Review
GPCR–Gα fusion proteins: molecular analysis of receptor–G-protein coupling

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Abstract

The efficiency of interactions between G-protein-coupled receptors (GPCRs) and heterotrimeric guanine nucleotide-binding proteins (G proteins) is greatly influenced by the absolute and relative densities of these proteins in the plasma membrane. The study of these interactions has been facilitated by the use of GPCR–Gα fusion proteins, which are formed by the fusion of GPCR to Gα. These fusion proteins ensure a defined 1:1 stoichiometry of GPCR to Gα and force the physical proximity of the signalling partners. Thus, fusion of GPCR to Gα enhances coupling efficiency can be used to study aspects of receptor–G-protein coupling that could not otherwise be examined by co-expressing GPCRs and G proteins as separate proteins. The results of studies that have made use of GPCR–Gα fusion proteins will be discussed in this article, along with the strengths and limitations of this approach.

Section snippets

Construction of fusion protein DNAs and structural properties of fusion proteins

Fusion proteins are generated by linking the GPCR C-terminus, which is located intracellularly, to the N-terminus of Gα (1, 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 18). This is achieved by fusing the open reading frames of the two proteins using DNA restriction enzyme or polymerase chain reaction (PCR)-based techniques, or both. Figure 1 illustrates the two-dimensional topology of GPCR–Gα fusion proteins in the plasma membrane. In most GPCRs, the second and third intracellular loops are crucial for

Evidence for highly efficient coupling

Strosberg's group was the first to construct and express a GPCR–Gα fusion protein. In their seminal paper, Bertin et al.1 showed that a fusion protein of the β2AR and GsαS was more efficient at stabilizing high-affinity agonist-binding and stimulating adenylate cyclase when expressed in Gsα-deficient S49 cyc lymphoma cells than non-fused β2AR expressed in S49 wild-type cells. These data were tantalizing in view of the fact that, in S49 wild-type cells, there is an ∼100-fold molar excess of Gsα

Structural properties and applications

The α2A-AR, A1R and 5-HT1A receptor were fused to pertussis toxin (PTX)-resistant mutants of various Giα/Goα proteins and expressed in mammalian cell lines (COS-7, Rat 1, HEK293)9, 10, 11, 12, 13, 15, 16, 17. The mutations in Giα/Goα proteins were introduced to differentiate between the PTX-sensitive GTPase activity of endogenous Gi-proteins of the host cells and the PTX-insensitive GTPase activity of the fusion protein9, 10, 12, 13, 15, 16, 17. However, the introduction of a point mutation at

Role of the length of the GPCR C-terminus for fusion-protein function

In fusion proteins, the GPCR C-terminus serves as tether between the GPCR core and Gα. The length of the C-terminus of different GPCRs is extremely variable53, 54. The C-termini of the α2AAR and β2AR comprise 25 and 72 amino acids, respectively55, 56. This marked difference in the length of the C-terminus could therefore have a significant impact on GPCR–G protein- and G protein–effector coupling in fusion proteins. To address this question, the properties of fusion proteins in which 26 [β2

Acknowledgements

When working at Stanford University, K. Wenzel-Seifert and R. Seifert were supported by a research fellowship of the Deutsche Forschungsgemeinschaft. The authors would like to thank Drs E. Sanders-Bush, V. T. Lam, U. Gether and T. W. Lee for their collaboration in the fusion protein project. The authors are also thankful to Drs M. L. Michaelis, E. K. Michaelis and R. Dobrowsky (Department of Pharmacology and Toxicology, The University of Kansas) and the reviewers of the manuscript for many

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