Results: 3

1.
Figure 3

Figure 3. From: G-protein-coupled receptor heterodimerization modulates receptor function.

Ligand binding and functional properties. a–c, Competition of 3H-diprenorphine binding by U69593 (square), norbinaltorphimine (triangle), diprenorphine (star), DPDPE (circle) and TIPPΨ (diamond) in membranes from cells expressing κ- (a), δ- (b) or κ- and δ- (c) receptors. d, Displacement of 3H-diprenorphine by U69593 in the presence of 10 µM DPDPE (triangle) or DPDPE in the presence of 10 µM U69593 (diamond). e, f, Decrease in intracellular cAMP (e) or increase in phospho-MAPK (f) by U69593 (square), DPDPE (circle) or U69593 + DPDPE (triangle). In e, the 50% inhibitory concentrations (nM) were: U69593, 1.3 ± 0.7; DPDPE, 0.9 ± 0.4; U69593 + DPDPE, 0.06 ± 0.03. Activation of homodimers in these cells could account for the effect seen by individual agonists. Error bars represent s.e.m. (n = 3–4).

Bryen A. Jordan, et al. Nature. ;399(6737):697-700.
2.
Figure 1

Figure 1. From: G-protein-coupled receptor heterodimerization modulates receptor function.

Characteristics of κ-opioid-receptor homodimers. a, Immunoblotting of lysates from cells expressing Flag–κ receptors or Flag–δ receptors. b, c, Myc-tagged κ-receptors can be co-precipitated only from cells expressing both myc and Flag-tagged receptors (b) under a variety of extraction conditions and not from a mixture of cells individually expressing these receptors (c). Expression of myc- or Flag-tagged receptors was confirmed by immunoblotting with the appropriate antisera (right panel). d, e, Treatment of cells expressing κ-receptors with 1 mM DTT for 30 min followed by 5 mM iodacetamide (IAM) or N-ethylmaleimide (NEM) results in monomerization (d), whereas treatment with 100 nM agonists for 60 min does not (e). Immunoblotting experiments used anti-Flag antibodies; immunoprecipitation experiments used anti-myc antibodies.

Bryen A. Jordan, et al. Nature. ;399(6737):697-700.
3.
Figure 2

Figure 2. From: G-protein-coupled receptor heterodimerization modulates receptor function.

Characterization of κ–δ heterodimers. a, κ–δ heterodimers can be immunoprecipitated only from myc–κ- and Flag-δ-expressing cells and not from myc–κ- and Flag–µ-expressing cells. b, κ–δ heterodimers can be immunoprecipitated under a variety of extraction conditions and not from a mixture of cells individually expressing these receptors. c, Expression of myc- or Flag-tagged receptors in each cell line was confirmed by immunoblotting with the appropriate antisera (right panel). Treatment with β-mercaptoethanol 5% (β–ME) for 5 min results in the destabilization of dimers. d, Internalization of receptors in response to 1 µM etorphine for 60 min. Stippled bars, myc–δ; shaded bars, Flag–κ. Significant differences from untreated controls are indicated; *P < 0.05; ***P < 0.005 (n = 3). Immunoblotting experiments used anti-Flag antibodies; immunoprecipitation experiments used anti-myc antibodies.

Bryen A. Jordan, et al. Nature. ;399(6737):697-700.

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