Mutation of important amino acid residue of Asp104Lys in human beta(1)-adrenergic receptor triggers functional and constitutive inactivation.

Based on our previous molecular modeling and radioligand binding study, we have demonstrated that aspartic acid of 104 in transmembrane helix (TMH) II of beta(1)-adrenergic receptor (beta(1)-AR) is important for functional characteristics of these receptors. We have also showed that mutation of negatively charged aspartic acid to neutral charged alanine exhibited constitutive activity of beta(1)-AR. However, the mutation of negatively charged aspartic acid to positively charged lysine is still remained to be examined, which is very important to know for fully understanding the characteristics of beta(1)-AR. At the present study, we mutated aspartic acid to lysine (Asp104Lys) residue in human beta(1)-AR. This resultant mutant (Asp104Lys) markedly reduced the binding affinity of isoproterenol and (-)-epinephrine. On the other hand, antagonist binding with this mutant was similar to the wild type receptor. Isoproterenol at its saturation concentrations produced lower amount of intracellular cyclic adenosine-3',5' cyclic monophosphate (cAMP) in HEK-293 cells expressing Asp104Lys mutant receptor as compared to cells expressing wild type receptor. Moreover, cAMP accumulation of Asp104Lys mutant was unchanged in the presence or absence of isoproterenol. Therefore, it has been demonstrated that Asp104Lys mutation in the human beta(1)-AR differentially affects the binding of antagonist and exhibits a functional uncoupling of G-protein-coupled receptors. Thus, we may suggest that mutation of negatively charged aspartic acid to positively charged lysine as well as neutral charged alanine may help to understand the mechanism of the activation or inactivation of beta(1)-AR by its conformational changes and this finding would be helpful for clarifying the functional responses mediated by beta(1)-AR.