Human β₂‐adrenergic receptor produced in stably transformed insect cells is functionally coupled via endogenous GTP‐binding protein to adenylyl cyclase

Abstract

Spodoptera frugiperda insect cells (Sf9) containing the stably integrated human P,-adrenergic receptor gene under the control of the baculovirus IE1 promoter expressed up to 350000 human receptorskell. 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 a subunit of stimulatory GTP-binding proteins cross-react with the insect a subunit of GTP-binding proteins, which also exhibits the same apparent molecular mass as its mammalian counterpart. The p subunit of GTP-binding proteins from insect cells reacts with anti-peptide serum directed against the Cterminal amino acids of the mammalian / 3 subunit of GTP-binding proteins, but is about =2 kDa larger than that of the P 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.Mammalian P-adrenergic receptors CB,AR) have been introduced into several heterologous expression systems for extensive pharmacological and biochemical analysis, including Escherichia coli (Marullo et al., 1989(Marullo et al., ,1990, yeast (King et al., 1990), Xenopus oocytes (Frielle et al., 1988;Kobilka et al., 1988), several mammalian cell lines Fraser et al., 1987;Hen et al., 1989;Lohse, 1992;Rands et al., 1990;Wang et al., 1989) and insect cells, using the baculovirus expression system (George et al., 1989;Reiliinder et al., 1991). Evidence has been presented that mammalian P,AR functionally couple to insect adenylyl cyclase in cells of Spodoptera frugiperda (Sf9) . However, little is known about the signal-transduction pathway in these invertebrate cells. Despite the several advantages which have made the baculovirus expression system a popular method of engineering richer sources of recombinant protein, analysis of insect proteins that are known to functionally interact with the foreign proteins has been hampered. This is because the transient expression that occurs after the cells have been infected with the recombi- nant baculovirus is a cytotoxic event that stops the transcription and translation of insect proteins and ultimately kills the cells. To facilitate further biochemical and biophysical characterization of P,AR, taking advantage of the stability and robust nature of insect ovarian cells, we have used the promoter of the IE1 gene (an immediate early gene of the baculovirus Aut...

Functional characterisation of the Drosophila 5‐HTdro1 and 5‐HTdro2B serotonin receptors in insect cells: activation of a Gαs‐like protein by 5‐HTdro1 but lack of coupling to inhibitory G‐proteins by 5‐HTdro2B

“…G~ specific antibody (RM/1) detected a 50 kDa protein in agreement with previous results [3] (Fig. 2).…”

“…2). However, the antibodies AS/7 (Gal/2) or EC/2 (Gcti3) failed to detect inhibitory G-proteins in Sf9 membranes in agreement with [3] and [4], respectively. Similarly, using the Gao-specific antibodies GC/2, IM1, OCI and ON1 we were unable to detect the G~o subtype in these cells.…”

“…However, it is unclear whether Sf9 cells synthesize a full complement of G-proteins to enable the use of this cell line for detailed studies on receptor-Gprotein interaction. The available reports [3,4] appear to highlight some inconsistencies in the class of G-protein subtypes that may be present in Sf9 cells.…”

Foreign Insect Hormones Stimulating the Transcription of theie-1Promoter ofBombyx moriNuclear Polyhedrosis Virusin Vivoandin Vitro

“…This nonlytic insect cell expression system provides a high-level expression of recombinant proteins under ie-1 control without viral infection, with the additional advantage of continuous production in a cellular environment in contrast to that generated by a baculovirus infection. [18][19][20][21] Insect hormones, especially ecdysone and juvenile hormone, mainly function as regulators of developmental events during the insect life cycle, to control the ecdysis and metamorphosis of the insect, and in‰uence the synthesis of DNA, RNA, and related proteins in larvae. 22) Co-treatment with ecdysteroid and juvenile hormone analogue (JHA) of silkworm larvae atˆfth instar increased the silk production up to 6z.…”

Extracellular Domains of the Bradykinin B2 Receptor Involved in Ligand Binding and Agonist Sensing Defined by Anti-peptide Antibodies

“…Sf9 cells (2 ϫ 10 6 /ml) were infected with the wild type or the recombinant baculovirus at a multiplicity of infection of 2-5. Cells were harvested 48 -72 h after infection (12).…”