Coexpression systems as models for the analysis of constitutive GPCR activity.

The investigation of constitutive activity of GPCRs in transfected mammalian cells is often hampered by the presence of other constitutively active receptors that generate a high background signal. This impairs the measurement of constitutive activity and of inverse agonistic effects, both of which often occur in a relatively small signal range. Moreover, constitutive activity of a GPCR depends on the interacting G-protein. Since the commonly used mammalian cells contain a set of several different G-protein types, it is very difficult to investigate the influence of specific Gα and Gβγ subunits on constitutive activity in more detail in these expression systems. Here, we show that the Sf9 cell/baculovirus expression system provides excellent conditions for the characterization of constitutively active GPCRs. Sf9 cells express a restricted set of G-protein subtypes that show only a limited capability of interacting with mammalian GPCRs. Moreover, the Sf9 cell/baculovirus expression system allows the combined expression of up to four different proteins encoded by the respective genetically modified baculoviruses. Using the highly constitutively active human histamine H₄R (hH₄R) as a paradigm, we demonstrate how the coexpression of hH₄R with different signaling proteins (Gα, Gβγ, and RGS-proteins) in combination with sensitive functional assays (high-affinity agonist binding and steady-state GTPase- and GTPγS-binding assays) allows in-depth studies of constitutive activity. The preparation of Sf9 cell membranes, coexpressing hH₄R and various additional proteins, is described in detail as well as the procedures of the different functional assays. Moreover, we show that coexpression of GPCRs with signal transduction components in Sf9 cells can also be applied to the characterization of other constitutively active receptors, for example, the formyl peptide receptor and β₂-adrenoceptor.