THE 3RD EXTRACELLULAR LOOP OF THE BETA(2)-ADRENERGIC RECEPTOR CAN MODULATE RECEPTOR G-PROTEIN AFFINITY

Chimeric receptors of the beta(2)-adrenergic receptor in which the ext racellular loops were replaced with the corresponding amino acids of t he alpha(1a)-adrenergic receptor were generated to measure changes in alpha(1)-antagonist affinity. Although no changes in alpha(1)-antagoni st affinity were measured in the beta(2)/alpha(1a) chimeras, a decreas ed IC50 (10-fold) for agonists as compared with wild type beta(2) cont rol was found because of the replacement of the third extracellular lo op (EX3). These agonist high affinity changes were because of a greate r proportion of high affinity sites (2-fold) that were convertible to low affinity sites with guanosine 5'-3-O-(thio)triphosphate. Adenylate cyclase activity evoked by the EX3 chimera showed commensurate increa ses in the basal signal transduction as well as the isoproterenol-stim ulated potency, suggesting constitutive activity. However, unlike othe r constitutively active adrenergic receptor mutants in which the mutat ion causes G protein-independent changes, the mechanism of the EX3 chi mera seems to be attributable to a greater ease with which the active ternary complex is formed because of a higher affinity/coupling of the G protein. Although the changes because of EX3 are indirect and most likely affect helical packing, they support an emerging hypothesis tha t G protein-coupled receptors have evolved their structure-function re lationships to constrain the receptor in an inactive state.