Chimeric receptor analysis of the ketanserin binding site in the human 5-hydroxytryptamine(1D) receptor: Importance of the second extracellular loop and fifth transmembrane domain in antagonist binding

The 5-hydroxytryptamine (5-HT)(1B/1D) receptor subtypes are involved in the regulation of 5-HT release and have gained particular interest because of their apparent role in migraine. Although selective antagonists for both re ceptor subtypes recently have been developed, the receptor domains involved in the pharmacological specificity of these antagonists are defined poorly . This was investigated with a chimeric 5-HT1B/1D receptor analysis and usi ng ketanserin as a selective antagonist of h5-HT1D (h5-HT1D) K-i = 24-27 nM ) as opposed to h5-HT1B (K-i = 2193-2902 nM) receptors. A domain of the h5- HT1D receptor encompassing the second extracellular loop and the fifth tran smembrane domain is necessary and sufficient to promote higher affinity bin ding (K-i = 65-115 nM) for ketanserin to the h5-HT1B receptor. The same dom ain of the h5-HT1B receptor, when exchanged in the h5-HT1D receptor, abolis hed high affinity binding of ketanserin (K-i = 364-1265 nM). A similar obse rvation was made with the antagonist ritanserin and seems specific because besides the unmodified binding affinities for 5-HT and zolmitriptan, only m inor modifications (2-4-fold) were observed for the agonists L 694247 and s umatriptan and the antagonists GR 127935 and SE 224239. Generating point mu tations of divergent amino acids compared with the h5-HT1B receptor did not demonstrate a smaller peptide region related to a significant modification of ketanserin binding. The antagonists ketanserin and ritanserin are likel y to bind the h5-HT1D receptor by its second extracellular loop, near the e xofacial surface of the fifth transmembrane domain, or both.