Resolution of Inverse Agonist-Induced Up-Regulation from Constitutive Activity of Mutants of the α_1b-Adrenoceptor

Abstract

Constitutively active forms of the hamster α_1b-adrenoceptor can be produced from the point mutations Asp¹⁴²Ala or Ala²⁹³Glu or exchange of a small segment of the third intracellular loop with the equivalent region of the β₂-adrenoceptor. Green fluorescent protein (GFP)-tagged forms of each of these mutants and of the wild type α_1b-adrenoceptor were expressed stably in HEK293 cells. The wild type α_1b-adrenoceptor-GFP was expressed both at the plasma membrane and with a distinctly perinuclear punctate pattern. Sustained treatment with a range of antagonist/inverse agonist ligands failed to modulate the cellular distribution or levels of expression of this construct. The form of the α_1b-adrenoceptor containing the β₂-adrenoceptor sequence substitution was predominantly located in punctate intracellular vesicles and sustained challenge with the same series of antagonists/inverse agonists produced a 5-fold up-regulation of protein levels with elevation of both plasma membrane and intracellular receptor. Quantification of these effects could be produced by spectrofluorometric analysis of cells grown in a 96-well microtiter plate. In contrast, both the Asp¹⁴²Ala and Ala²⁹³Glu forms of the α_1b-adrenoceptor-GFP were located predominantly at the plasma membrane. Levels of these two point mutants were not increased by any of the antagonist/inverse agonist ligands tested, although the sequence substitution mutation encompasses codon 293. Resolution of constitutive activity and ligand-induced up-regulation was further exemplified by a mutant lacking eight serine residues in the C-terminal tail that displayed little constitutive activity but was up-regulated by sustained ligand challenge. These results demonstrate the nonequivalence of mutations in their regulation by antagonist/inverse agonist ligands.