title: Resolution of inverse agonist-induced up-regulation from constitutive activity of mutants of the alpha(1b)-adrenoceptor.
abstract: Constitutively active forms of the hamster alpha(1b)-adrenoceptor can be produced from the point mutations Asp(142)Ala or Ala(293)Glu or exchange of a small segment of the third intracellular loop with the equivalent region of the beta(2)-adrenoceptor. Green fluorescent protein (GFP)-tagged forms of each of these mutants and of the wild type alpha(1b)-adrenoceptor were expressed stably in HEK293 cells. The wild type alpha(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 alpha(1b)-adrenoceptor containing the beta(2)-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 Asp(142)Ala and Ala(293)Glu forms of the alpha(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.
authors.affiliation: Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland.
authors.forename: P A
authors.initials: PA
authors.lastname: Stevens
authors.forename: N
authors.initials: N
authors.lastname: Bevan
authors.forename: S
authors.initials: S
authors.lastname: Rees
authors.forename: G
authors.initials: G
authors.lastname: Milligan
mesh_terms: D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D011224:Prazosin / Q000494:pharmacology; D011487:Protein Conformation; D019275:Radiopharmaceuticals / Q000494:pharmacology; D018340:Receptors, Adrenergic, alpha-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014316:Tritium; D015854:Up-Regulation
chemical_list: C547716:ADRA1B protein, human; D000317:Adrenergic alpha-Antagonists; D019275:Radiopharmaceuticals; D018340:Receptors, Adrenergic, alpha-1; D014316:Tritium; D011224:Prazosin