RT Journal Article SR Electronic T1 Resolution of Inverse Agonist-Induced Up-Regulation from Constitutive Activity of Mutants of the α1b-Adrenoceptor JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP 438 OP 448 DO 10.1124/mol.58.2.438 VO 58 IS 2 A1 Patricia A. Stevens A1 Nicola Bevan A1 Stephen Rees A1 Graeme Milligan YR 2000 UL http://molpharm.aspetjournals.org/content/58/2/438.abstract AB Constitutively active forms of the hamster α1b-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 α1b-adrenoceptor were expressed stably in HEK293 cells. The wild type α1b-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 α1b-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 α1b-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.