Amino acids of the human α1d-adrenergic receptor involved in antagonist binding

Computer simulations of the human alpha(1d)-adrenergic receptor (alpha(1d)-AR) based on the crystal structure of rhodopsin have been combined with experimental site-difected mutagenesis to investigate the role of residues in the transmembrane domains in antagonist binding. Our results indicate that the amino acids Asp 176 in the third transmembrane domain (TMD), Glu237 in TMD IV, and Ser258 in TMD V Of alpha(1d)-AR were directly involved in prazosin and tamsulosin binding. The Asp176Ala mutant did not exhibit any affinity for [H-3]prazosin and neither did it show agonist-stimulated inositol phosphates (IP) formation. On the other hand, the Glu237Ala and Ser258Ala mutant alpha(1d)-AR showed increased binding affinity for [H-3]prazosin. Competition binding experiments showed that prazosin affinity had increased to 5-fold and 3-fold in the Glu237Ala and Ser258Ala mutants, respectively, versus wild-type; and tamsulosin affinity only increased in the Ser258Ala mutant (2-fold vs wild-type). It seems that these two residues constrain the receptor by interaction with other residues and this disruption of the interaction increased the receptor's binding affinity towards antagonists. However, the Glu237Ala and Ser258Ala mutant receptors retained the ability to stimulate the formation of myo-[H-3]inositol but had activities lower than that of the wild-type receptor. The present results provide direct evidence that these amino acid residues are responsible for the interactions between alpha(1d)-AR and the radioligand [H-3]prazosin as well as tamsulosin.