Radiolabelling of the human 5-HT2A receptor with an agonist, a partial agonist and an antagonist: effects on apparent agonist affinities.

Previous work has shown that 5-hydroxytryptamine (5-HT)2A receptors can be radiolabelled with various radioligands, including partial agonists, such as [125I]-DOI and [3H]-DOB, and antagonists, such as [3H]-ketanserin and [3H]-spiperone. Because 5-HT has high affinity for the 5-HT2A receptor when displacing [3H]-DOB, the purpose of the present study was to determine whether or not the receptor could be labelled with [3H]-5-HT and what would be the effect of labelling the receptor with various radioligands having differing efficacies at the receptor. Consequently, the human 5-HT2A receptor stably expressed in NIH 3T3 cells was radiolabelled with the endogenous agonist [3H]-5-HT, the partial agonist [3H]-DOB, and the antagonist [3H]-ketanserin. The receptor could be radiolabelled with [3H]-5-HT with a Kd value of 1.3 +/- 0.1 nM and a Bmax value of 3461 +/- 186 fmoles/mg protein and the radiolabelling was sensitive to the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate (GMP-PNP). Ketanserin labeled significantly more receptors (Kd = 1.1 +/- 0.1 nM: Bmax = 27,684 +/- 1500 fmoles/mg protein) than [3H]-DOB (Kd = 0.8 +/- 0.08 nM: Bmax = 8332 +/- 16 fmoles/mg protein) which, in turn, labelled significantly more receptors than [3H]-5-HT. The apparent affinity of antagonists did not change when the receptor was radiolabelled with either [3H]-agonists or [3H]-antagonists; however, agonists had a higher apparent affinity for [3H]-agonist-labeled receptors than for [3H]-antagonist-labeled receptors. Therefore, the apparent affinity of agonists for the 5-HT2A receptor estimated from displacement experiments depends on the intrinsic efficacy of the radioligand used.

[1]  F. Szele,et al.  High affinity agonist binding to cloned 5-hydroxytryptamine2 receptors is not sensitive to GTP analogs. , 1993, Molecular pharmacology.

[2]  T. de Boer,et al.  Genomic organisation and functional expression of the gene encoding the human serotonin 5-HT2C receptor. , 1994, European journal of pharmacology.

[3]  S. Peroutka,et al.  Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]mesulergine in a clonal 5-hydroxytryptamine1C cell line , 1992, Brain Research.

[4]  P. Conn,et al.  Regulation of serotonin-stimulated phosphoinositide hydrolysis: relation to the serotonin 5-HT-2 binding site , 1986, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[5]  S H Snyder,et al.  Multiple serotonin receptors: differential binding of [3H]5-hydroxytryptamine, [3H]lysergic acid diethylamide and [3H]spiroperidol. , 1979, Molecular pharmacology.

[6]  B. Roth,et al.  A single point mutation (Phe340-->Leu340) of a conserved phenylalanine abolishes 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]mesulergine but not [3H]ketanserin binding to 5-hydroxytryptamine2 receptors. , 1993, Molecular pharmacology.

[7]  D. Hoyer,et al.  Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin. , 1985, European journal of pharmacology.

[8]  R. Glennon,et al.  [3H]DOB: a specific agonist radioligand for 5-HT2 serotonin receptors. , 1985, European journal of pharmacology.

[9]  P. Conn,et al.  Serotonin-stimulated phosphoinositide turnover: mediation by the S2 binding site in rat cerebral cortex but not in subcortical regions. , 1985, The Journal of pharmacology and experimental therapeutics.

[10]  B. Hoffman,et al.  Molecular pharmacological differences in the interaction of serotonin with 5-hydroxytryptamine1C and 5-hydroxytryptamine2 receptors. , 1992, Molecular pharmacology.

[11]  P. Conn,et al.  Selective 5ht-2 antagonists inhibit serotonin stimulated phosphatidylinositol metabolism in cerebral cortex , 1984, Neuropharmacology.

[12]  D Rodbard,et al.  Ligand: a versatile computerized approach for characterization of ligand-binding systems. , 1980, Analytical biochemistry.

[13]  S. Peroutka,et al.  Differentiation of 5-hydroxytryptamine2 receptor subtypes using 125I-R- (-)2,5-dimethoxy-4-iodo-phenylisopropylamine and 3H-ketanserin , 1989, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[14]  S. Peroutka,et al.  Antagonism of 5-hydroxytryptamine2 receptor-mediated phosphatidylinositol turnover by d-lysergic acid diethylamide. , 1988, The Journal of pharmacology and experimental therapeutics.

[15]  J. Leysen,et al.  Evidence that phospholipid turnover is the signal transducing system coupled to serotonin-S2 receptor sites. , 1985, Journal of Biological Chemistry.

[16]  F. van Huizen,et al.  Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes. , 1992, European journal of pharmacology.

[17]  D. Hoyer,et al.  A proposed new nomenclature for 5-HT receptors. , 1993, Trends in pharmacological sciences.

[18]  I. Martin,et al.  Molecular biology of 5-HT receptors , 1994, Neuropharmacology.

[19]  P P Humphrey,et al.  International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (Serotonin). , 1994, Pharmacological reviews.

[20]  R. Loncharich,et al.  Species variations in transmembrane region V of the 5-hydroxytryptamine type 2A receptor alter the structure-activity relationship of certain ergolines and tryptamines. , 1994, Molecular pharmacology.

[21]  B. Hoffman,et al.  4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine and [3H]ketanserin labeling of 5-hydroxytryptamine2 (5HT2) receptors in mammalian cells transfected with a rat 5HT2 cDNA: evidence for multiple states and not multiple 5HT2 receptor subtypes. , 1990, Molecular pharmacology.