Asp125 and Thr130 in Transmembrane Domain 3 Are Major Sites of α_<1b>-Adrenergic Receptor Antagonist Binding(Pharmacology)

概要

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Site-directed mutagenesis was used to investigate the molecular interactions involved in prazosin binding to the human α_<1b>-adrenergic receptor (α_<1b>-AR) receptor. Based on molecular modeling studies, Thr130 and Asp125 in transmembrane region III of the α_<1b>-AR receptor were found to interact with prazosin. Thr130 and Asp125 were mutated to alanine (Ala) and expressed in HEK293 cells. The radioligand [^3H] prazosin did not show any binding to Asp125Ala mutant of α_<1b>-AR. Therefore, it was not possible to find any prazosin affinity to the mutant using the radioligand [^3H] prazosin. The mutation also abolished phenylephrine-stimulated inositol phosphate (IP) formation of [^3H] myo-inositol. On the other hand, the Thr130Ala mutant showed reduced binding affinity for [^3H] prazosin (dissociation constant, K_d 674.27 pM versus 90.27 pM for the wild-type receptor) and had reduced affinity for both tamsulosin and prazosin (11-fold and 9-fold, respectively). However, the Thr130Ala mutant receptor retained the ability to stimulate the formation of [^3H] myo-inositol. The results provide direct evidence that Asp125 and Thr130 are responsible for the interactions between α_<1b>-AR receptor and radioligand [^3H] prazosin as well as tamsulosin.
公益社団法人日本薬学会の論文
2007-10-01