Reconstitution of G protein-coupled receptors with recombinant G protein α and βγ subunits
References (40)
Cell
(1995)J. Biol. Chem.
(1998)- et al.
Genomics
(1999) - et al.
J. Biol. Chem.
(1997) - et al.
J. Biol. Chem.
(1998) Trends Pharm. Sci.
(1999)- et al.
J. Biol. Chem.
(1999) - et al.
J. Biol. Chem.
(1996) - et al.
Methods Enzymol.
(1994) - et al.
Methods Neurosci.
(1996)
J. Biol. Chem.
Annu. Rev. Micro.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
J. Biol. Chem.
Annu. Rev. Biochem.
Cited by (13)
Chapter 11 Subsecond Analyses of G-Protein Coupled-Receptor Ternary Complex Dynamics by Rapid Mix Flow Cytometry
2009, Methods in EnzymologyCitation Excerpt :The advantages of modular assemblies on beads to study GPCR ternary complex dynamics in modular systems have been recently explained in a review chapter (Buranda et al., 2007). The assembly of specific isotypes of promiscuous G‐protein subunits can be clearly defined and has the potential of explaining the differences in dynamic and equilibrium roles played by different isotypes of α and βγ‐subunits (Kukkonen et al., 2001; Lindorfer et al., 1998; Mayeenuddin et al., 2006; McIntire et al., 2001, 2002; Simons et al., 2003b). As shown in Table 11.1, the substitution of β1 for β4 in the Gβγ dimer has a small but measurable effect (τ1/2 = 18.3 sec vs 12.4 sec) on the dissociation rate of LF, after the receptor uncouples from the G‐protein (τ1/2 < 1sec).
Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides
2007, Analytical BiochemistryCitation Excerpt :The cloning of the FPR, its expression in U937 cells, generation of FPR–Gαi2 and FPR–GFP fusion constructs, and the membrane preparations have been described [13]. G protein βγ dimers were prepared and purified as previously described [20,23]. Beads coated with ≈4 million M2 anti-FLAG antibodies were prepared as previously described [22].
Some Mechanistic Insights into GPCR Activation from Detergent-Solubilized Ternary Complexes on Beads
2007, Advances in Protein ChemistryCitation Excerpt :Much of the advantage of this platform is the enabling of the practitioner to define reagent stoichiometry and identity, for example, isotypes of promiscuous G protein subunits can be clearly defined. The assembly of ternary complexes on beads has the potential of meeting the challenge of a systematic study that could lead to the elucidation of differences in dynamic and equilibrium roles played by different isotypes of α and βγ subunits (Kukkonen et al., 2001; Lindorfer et al., 1998; Mayeenuddin et al., 2006; McIntire et al., 2001, 2002; Simons et al., 2003). The studies described here have been limited by availability of purified protein targets and have so far used the limited isotypes available to the authors.
Heterologous expression and biophysical characterization of soluble oligosaccharyl transferase subunits
2004, Archives of Biochemistry and BiophysicsDifferential sensitivity of phosphatidylinositol 3-kinase p110γ to isoforms of G protein βγ dimers
2004, Journal of Biological ChemistryCitation Excerpt :As expected, the Gs, Gi, and Go α subunits did not activate PLCβ (44). Each of the preparations of G α subunit used in these experiments was tested for its ability to couple to the appropriate recombinant receptors expressed in Sf9 cell membranes (31, 45). All of the Gα subunits were active proteins in this assay.
G protein activation by G protein coupled receptors: Ternary complex formation or catalyzed reaction?
2004, Biochemical PharmacologyCitation Excerpt :This allows both subunits to interact with effectors or regulators, as outlined below. Both G protein subunits are necessary to reconstitute the stable (high affinity) “ternary complex”, HRG, in the absence of nucleotides [7]. GPCRs are unlikely to contact the “switch regions”, which are at least 30 Å from the membrane surface [1].