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.
Evidence has accumulated indicating that the radioactive hallucinogens 4-bromo-[3H](2,5-dimethoxy)phenylisopropylamine ([3H]DOB) and 4-[125I]iodo-(2,5-dimethoxy)phenylisopropylamine ([125I]DOI) label an agonist high affinity state of the 5-hydroxytryptamine2 (5HT2) receptor and [3H]ketanserin labels both agonist high and low affinity states. Recently, an alternative hypothesis has been put forward proposing that the radioactive hallucinogens are labeling a 5HT2 receptor subtype distinct from the receptor labeled by [3H]ketanserin. In order to provide definitive evidence as to which of these hypotheses is correct, the rat 5HT2 receptor gene was transfected into NIH-3T3 (mammalian fibroblast) cells and COS (green monkey kidney) cells. Neither nontransfected cell type expresses 5HT2 receptors; the transfected cells expressed high affinity binding sites for both [125I] DOI (KD = 0.8 nM and Bmax = 363 fmol/mg in NIH-3T3 cells; KD = 0.2 nM and Bmax = 26 fmol/mg in COS cells) and [3H]ketanserin (KD = 0.4 nM and Bmax = 5034 fmol/mg in NIH-3T3 cells; KD = 1.0 nM and Bmax = 432 fmol/mg in COS cells). The affinities of agonists and antagonists for the [125I]DOI-labeled receptor were significantly higher than for the [3H]ketanserin-labeled receptor. The affinities of agonists and antagonists for these binding sites were essentially identical to their affinities for the sites radiolabeled by these radioligands in mammalian brain homogenates. The [125I]DOI binding was guanyl nucleotide sensitive, indicating a coupling to a GTP-binding protein. These data indicate that the 5HT2 receptor gene product contains both the guanyl nucleotide-sensitive [125I]DOI binding site and the [3H]ketanserin binding site. Therefore, these data indicate that the 5HT2 receptor gene product can produce a high affinity binding site for the phenylisopropylamine hallucinogen agonists as well as for the 5HT2 receptor antagonists. These results strongly support the two-state hypothesis for the 5HT2 receptor and do not support the multiple 5HT2 receptor subtype hypothesis.
Teitler
M
M
Department of Pharmacology and Toxicology, Albany Medical College, New York 12208.
Leonhardt
S
S
Weisberg
E L
EL
Hoffman
B J
BJ
D000662:Amphetamines / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects; D002522:Chlorocebus aethiops; D004247:DNA / Q000502:physiology; D007457:Iodine Radioisotopes; D007650:Ketanserin / Q000494:pharmacology*; D051379:Mice; D008954:Models, Biological; D051381:Rats; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D000662:Amphetamines; D007457:Iodine Radioisotopes; D011985:Receptors, Serotonin; D014316:Tritium; D004247:DNA; D007650:Ketanserin; C015952:4-iodo-2,5-dimethoxyphenylisopropylamine