Human beta 2-adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP-binding protein to adenylyl cyclase.
Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human beta 2-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350,000 human receptors/cell. The number of receptors did not change with cell density or age of culture. The adrenergic receptors overexpressed in the insect cells were functional with respect to their ligand binding and signalling properties. Coupling of the receptors to endogenous GTP-binding proteins is demonstrated by hormone-dependent stimulation of GTPase and adenylyl cyclase activity in the transformed insect cells. Western-blot analysis revealed that the endogenous GTP-binding protein appears to be of the heterotrimeric type. Antibodies raised against the mammalian alpha subunit of stimulatory GTP-binding proteins cross-react with the insect alpha subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The beta subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the C-terminal amino acids of the mammalian beta subunit of GTP-binding proteins, but is about approximately 2 kDa larger than that of the beta subunit of GTP-binding proteins from bovine brain. Exposure of the transformed insect cells to L-isoproterenol rapidly induces uncoupling and internalization of 30% of the heterologously expressed receptors. In contrast to the situation in mammalian cells, prolonged exposure of the agonist (24 h) does not result in down regulation of the remaining 70% of the receptors.
Kleymann
G
G
Max-Planck-Institut für Biophysik, Abt. Molekulare Membranbiologie, Frankfurt/M, Germany.
Boege
F
F
Hahn
M
M
Hampe
W
W
Vasudevan
S
S
Reiländer
H
H
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009036:Moths; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D008024:Ligands; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol