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Vol. 58, Issue 2, 438-448, August 2000
1b-Adrenoceptor
Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Scotland (P.A.S., G.M.); and Molecular Discovery
Department, Glaxo-Wellcome Research and Development, Stevenage,
England (N.B., S.R.)
Constitutively active forms of the hamster
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 
2-adrenoceptor. Green fluorescent protein
(GFP)-tagged forms of each of these mutants and of the wild type
1b-adrenoceptor were expressed stably in HEK293 cells.
The wild type 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 1b-adrenoceptor containing the 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 Asp142Ala
and Ala293Glu forms of the
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.
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