title: Asp125 and Thr130 in transmembrane domain 3 are major sites of alpha1b-adrenergic receptor antagonist binding.
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.
authors.affiliation: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Aoba, Niigata, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Takahashi
authors.forename: Murad
authors.initials: M
authors.lastname: Hossain
authors.forename: Maruf
authors.initials: M
authors.lastname: Ahmed
authors.forename: Mohiuddin Ahmed
authors.initials: MA
authors.lastname: Bhuiyan
authors.forename: Toshio
authors.initials: T
authors.lastname: Ohnuki
authors.forename: Takafumi
authors.initials: T
authors.lastname: Nagatomo
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000378:metabolism*; D000409:Alanine / Q000737:chemistry / Q000235:genetics; D001224:Aspartic Acid / Q000737:chemistry*; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D008024:Ligands; D008958:Models, Molecular; D010716:Phosphatidylinositols / Q000378:metabolism; D011224:Prazosin / Q000378:metabolism; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry*; D014162:Transfection
chemical_list: C547716:ADRA1B protein, human; D000317:Adrenergic alpha-Antagonists; D018076:DNA, Complementary; D008024:Ligands; D010716:Phosphatidylinositols; D018340:Receptors, Adrenergic, alpha-1; D013912:Threonine; D001224:Aspartic Acid; D000409:Alanine; D011224:Prazosin