CHIMERAS OF ALPHA(1)-ADRENERGIC RECEPTOR SUBTYPES IDENTIFY CRITICAL RESIDUES THAT MODULATE ACTIVE STATE ISOMERIZATION

We have identified previously two amino acids, one in each of the fift h and sixth transmembrane segments of both the alpha(1a)-adrenergic re ceptor and the alpha(1b)-adrenergic receptor (AR), that account almost entirely for the selectivity of agonist binding by these receptor sub types (Hwa, J., Graham, R. M., and Perez, D. M. (1995) J. Biol. Chem. 270, 23189-23195). Thus reversal of these two residues, from those fou nd in the native receptor of one subtype to those in the other subtype , produces complementary changes in subtype selectivity of agonist bin ding. Here we show that mutating only one of these residues in either the alpha(1b)-AR or the alpha(1a)-AR to the corresponding residue in t he other subtype (Ala(204) --> Val for the alpha(1b); Met(292) --> Leu for the alpha(1a)-AR) results in chimeras that are constitutively act ive for signaling by both the phospholipase C and phospholipase A(2) p athways. This is evident by an increased affinity for agonists, increa sed basal phospholipase C and phospholipase A(2) activation, and incre ased agonist potency. Although mutation of the other residue involved in agonist binding selectivity, to the corresponding residue in the ot her subtype (Leu(314) --> Met for the alpha(1b)-AR; Val(185) --> Ala f or the alpha(1a)-AR) does not alter receptor binding or signaling, per se, when combined with the corresponding constitutively activating mu tations, the resulting chimeras, Ala(204) --> Val/Leu(314) --> Met (al pha(1b)-AR) and Val(185) --> Ala/Met(292) --> Leu ((alpha(1a)-AR), dis play wild type ligand binding and signaling. A simple interpretation o f these results is that the alpha(1a)- and alpha(1b)-ARs possess resid ues that critically modulate isomerization from the basal state, R, to the active state R, and that the native receptor structures have evo lved to select residues that repress active state isomerization. It is likely that the residues identified here modulate important interheli cal interactions between the fifth and sixth transmembrane segments th at inhibit or promote receptor signaling.