radioligand binding experiments was not observed in the functional assay. According to its ability to inhibit agonist stimulated binding of [35S]GTPgammaS at all three opioid receptors, compound 8 behaves as a mu/kappa opioid receptor pure antagonist with negligible affinity for the delta receptor.     

The history of radium in medicine in Manchester

An established senescence-accelerated model mouse strain, SAMP8, shows the deterioration of learning and memory compared with a normal control strain, SAMR1. D-Serine binds to strychnine-insensitive glycine binding sites of the N-methyl-D-aspartate (NMDA) receptor complex, and enhances glutamate binding to the receptor complex. To investigate the relationship of endogenous brain D-serine and the brain dysfunction caused by aging, the level of brain free D-serine and the D-[3H]serine binding to the brain samples were examined using the SAMP8 and SAMR1 mice. The free D-serine level was highest in the cerebral frontal and occipital cortices in both the SAMP8 and SAMR1; no difference in the D-serine level was shown between the two strains. A receptor autoradiographical analysis showed that the D-[3H]serine binding to the brain section was highest in the hippocampus, and the binding in the SAMP8 brains was lower than that of the SAMR1. The D-[3H]serine binding to the crude cerebral membranes indicated that the value of the total binding sites for the SAMP8 was lower than that for the SAMR1, whereas the value of the dissociation constant Kd for the SAMP8 was similar to that of the SAMR1. These results suggest that the number of D-[3H]serine binding sites was decreased in the SAMP8 compared to the SAMR1, but the affinity of D-[3H]serine to the binding sites was not altered. These results support the view that a decrease of NMDA receptor complex is involved in the age-related neural dysfunction of SAMP8 mice.     

Characterization of 5,7-dichlorokynurenate-insensitive D-[3H]serine binding to synaptosomal fraction isolated from rat brain tissues

To explore target sites for endogenous D-serine that are different from the glycine site of the N-methyl-D-aspartate (NMDA) type glutamate receptor, we have studied the binding of D-[3H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 microM) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 mM unlabeled D-serine. Association, dissociation, and saturation experiments indicated that D-[3H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K(D) of 614 nM and a Bmax of 2.07 pmol/mg of protein. D-Serine, L-serine, and glycine produced a total inhibition of the specific DCK-insensitive D-[3H]serine binding to the cerebellum with similar Ki values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 microM. The profiles of displacement of the DCK-insensitive D-[3H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive D-[3H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive D-[3H]serine binding site could be a novel candidate for a target for endogenous D-serine in mammalian brains.     

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol(-1). Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg(-1) of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs = 0.104 min(-1); K1 =0.034 min(-1): T1/2=20 min; K(+1)=0.07 min nM(-1). Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(delta), OP2(kappa) and OP3(mu). Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)-NH2([F/G]NC(1-13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH = NC-NH2-NC(1-13)-NH2 > > NC(1-12)-NH2 > NC(1-13)-OH > > NC(1-11)-NH2, and for NC and NC(1-13)-NH2 analogues: [Tyr1]NC-NH2 > or = [Leu1]NC(1-13)-NH2 > or = [Tyr1]NC(1-13)-NH2 > or = [F/G]NC(1-13)-NH2 > > [Phe3]NC(1-13)-NH2 > [DF/G]NC(1-13)-NH2. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 microM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.     

Functional analysis of GnRH receptor ligand binding using biotinylated GnRH derivatives

The objective of this study was to determine whether the gonadotrophin-releasing hormone (GnRH) ligand binds to the GnRH receptor (GnRH-R) with either the N- and C-termini or the beta-II turn pointing towards the cell. The functionality of GnRH and two biotinylated GnRH derivatives, biotin [D-Lys6]GnRH and biotin [Gln1]GnRH biotinylated at positions 6 and 1, respectively was assessed. Streptavidin was also used in combination with these peptides to investigate the effects of the steric hindrance caused by this molecule on ligand binding when bound to the biotin molecules at the two positions. GnRH bound to the receptor with high affinity, which was not affected by the addition of streptavidin. Both the biotinylated derivatives bound to the receptor though with lower affinities than GnRH. The biotin [D-Lys6]GnRH-streptavidin complex bound to the receptor albeit with lower affinity compared to biotin [D-Lys6]GnRH only, although it maintained its ability to cause receptor internalisation. The ability of the biotin [Gln1]GnRH to bind to the receptor was abolished in the presence of excess streptavidin. Both GnRH and biotin [D-Lys6]GnRH stimulated total inositol phosphate production whereas biotin [Gln1]GnRH exhibited GnRH antagonist activity. It appears that the small biotin molecule can be accommodated within the binding pore when attached to position 1 of the ligand but not when complexed to streptavidin. The fact that biotin [D-Lys6]GnRH maintains functionality when complexed to streptavidin while biotin [Gln1]GnRH does not, suggests that the N- and possibly the C-termini are required for receptor binding. Thus the most likely binding orientation for the ligand is with the N- and C-termini pointing inwards with the residue at position 6 pointing away from the binding site.     

Comparison of inversion-recovery gradient- and spin-echo and fast spin-echo techniques in the detection and characterization of liver lesions

For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1 angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1, Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.     

Biological and conformational examination of stereochemical modifications using the template melanotropin peptide, Ac-Nle-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2, on human melanocortin receptors

Examination of conformationally constrained melanotropin peptide (Ac-Nle4-c[Asp5-His-Phe7-Arg-Trp9-Ala-Lys]-NH2) on four human melanotropin receptors (hMC1R, hMC3R, hMC4R, and hMC5R) resulted in identifying the importance of ligand stereochemistry at positions 5, 7, and 9 for agonist binding affinity and receptor selectivity. A trend in ligand structure-activity relationships emerged for these peptides, with the hMC1R and hMC4R possessing similar tendencies, as did the hMC3R and hMC5R. alpha-MSH (Ac-Ser-Tyr-Ser-Met4-Glu-His-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), NDP-MSH (Ac-Ser-Tyr-Ser-Nle4-Glu-His-D-Phe7-Arg-Trp-Gly-Lys-Pro-Val-NH2), and MTII (Ac-Nle4-c[Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)-NH2) were also examined at each of these melanocortin receptors. Interestingly, the linear NDP-MSH possessed greater binding affinity for the hMC3R and hMC5R than did the cyclic analogue MTII. The peptide Ac-Nle-c[Asp-His-Phe-Arg-D-Trp9-Ala-Lys]-NH2 demonstrated the greatest differentiation in binding affinity between the hMC1R and hMC4R (78-fold). Analogue Ac-Nle-c[Asp-His-Phe7-Arg-Trp-Ala-Lys]-NH2 resulted in micromolar binding affinity (or greater) at the hMC3R and hMC5R, demonstrating the importance of D-Phe7 for ligand binding potency at these receptors. Ac-c[Asp-His-Phe-Arg-Trp-Ala-Lys]-NH2 resulted in loss of binding affinity at the hMC5R, implicating the importance of Nle4 (or a hydrophobic residue in this position) for binding to this receptor. Ac-Nle-c[D-Asp5-His-Phe-Arg-Trp-Ala-Lys]-NH2 was unable to competitively displace [125I]NDP-MSH binding at micromolar concentrations on the hMC3R and hMC5R, suggesting the importance of chirality of Asp5 either for ligand-receptor interactions or for orientation of the side chain lactam bridge and the structural integrity of the peptide conformation. Energy calculations performed for these peptides resulted in the identification of a low-energy ligand conformer family that is common to all the ligands. The differences in ligand binding affinities observed in this study are postulated to be a result of different ligand-receptor complexed interactions and not solely to the ligand structure.     

Polyneuropathy due to ethylene oxide, propylene oxide, and butylene oxide

Radioligand exchange was examined for its ability to derive the dissociation constant (Kd) of the rat ventral prostate nuclear androgen receptor. In one 24-h, 12 degrees C incubation, Scatchard plot analysis of [3H]dihydrotestosterone ([3H]DHT) exchange binding produced a Kd of 6.9 x 10(-9) M. Specific binding of [3H]DHT ranged from 114 to 758 pM, and the extrapolated value for the total number of binding sites (n) was 1320 pM. When aliquots from the same receptor pool were incubated with unlabeled DHT, and bound androgen was measured by radioimmunoassay, each titration point held a concentration of specifically bound unlabeled DHT little different from the preincubation value of bound endogenous ligand (1338 pM), suggesting that few, if any, unoccupied sites were created during the incubation. In a second radioligand exchange assay, unoccupied receptor sites were measured at the end of incubation. Virtually no unoccupied sites were found, though the range of predicted values was 124 to 383 pM (n = 425 pM). The data, in toto, suggest that although radioactive ligand exchanges with bound unlabeled ligand, the dynamics of the process do not include the creation of unoccupied sites. Since the Kd is determined by measuring the concentrations of unoccupied sites, free ligand, and receptor sites bound to ligand, the absence of unoccupied sites suggests that radioligand exchange cannot be used to directly determine the Kd of the prostate nuclear androgen receptor. The numerical value obtained from radioligand exchange, therefore, instead of being a Kd, is very likely the result of a graphic plot of the increase in specific activity of bound radioligand as [3H]DHT is titrated to higher levels. In the last phase of the study a technique was developed which allows for the correct determination of the Kd of the rat ventral prostate nuclear androgen receptor. For the determination, data from an experiment measuring uptake binding into unoccupied sites were combined with data obtained from radioligand exchange binding. From this, the Kd of the receptor was calculated to be 1 x 10(-12) M.     

3H]-BIBP3226 and [3H]-NPY binding to intact SK-N-MC cells and CHO cells expressing the human Y1 receptor

We have studied the binding of [3H]-NPY and the newly developed non-peptide Y1 receptor antagonist [3H]-BIBP3226 to intact SK-N-MC cells and CHO-K1 cells transfected with the human NPY Y1 receptor gene i.e. CHO-Y1 cells. Whereas the association and dissociation of the specific [3H]-NPY binding was slow, the binding kinetics of [3H]-BIBP3226 binding was very rapid. Saturation binding of both radioligands reveal the presence of an apparently homogeneous population of high affinity binding sites in both cell lines. The corresponding equilibrium dissociation constants are similar for the two cell lines and are close to those obtained from previous competition binding experiments. The specific binding of both radioligands was completely and with high affinity displaced by BIBP3226 and its inactive (S)-enantiomer BIBP3435 was much less potent. Whilst the NPY Y1 agonists NPY, PYY and [Leu31-Pro34]-NPY completely and potently displaced [3H]-NPY binding, they could only displace 70 to 80% of the [3H]-BIBP3226 binding sites in CHO-Y1 and SK-N-MC cells. A possible explanation can be that only part of the receptors are G-protein coupled. In agreement pertussis toxin was found to reduce high affinity [3H]-NPY binding sites in CHO-Y1 cells whereas [3H]-BIBP3226 binding parameters remained unchanged.