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) invo lves hydrogen bonding of serine residues in the fifth transmembrane se gment (TMV) to the catechol hydroxyls of the endogenous agonists, epin ephrine 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 an d full agonist activity, However, with the alpha(2a)-AR only one of tw o TMV serines (Ser(204), equivalent to Ser(207) in the beta-AR) appear s to contribute partially 60 agonist-binding and activation, Because t he alpha(1a)-AR uniquely contains only two TMV serines, this subtype w as used to systematically evaluate the role of hydrogen bonding in alp ha(1)-AR activation, Binding of epinephrine or its monohydroxyl congen ers, 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 se rines in the double mutant, Ser(188/192) --> Ala, binding of all three ligands was significantly reduced (10-100-fold) consistent with a sin gle hydrogen bond interaction, However, receptor-mediated inositol pho sphate production was markedly attenuated only with the Ser(188) --> A la mutation and not with Ser(192) --> Ala. In support of the importanc e of Ser(188), binding of phenylephrine (meta-hydroxyl only) by Ser(19 2) --> Ala increased 7-fold over that observed with either the wild ty pe receptor or the Ser(188) --> Ala mutation. Binding of synephrine (p ara-hydroxyl only) was unchanged with the Ser(192) --> Ala mutation, I n addition, when combined with a recently described constitutively act ive alpha(1a)-AR mutation (Met(292) --> Leu), only the Ser(188) --> Al a mutation and not Ser(192) --> Ala relieved the high affinity binding and increased agonist potency observed with the Met(292) --> Leu muta tion. A simple interpretation of these findings is that the meta-hydro xyl of the endogenous agonists preferentially binds to Ser(188), and i t is this hydrogen bond interaction, and not that between the para-hyd roxyl and Ser(192), that allows receptor activation, Furthermore, sinc e Ser(188) and Ser(192) are separated by three residues on the TMV alp ha-helix, whereas Ser(204) and Ser(207) of the beta(2)-AR are separate d by only two residues, the orientation of the catechol ring in the al pha(1)-AR binding pocket appears to be unique and rotated approximatel y 120 degrees to that in the beta(2)-AR.