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. 2004 Sep;87(3):1406-16.
doi: 10.1529/biophysj.103.035253.

A Signal Transduction Pathway Model Prototype I: From Agonist to Cellular Endpoint

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Free PMC article

A Signal Transduction Pathway Model Prototype I: From Agonist to Cellular Endpoint

Thomas J Lukas. Biophys J. .
Free PMC article

Abstract

The postgenomic era is providing a wealth of information about the genes involved in many cellular processes. However, the ability to apply this information to understanding cellular signal transduction is limited by the lack of tools that quantitatively describe cellular signaling processes. The objective of the current studies is to provide a framework for modeling cellular signaling processes beginning at a plasma membrane receptor and ending with a measurable endpoint in the signaling process. Agonist-induced Ca(2+) mobilization coupled to down stream phosphorylation events was modeled using knowledge of in vitro and in vivo process parameters. The simulation process includes several modules that describe cellular processes involving receptor activation phosphoinositide metabolism, Ca(2+)-release, and activation of a calmodulin-dependent protein kinase. A Virtual Cell-based simulation was formulated using available literature data and compared to new and existing experimental results. The model provides a new approach to facilitate hypothesis-driven investigation and experimental design based upon simulation results. These investigations may be directed at the timing of multiple phosphorylation/dephosphorylation events affecting key enzymatic activities in the signaling pathway.

Figures

FIGURE 1
FIGURE 1
Three-compartment cell model containing plasma membrane, cytoplasmic reticulum, and ER. The primary activities modeled using the Virtual Cell are the GPCR-mediated activation of phospholipase C, Ca2+ release and uptake in the ER, and activation of the CaM-MLCK complex.
FIGURE 2
FIGURE 2
Generic GPCR receptor model details. The model is based upon the precoupled receptor concept (Shea and Linderman, 1997) including high-affinity coupled (R*) and low-affinity uncoupled (R) receptors for ligand (L) binding.
FIGURE 3
FIGURE 3
Phospholipid synthesis cascade. In the model, the phosphatidylinositol (PI) and phosphatidyl 4-phosphate (PIP) are fixed. The PLC substrate, phosphatidylinositol 4,5-bisphosphate (PIP2) is synthesized at a combined basal and stimulated rate from PIP. (See Eqs. 6–7).
FIGURE 4
FIGURE 4
Activation of PLC by dual pathways. The Ga subunit Gq-GTP interacts with PLC to form active complex PLCβ-Gq-GTP that has high affinity for Ca2+ thus creating the active enzyme Ca2+-PLCβ-Gq-GTP that hydrolyzes PIP2 to DAG and IP3 (Fig. 3). Ca2+-PLCβ provides the basal activity in the absence of activated Gq proteins.
FIGURE 5
FIGURE 5
Ca2+-calmodulin-associated signaling elements. Shown are the details of the MLC phosphorylation/dephosphorylation module. The model and simulation system contain dynamic regulation of MLCK and MLCP activities through second messenger-mediated phosphorylation/dephosphorylation events. The effectors are shown in boxes and second messengers in circles.
FIGURE 6
FIGURE 6
Simulation results of a time course of ligand-GPCR-activated processes. (A) Shown are curves for the stimulation of cells with 100 nM agonist (10× Kd) yielding Ca2+-CaM-MLCK complex formation (solid line), Ca2+ release (dotted line), and IP3 (dot-dash line). (B) Prediction of the ligand dependence of the peak of IP3 (squares), Ca2+ (triangles), and Ca2+-CaM-MLCK complex (circles) through activation of the receptor-linked downstream processes. Inset has an experimental example of the dependence of Ca2+ peak on bradykinin concentration in stimulation of smooth muscle cells (adapted from Marsh and Hill, 1993).
FIGURE 7
FIGURE 7
Comparison of the predicted Ca2+ response with experimental results. (A) Output from simulations of the model GPCR-coupled receptor with 100 nM agonist (10× Kd) . IP3 (dashed line)-induced Ca2+ release (dotted line) in the model containing precoupled receptors is faster than that from the model without receptor precoupling (dot-dash line). The experimental data (solid line with points) is the Ca2+ response from smooth muscle cells stimulated with Arg-vasopressin (adapted from Simpson and Ashley, 1989). (B) Profile of IP3 generation (dashed line), PIP2 (dotted line), and activated PLC (solid line) from the precoupled receptor model initiated with 100 nM agonist. The peak of IP3 response corresponds to the peak of PLC activity and the consumption of PIP2.
FIGURE 8
FIGURE 8
Effects of GPCR ligand/agonist concentration on the profile of MLC phosphorylation predictions. Using the generic model with receptor precoupling, the agonist concentration was varied (3–300 nM). The MLC phosphorylation profile is shown with the thickest line corresponding to 3 nM, and lines of decreasing thickness represent 10, 30, 100, and 300 nM respectively. The greatest shift in peak time occurs between the application of 3 and 30 nM agonist.
FIGURE 9
FIGURE 9
Predicted Ca2+-CaM-MLCK complex formation (circles) and peak Ca2+ concentration (boxes) for different bradykinin receptors. (A) Simulated dose-response curves for the high-affinity B2 receptor using the published parameters (Table 1). (B) Simulated dose-response curves for the low-affinity B1 receptor with no change in receptor kinetics. (C) Simulated dose-response curves for the B1 receptor with a fivefold increase in the on-rate parameter.
FIGURE 10
FIGURE 10
Predicted Ca2+ and myosin light chain phosphorylation profiles for a cell containing equal numbers of bradykinin B1 and B2 receptors. (A) Predicted Ca2+ response by application of 3 μM (dotted line) and 30 μM (solid line) of bradykinin. (B) Predicted MLC phosphorylation profile as a function of time with the same treatments. Note the elevated baseline (particularly at 30 μM) after the peak of Ca2+ or MLC phosphorylation.

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