Sf9 cells: a versatile model system to investigate the pharmacological properties of G protein-coupled receptors.
The Sf9 cell/baculovirus expression system is widely used for high-level protein expression, often with the purpose of purification. However, proteins may also be functionally expressed in the defined Sf9 cell environment. According to the literature, the pharmacology of G-protein-coupled receptors (GPCRs) functionally reconstituted in Sf9 cells is similar to the receptor properties in mammalian cells. Sf9 cells express both recombinant GPCRs and G-proteins at much higher levels than mammalian cells. Sf9 cells can be grown in suspension culture, providing an inexpensive way of obtaining large protein amounts. Co-infection with various baculoviruses allows free combination of GPCRs with different G-proteins. The absence of constitutively active receptors in Sf9 cells provides an excellent signal-to background ratio in functional assays, allowing the detection of agonist-independent receptor activity and of small ligand-induced signals including partial agonistic and inverse agonistic effects. Insect cell Gα(i)-like proteins mostly do not couple productively to mammalian GPCRs. Thus, unlike in mammalian cells, Sf9 cells do not require pertussis toxin treatment to obtain a Gα(i)-free environment. Co-expression of GPCRs with Gα(i1), Gα(i2), Gα(i3) or Gα(o) in Sf9 cells allows the generation of a selectivity profile for these Gα(i/o)-isoforms. Additionally, GPCR-G-protein combinations can be compared with defined 1:1 stoichiometry by expressing GPCR-Gα fusion proteins. Sf9 cells can also be employed for ligand screening in medicinal chemistry programs, using radioligand binding assays or functional assays, like the steady-state GTPase- or [(35)S]GTPγS binding assay. This review shows that Sf9 cells are a versatile model system to investigate the pharmacological properties of GPCRs.
Schneider
Erich H
EH
Laboratory of Molecular Immunology, NIAID/NIH, Bethesda, MD 20892-1886, USA. schneidere@mail.nih.gov
Seifert
Roland
R
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008954:Models, Biological; D011485:Protein Binding; D011869:Radioligand Assay; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera
D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go