The unique nature of the serine interactions for α1-adrenergic receptor agonist binding and activation

Abstract

Activation of the β2- and α2-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 β2-AR both Ser204 and Ser207 but not a third TMV serine (Ser203) are required for binding and full agonist activity. However, with the α(2a)-AR only one of two TMV serines (Ser204, equivalent to Ser207 in the β-AR) appears to contribute partially to agonist-binding and activation, Because the α(1a)-AR uniquely contains only two TMV serines, this subtype was used to systematically evaluate the role of hydrogen bonding in α1-AR activation. Binding of epinephrine or its monohydroxyl congeners, phenylephrine and synephrine, was not decreased when tested with alanine-substitution mutants that lacked either Ser188 (Ser188 → Ala) or Ser192 (Ser192 → Ala). With the substitution of both serines in the double mutant, Ser188-192 → 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 Ser188 → Ala mutation and not with Ser192 → Ala. In support of the importance of Ser188, binding of phenylephrine (meta-hydroxyl only) by Ser192 → Ala increased 7-fold over that observed with either the wild type receptor or the Ser188 → Ala mutation. Binding of synephrine (para-hydroxyl only) was unchanged with the Ser192 → Ala mutation. In addition, when combined with a recently described constitutively active α(1a)-AR mutation (Mer292 → Leu), only the Ser188 → Ala mutation and not Ser192 → Ala relieved the high affinity binding and increased agonist potency observed with the Mer292 → Leu mutation. A simple interpretation of these findings is that the meta-hydroxyl of the endogenous agonists preferentially binds to Ser188, and it is this hydrogen bond interaction, and not that between the para-hydroxyl and Ser192, that allows receptor activation. Furthermore, since Ser188 and Ser192 are separated by three residues on the TMV α- helix, whereas Ser204 and Ser207 of the β2-AR are separated by only two residues, the orientation of the catechol ring in the α1-AR binding pocket appears to be unique and rotated approximately 120° to that in the β2-AR.