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Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells.

Falkenburger BH, Jensen JB, Hille B - J. Gen. Physiol. (2010)

Bottom Line: Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP(2)-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010.By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with approximately 3,000 fluorescently labeled receptors, G proteins, or PLC molecules per microm(2) of plasma membrane.In agreement with previous work, the model suggests that binding of PLC to Galpha(q) greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Galpha(q) subunits bound to PLC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
G protein-coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Galpha subunit, are common for many different GPCRs. For G(q)-coupled M(1) muscarinic (acetylcholine) receptors (M(1)Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M(1)R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M(1)R signaling through G(q) to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP(2)-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with approximately 3,000 fluorescently labeled receptors, G proteins, or PLC molecules per microm(2) of plasma membrane. Endogenous levels are much lower, 1-40 per microm(2). Our kinetic model reproduces the time courses and concentration-response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M(1)R and Gbeta FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Galpha(q) greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Galpha(q) subunits bound to PLC.

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The model reproduces Oxo-M concentration–response curves. Maximum formation of R–Gβ complexes (A), maximum depletion of Gαβγ trimers (B), and maximum formation of Gα–PLC complexes (C) predicted by the model for different concentrations of Oxo-M (lines) were compared with averaged and normalized Oxo-M concentration data (symbols; from Jensen et al., 2009) for M1R/Gβ FRET (A), Gα/Gβ FRET (B), and Gα/PLC FRET (C). Initial conditions (in µm−2): R, 3,000; G proteins, 3,000; PLC, 10 (A and B) or 3,000 (C). (D) Superimposed, normalized concentration–response curves predicted for maximum formation of R–Gβ complexes (red), maximum depletion of Gαβγ trimers (blue; inverted), and maximum formation of Gα–PLC complexes (green) predicted by the model for native cells. Initial conditions (in µm−2): R, 1; G proteins, 40; PLC, 10. Vertical lines indicate dissociation constants KL1 and KL2 for receptors without and with G proteins bound.
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fig7: The model reproduces Oxo-M concentration–response curves. Maximum formation of R–Gβ complexes (A), maximum depletion of Gαβγ trimers (B), and maximum formation of Gα–PLC complexes (C) predicted by the model for different concentrations of Oxo-M (lines) were compared with averaged and normalized Oxo-M concentration data (symbols; from Jensen et al., 2009) for M1R/Gβ FRET (A), Gα/Gβ FRET (B), and Gα/PLC FRET (C). Initial conditions (in µm−2): R, 3,000; G proteins, 3,000; PLC, 10 (A and B) or 3,000 (C). (D) Superimposed, normalized concentration–response curves predicted for maximum formation of R–Gβ complexes (red), maximum depletion of Gαβγ trimers (blue; inverted), and maximum formation of Gα–PLC complexes (green) predicted by the model for native cells. Initial conditions (in µm−2): R, 1; G proteins, 40; PLC, 10. Vertical lines indicate dissociation constants KL1 and KL2 for receptors without and with G proteins bound.

Mentions: Fig. 6 shows predicted FRET time courses on an absolute scale and with a linear time axis (solid traces). In addition to time courses, we reproduced concentration–response data from the FRET measurements of Jensen et al. (2009). Fig. 7 overlays experimental Oxo-M concentration–response curves (symbols) with model curves (solid traces) for G protein binding to receptor (Fig. 7 A), G protein dissociation (Fig. 7 B), and Gαq binding to PLC (Fig. 7 C). Again, the match is generally reasonable.


Kinetics of M1 muscarinic receptor and G protein signaling to phospholipase C in living cells.

Falkenburger BH, Jensen JB, Hille B - J. Gen. Physiol. (2010)

The model reproduces Oxo-M concentration–response curves. Maximum formation of R–Gβ complexes (A), maximum depletion of Gαβγ trimers (B), and maximum formation of Gα–PLC complexes (C) predicted by the model for different concentrations of Oxo-M (lines) were compared with averaged and normalized Oxo-M concentration data (symbols; from Jensen et al., 2009) for M1R/Gβ FRET (A), Gα/Gβ FRET (B), and Gα/PLC FRET (C). Initial conditions (in µm−2): R, 3,000; G proteins, 3,000; PLC, 10 (A and B) or 3,000 (C). (D) Superimposed, normalized concentration–response curves predicted for maximum formation of R–Gβ complexes (red), maximum depletion of Gαβγ trimers (blue; inverted), and maximum formation of Gα–PLC complexes (green) predicted by the model for native cells. Initial conditions (in µm−2): R, 1; G proteins, 40; PLC, 10. Vertical lines indicate dissociation constants KL1 and KL2 for receptors without and with G proteins bound.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812500&req=5

fig7: The model reproduces Oxo-M concentration–response curves. Maximum formation of R–Gβ complexes (A), maximum depletion of Gαβγ trimers (B), and maximum formation of Gα–PLC complexes (C) predicted by the model for different concentrations of Oxo-M (lines) were compared with averaged and normalized Oxo-M concentration data (symbols; from Jensen et al., 2009) for M1R/Gβ FRET (A), Gα/Gβ FRET (B), and Gα/PLC FRET (C). Initial conditions (in µm−2): R, 3,000; G proteins, 3,000; PLC, 10 (A and B) or 3,000 (C). (D) Superimposed, normalized concentration–response curves predicted for maximum formation of R–Gβ complexes (red), maximum depletion of Gαβγ trimers (blue; inverted), and maximum formation of Gα–PLC complexes (green) predicted by the model for native cells. Initial conditions (in µm−2): R, 1; G proteins, 40; PLC, 10. Vertical lines indicate dissociation constants KL1 and KL2 for receptors without and with G proteins bound.
Mentions: Fig. 6 shows predicted FRET time courses on an absolute scale and with a linear time axis (solid traces). In addition to time courses, we reproduced concentration–response data from the FRET measurements of Jensen et al. (2009). Fig. 7 overlays experimental Oxo-M concentration–response curves (symbols) with model curves (solid traces) for G protein binding to receptor (Fig. 7 A), G protein dissociation (Fig. 7 B), and Gαq binding to PLC (Fig. 7 C). Again, the match is generally reasonable.

Bottom Line: Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP(2)-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010.By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with approximately 3,000 fluorescently labeled receptors, G proteins, or PLC molecules per microm(2) of plasma membrane.In agreement with previous work, the model suggests that binding of PLC to Galpha(q) greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Galpha(q) subunits bound to PLC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.

ABSTRACT
G protein-coupled receptors (GPCRs) mediate responses to external stimuli in various cell types. Early events, such as the binding of ligand and G proteins to the receptor, nucleotide exchange (NX), and GTPase activity at the Galpha subunit, are common for many different GPCRs. For G(q)-coupled M(1) muscarinic (acetylcholine) receptors (M(1)Rs), we recently measured time courses of intermediate steps in the signaling cascade using Förster resonance energy transfer (FRET). The expression of FRET probes changes the density of signaling molecules. To provide a full quantitative description of M(1)R signaling that includes a simulation of kinetics in native (tsA201) cells, we now determine the density of FRET probes and construct a kinetic model of M(1)R signaling through G(q) to activation of phospholipase C (PLC). Downstream effects on the trace membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP(2)-dependent KCNQ2/3 current are considered in our companion paper in this issue (Falkenburger et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.200910345). By calibrating their fluorescence intensity, we found that we selected transfected cells for our experiments with approximately 3,000 fluorescently labeled receptors, G proteins, or PLC molecules per microm(2) of plasma membrane. Endogenous levels are much lower, 1-40 per microm(2). Our kinetic model reproduces the time courses and concentration-response relationships measured by FRET and explains observed delays. It predicts affinities and rate constants that align well with literature values. In native tsA201 cells, much of the delay between ligand binding and PLC activation reflects slow binding of G proteins to receptors. With M(1)R and Gbeta FRET probes overexpressed, 10% of receptors have G proteins bound at rest, rising to 73% in the presence of agonist. In agreement with previous work, the model suggests that binding of PLC to Galpha(q) greatly speeds up NX and GTPase activity, and that PLC is maintained in the active state by cycles of rapid GTP hydrolysis and NX on Galpha(q) subunits bound to PLC.

Show MeSH
Related in: MedlinePlus