Fusion proteins as model systems for the analysis of constitutive GPCR activity.

In many cases, the coexpression of GPCRs with G-proteins and/or regulators of G-protein signaling (RGS-proteins) allows a successful reconstitution of high-affinity agonist binding and functional responses. However, in some cases, coexpressed GPCRs and G-proteins interact inefficiently, resulting in weak [³⁵S]GTPγS- and steady-state GTPase assay signals. This may be, for example, caused by a rapid dissociation of the G-protein from the plasma membrane, as has been reported for Gα(s). Moreover, for a detailed characterization of GPCR/G-protein interactions, it may be required to work with a defined GPCR/G-protein stoichiometry and to avoid cross-interaction with endogenous G-proteins. Cross-talk to endogenous G-proteins has been shown to play a role in some mammalian expression systems. These problems can be addressed by the generation of GPCR-Gα fusion proteins and their expression in Sf9 insect cells. When the C-terminus of the receptor is fused to the N-terminus of the G-protein, a 1:1 stoichiometry of both proteins is achieved. In addition, the close proximity of GPCR and G-protein in fusion proteins leads to enhanced interaction efficiency, resulting in increased functional signals. This approach can also be extended to fusion proteins of GPCRs with RGS-proteins, specifically when steady-state GTP hydrolysis is used as read-out. GPCR-RGS fusion proteins optimize the interaction of RGS-proteins with coexpressed Gα subunits, since the location of the RGS-protein is close to the site of receptor-mediated G-protein activation. Moreover, in contrast to coexpression systems, GPCR-Gα and GPCR-RGS fusion proteins provide a possibility to imitate physiologically occurring interactions, for example, the precoupling of receptors and G-proteins or the formation of complexes between GPCRs, G-proteins and RGS-proteins (transducisomes). In this chapter, we describe the technique for the generation of fusion proteins and show the application of this approach for the characterization of constitutively active receptors.