GPCR-induced dissociation of G-protein subunits in early stage signal transduction

Figures & data

Figure 1.  Reconstitution of α_2A-AR with (A), Gα_i1his + Gβ₁γ₂ or (B), Gα_i1+Gβ₁γ₂his in suspension. G-protein subunits (20 nM) were combined with 0.1 mg/ml of α_2A-AR membranes, 5 µM GDP, 10 µM AMP-PNP (‘reconstitution mix’), 0.2 nM [³⁵S]GTPγS and 100 µM yohimbine (where shown) in TMND buffer. The reaction was started by addition of UK14304 (10 µM final concentration). The reaction was incubated for 90 min at 27°C with shaking. The final volume was 100 µl and the entire reaction mix was filtered over a GF/C filter and washed with 3×4 ml with ice-cold TMN buffer (n=3, mean±SEM). Abbreviations; UK, UK14304, Yoh, yohimbine.

Figure 2.  Ni^2 +-bead capture of α_2A-AR-activated [³⁵S]GTPγS:Gα_i1his. 20 nM of both Gα_i1his and Gβ₁γ₂ were combined with 0.1 mg/ml of α_2A-AR membranes and 5 µM GDP and 10 µM AMP-PNP (‘reconstitution mix’), 0.2 nM [³⁵S]GTPγS, in TMND buffer. Various volumes of Ni(NTA) agarose beads were added to the reconstitution mix as indicated, in the absence (○) or presence (•) of 10 µM UK14304 [final]. The mix was incubated for 90 min at 27°C with shaking. Final volume was 100 µl. The entire reaction was filtered over a Whatman #1 filter paper and washed with 3×4 ml with ice-cold TMN buffer. A representative experiment is shown.

Figure 3.  Agonist dose–response curves with and without Ni^2 +-beads. Various concentrations of UK14304 were incubated with 0.2 mg/ml α_2A-AR containing reconstituted Gα_i1his (50 nM) and β₁γ₂ (50 nM), 5 µM GDP; 10 µM AMP-PNP and 0.2 nM [³⁵S]GTPγS in the absence (□) or presence (▪) of 10 µl Ni(NTA) agarose beads and the basal binding was 40 and 42 fmol/mg, respectively, whilst the maximal (UK14304-stimulated) binding was 300 and 275 fmol/mg, respectively. Data are presented as percent of maximum bound. The mix was incubated for 90 min at 27°C with shaking. Final volume was 100 µl and the entire reaction was filtered over a Whatman #1 filter and washed with 3×4 ml with ice-cold TMN buffer. The EC₅₀ values were 24 nM and 11 nM in the absence and presence of Ni(NTA) agarose beads, respectively. A representative experiment is shown.

Figure 4.  Antagonist competition curves. To determine IC₅₀ values at the α_2A-AR, the following adrenergic receptor subtype-specific antagonists were used: rauwolscine (•), yohimbine (○), prazosin (▪) or propranolol (□) as indicated. (A) Direct receptor binding of [³H]MK912 (1 nM) with various concentrations of different adrenergic receptor subtype-specific antagonists as indicated. The reaction mix was filtered over GF/C filters and washed with 3×4 ml with ice-cold TMN buffer. The calculated IC₅₀ values for rauwolscine, yohimbine, prazosin or propranolol for the α_2A-AR membrane preparation were 0.091 µM, 0.141 µM, ≈73 µM and ≈275 µM, respectively. (B) Receptor-stimulated binding of [³⁵S]GTPγS in the presence of UK14304 and the indicated amounts of selected antagonists. The experiment was carried out using 20 nM of both Gα_i1his and Gβ₁γ₂ combined with 0.1 mg/ml of α_2A-AR membranes, 5 µM GDP and 10 µM AMP-PNP (‘reconstitution mix’), 0.2 nM [³⁵S]GTPγS in the presence of various concentrations of the different adrenergic receptor subtype-specific antagonists as indicated and 10 µl Ni(NTA) agarose beads (n=3, mean±SEM). UK14304 (1 µM final concentration) was added to start the reactions and the mix was incubated for 90 min at 27°C with shaking. Final volume was 100 µl and the entire reaction was filtered over a Whatman #1 filter and washed with 3×4 ml with ice-cold TMN buffer. The IC₅₀ values for each of the antagonists was determined as 0.051 µM (rauwolscine: selective α₂-AR antagonist); 0.080 µM (yohimbine: selective α₂-AR antagonist); 8.3 µM (prazosin: selective α₁-AR antagonist) and 86.9 µM (propranolol: β-AR antagonist).

Table I.  Demonstration of his-tag specific capture of G-proteins on Ni^2 +-beads. Purified samples of (50 nM) Gα_i1, Gα_i1his, β₁γ₂his, Gα_i1hisβ₁γ₂ or Gα_i1β₁γ₂his were incubated with 5 nM [³⁵S]GTPγS in the absence or presence of Ni(NTA) beads (±200 mM imidazole) for 90 min at 27°C. The samples were then filtered over a filter paper stack as described in Materials and Methods, and depicted in Figure 7. Each filter was counted by liquid scintillation counting separately and the results are shown (in the table, ‘–’ indicates negligible binding associated with the filter, whereas ‘ + ’ indicates significant binding was associated with that filter, by measurement of radioactivity). The Whatman #1 ‘Paper’ filters retain the relatively large (approximately 45–165 µm diameter) Ni(NTA) beads and associated G-proteins if they contain a histidine tag, whilst GF/C filters bind all proteins that are not immobilized on the surface of Ni-NTA beads.

	Gαi1		Gαi1his		β1γ2his		Gαi1hisβ1γ2		Gαi1β1γ2his
	Paper	GF/C	Paper	GF/C	Paper	GF/C	Paper	GF/C	Paper	GF/C
No Ni-NTA beads	−	+	−	+	−	−	−	+	−	+
Ni-NTA beads	−	+	+	−	−	−	+	−	+	+
Ni-NTA beads + imidazole	−	+	−	+	−	−	−	+	−	+

Figure 5.  Reconstitution of α_2A-AR with Gα_i1his + Gβ₁γ₂ in suspension. 20 nM of both Gα_i1his and Gβ₁γ₂ were combined with 0.1 mg/ml of α_2A-AR membranes, 5 µM GDP, 10 µM AMP-PNP (‘reconstitution mix’), 0.2 nM [³⁵S]GTPγS and 100 µM yohimbine (where shown) in TMND buffer. The reaction was started with 10 µM UK14304 (final) and the reaction was incubated for 90 min at 27°C with shaking. Final volume was 100 µl. The entire reaction was filtered over a filter ‘stack’ that was comprised of a Whatman #1 paper filter on top of a GF/C filter. The sample was washed with 3×4 ml with ice-cold TMN buffer. (A) The Whatman #1 filter data set and (B) GF/C filter data set were counted (by scintillation) separately (n=3, mean±SEM). Abbreviations; UK, UK14304, Yoh, yohimbine.

Figure 6.  α_2A-AR-activated [³⁵S]GTPγS:G-protein subunit dissociation. (A), Ni^2 +-bead capture of α_2A-AR-activated [³⁵S]GTPγS:Gα_i1his. 20 nM of both Gα_i1his and Gβ₁γ₂ were used and (B), α_2A-AR-activated [³⁵S]GTPγS:Gα_i1/β₁γ₂his dissociation. 20 nM of both Gα_i1 and β₁γ₂his were used. In both (A) and (B) the appropriate combination of G-proteins were combined with 0.1 mg/ml of α_2A-AR membranes and 5 µM GDP and 10 µM AMP-PNP (‘reconstitution mix’), 0.2 nM [³⁵S]GTPγS, 100 µM Yohimbine or 100 mM imidazole (where shown) in TMND buffer. 10 µl of Ni(NTA) agarose beads were added to the reconstitution mix and the reaction was started with 10 µM UK14304 [final]. The mix was incubated for 90 min at 27°C with shaking. Final volume was 100 µl. The entire reaction was filtered over a ‘filter stack’ that was comprised of a Whatman #1 filter paper on top with a GF/C filter beneath it. The bead sample was washed with 3×4 ml with ice-cold TMN buffer. Whatman #1 paper filters and GF/C filters were counted (scintillation) separately as indicated (n=3, mean±SEM). Abbreviations; UK, UK14304.

Figure 7.  Schematic demonstrating the specificity of the ‘filter stack’. Following α_2A-AR-stimulated G-protein activation, as measured by [³⁵S]GTPγS binding to Gα subunits, the reaction mixture is filtered over a ‘filter stack’ comprising a top layer of Whatman #1 filter paper (to collect Ni^2 +-coated (Ni(NTA)) agarose beads containing hexahistidine-tagged G-proteins), with a GF/C directly beneath it (to collect G-proteins that are not captured by the Ni(NTA) agarose). (A), The [³⁵S]GTPγS bound to Gα_i1his is attached to the Ni(NTA) bead via the heaxahistidine-Ni^2 + interaction, and is thus retained on the upper Whatman #1 filter in the stack. (B), Following α_2A-AR-stimulated G-protein activation using Gα_i1 and β₁γ₂his, the activated Gα_i1:[³⁵S]GTPγS complex is not retained on the Ni(NTA) beads but is captured by the GF/C filter.