Abstract
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.
Footnotes
- Received March 1, 2000.
- Accepted May 18, 2000.
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Send reprint requests to: Graeme Milligan, Davidson Building, University of Glasgow, University Ave., Glasgow G12 8QQ, Scotland, UK. E-mail:g.milligan{at}bio.gla.ac.uk
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Financial support for this work was provided by the Medical Research Council, the Biotechnology and Biosciences Research Council, and the European Union Biomed II programme “Inverse agonism: Implications for drug design”.
- The American Society for Pharmacology and Experimental Therapeutics
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