Abstract
Site-directed mutagenesis was used to investigate the molecular interactions involved in prazosin binding to the human alpha(1b)-adrenergic receptor (alpha(1b)-AR) receptor. Based on molecular modeling studies, Thr130 and Asp125 in transmembrane region III of the alpha(1b)-AR receptor were found to interact with prazosin. Thr130 and Asp125 were mutated to alanine (Ala) and expressed in HEK293 cells. The radioligand [(3)H]prazosin did not show any binding to Asp125Ala mutant of alpha(1b)-AR. Therefore, it was not possible to find any prazosin affinity to the mutant using the radioligand [(3)H]prazosin. The mutation also abolished phenylephrine-stimulated inositol phosphate (IP) formation of [(3)H]myo-inositol. On the other hand, the Thr130Ala mutant showed reduced binding affinity for [(3)H]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 [(3)H]myo-inositol. The results provide direct evidence that Asp125 and Thr130 are responsible for the interactions between alpha(1b)-AR receptor and radioligand [(3)H]prazosin as well as tamsulosin.
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