Trends in Pharmacological Sciences
ReviewGPCR–Gα fusion proteins: molecular analysis of receptor–G-protein coupling
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
References (58)
- et al.
FEBS Lett.
(1997) - et al.
J. Biol. Chem.
(1997) J. Biol. Chem.
(1998)FEBS Lett.
(1998)- et al.
FEBS Lett.
(1998) Neuropharmacology
(1999)- et al.
Biochim. Biophys. Acta
(1990) Trends Pharmacol. Sci.
(1997)- et al.
Trends Pharmacol. Sci.
(1993) - et al.
Trends Pharmacol. Sci.
(1998)
Trends Pharmacol. Sci.
Cell
Biochem. Pharmacol.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
Methods Enzymol.
Biochim. Biophys. Acta
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
Cell
J. Biol. Chem.
Proc. Natl. Acad. Sci. U. S. A.
Recept. Channels
Int. J. Cancer
J. Biol. Chem.
Eur. J. Biochem.
Cited by (132)
A rapid, tag-free way to purify functional GPCRs
2024, Journal of Biological ChemistryReversing thromboxane A2 receptor activity from calcium to cAMP signaling by shifting Gαq to Gαs covalently linked to the receptor
2022, Biochemical Engineering JournalNovel fluorescent GPCR biosensor detects retinal equilibrium binding to opsin and active G protein and arrestin signaling conformations
2020, Journal of Biological ChemistryAllosteric modulation of β-arrestin-biased angiotensin II type 1 receptor signaling by membrane stretch
2014, Journal of Biological ChemistryDivergent transducer-specific molecular efficacies generate biased agonism at a G protein-coupled receptor (GPCR)
2014, Journal of Biological ChemistryCitation Excerpt :To bypass the problem, we adopted an alternative strategy. As shown previously, chimeric proteins consisting of receptors fused to different transduction proteins are functionally coupled and maintain the same efficacy profiles of the native receptor (42). Under such conditions, it is assumed that the presence of a covalently tethered transducer on the receptor can emulate the situation in which a transduction protein is expressed in the membrane at large stoichiometric excess over receptor.