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.
Laboratory of Molecular Immunology, NIAID/NIH, Bethesda, Maryland, USA.
Erich H
EH
Schneider
Roland
R
Seifert
D000818:Animals; D002460:Cell Line; D020558:GTP Phosphohydrolases / Q000378:metabolism; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D020710:RGS Proteins / Q000235:genetics* / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*
D044385:GTP-Binding Protein alpha Subunits; D020710:RGS Proteins; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020558:GTP Phosphohydrolases