The unique nature of the serine interactions for alpha(1)-adrenergic receptor agonist binding and activation

Activation of the beta(2)- and alpha(2)-adrenergic receptors (AR) involves hydrogen bonding of serine residues in the fifth transmembrane segment (TMV) to the catechol hydroxyls of the endogenous agonists, epinephrine and norepinephrine, With the beta(2)-AR both Ser(204) and Ser(207) but not a third TMV serine (Ser(203)) are required for binding and full agonist activity, However, with the alpha(2a)-AR only one of two TMV serines (Ser(204), equivalent to Ser(207) in the beta-AR) appears to contribute partially 60 agonist-binding and activation, Because the alpha(1a)-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in alpha(1)-AR activation, Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine-substitution mutants that lacked either Ser(188) (Ser(188) --> Ala) or Ser(192) (Ser(192) --> Ala). With the substitution of both serines in the double mutant, Ser(188/192) --> Ala, binding of all three ligands was significantly reduced (10-100-fold) consistent with a single hydrogen bond interaction, However, receptor-mediated inositol phosphate production was markedly attenuated only with the Ser(188) --> Ala mutation and not with Ser(192) --> Ala. In support of the importance of Ser(188), binding of phenylephrine (meta-hydroxyl only) by Ser(192) --> Ala increased 7-fold over that observed with either the wild type receptor or the Ser(188) --> Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser(192) --> Ala mutation, In addition, when combined with a recently described constitutively active alpha(1a)-AR mutation (Met(292) --> Leu), only the Ser(188) --> Ala mutation and not Ser(192) --> Ala relieved the high affinity binding and increased agonist potency observed with the Met(292) --> Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser(188), and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser(192), that allows receptor activation, Furthermore, since Ser(188) and Ser(192) are separated by three residues on the TMV alpha-helix, whereas Ser(204) and Ser(207) of the beta(2)-AR are separated by only two residues, the orientation of the catechol ring in the alpha(1)-AR binding pocket appears to be unique and rotated approximately 120 degrees to that in the beta(2)-AR.