[3H]-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and [3H] ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor.
The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to [3H]ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding the serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both [3H]DOB and [3H]ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate [Gpp(NH)p] to this system caused a rightward shift and steepening of agonist competition curves for [3H] ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity [3H]DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that [3H]DOB and [3H]ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein. These observations are consistent with the classical view of interconvertible agonist affinity states of GTP-binding protein-coupled receptors and strongly support the "two state" over the "two receptor" model for DOB binding to the 5-HT2 receptor.
Branchek
T
T
Neurogenetic Corporation, Paramus, New Jersey 07652.
Adham
N
N
Macchi
M
M
Kao
H T
HT
Hartig
P R
PR
D004290:DOM 2,5-Dimethoxy-4-Methylamphetamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007650:Ketanserin / Q000494:pharmacology*; D051379:Mice; D011985:Receptors, Serotonin / Q000145:classification / Q000187:drug effects* / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D000345:Affinity Labels; D011985:Receptors, Serotonin; D014316:Tritium; D004290:DOM 2,5-Dimethoxy-4-Methylamphetamine; C003943:2,5-dimethoxy-4-bromoamphetamine; D006165:Guanylyl Imidodiphosphate; D007650:Ketanserin