A Dual Receptor Crosstalk Model of G-Protein-Coupled Signal Transduction

Macrophage cells that are stimulated by two different ligands that bind to G-protein-coupled receptors (GPCRs) usually respond as if the stimulus effects are additive, but for a minority of ligand combinations the response is synergistic. The G-protein-coupled receptor system integrates signaling cues from the environment to actuate cell morphology, gene expression, ion homeostasis, and other physiological states. We analyze the effects of the two signaling molecules complement factors 5a (C5a) and uridine diphosphate (UDP) on the intracellular second messenger calcium to elucidate the principles that govern the processing of multiple signals by GPCRs. We have developed a formal hypothesis, in the form of a kinetic model, for the mechanism of action of this GPCR signal transduction system using data obtained from RAW264.7 macrophage cells. Bayesian statistical methods are employed to represent uncertainty in both data and model parameters and formally tie the model to experimental data. When the model is also used as a tool in the design of experiments, it predicts a synergistic region in the calcium peak height dose response that results when cells are simultaneously stimulated by C5a and UDP. An analysis of the model reveals a potential mechanism for crosstalk between the Gαi-coupled C5a receptor and the Gαq-coupled UDP receptor signaling systems that results in synergistic calcium release.

[1]  L. Stryer,et al.  Molecular model for receptor-stimulated calcium spiking. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[2]  DIANQING Wu,et al.  Activation of phospholipase C 132 by the av and 13 y subunits of trimeric GTP-binding protein , 2005 .

[3]  T. Lukas.  A signal transduction pathway model prototype I: From agonist to cellular endpoint. , 2004, Biophysical journal.

[4]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[5]  D. Rubin,et al.  Inference from Iterative Simulation Using Multiple Sequences , 1992 .

[6]  J. Keizer,et al.  A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agonist-stimulated oscillations in Ca2+ concentration. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Kehrl,et al.  RGS2: a multifunctional regulator of G-protein signaling. , 2002, The international journal of biochemistry & cell biology.

[8]  P. Casey,et al.  G protein involvement in receptor-effector coupling. , 1988, The Journal of biological chemistry.

[9]  G. Lemon,et al.  Metabotropic receptor activation, desensitization and sequestration-I: modelling calcium and inositol 1,4,5-trisphosphate dynamics following receptor activation. , 2003, Journal of theoretical biology.

[10]  E M Ross,et al.  Rapid GTP binding and hydrolysis by G(q) promoted by receptor and GTPase-activating proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[11]  S. Rhee,et al.  Regulation of phosphoinositide-specific phospholipase C. , 2001, Annual review of biochemistry.

[12]  Upinder S Bhalla,et al.  Simulations of inositol phosphate metabolism and its interaction with InsP(3)-mediated calcium release. , 2002, Biophysical journal.

[13]  Martin Oppermann,et al.  Ligand‐induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by G protein‐coupled receptor kinases , 1999, European journal of immunology.

[14]  Carol S. Woodward,et al.  Enabling New Flexibility in the SUNDIALS Suite of Nonlinear and Differential/Algebraic Equation Solvers , 2020, ACM Trans. Math. Softw..

[15]  C. Ribas,et al.  Mechanisms of regulation of the expression and function of G protein-coupled receptor kinases. , 2003, Cellular signalling.

[16]  Martin J. Lohse,et al.  Crosstalk between Gαi- and Gαq-coupled receptors is mediated by Gβγ exchange , 1999 .

[17]  Michelle A. Digman,et al.  Activation Mechanisms of Conventional Protein Kinase C Isoforms Are Determined by the Ligand Affinity and Conformational Flexibility of Their C1 Domains* , 2003, Journal of Biological Chemistry.

[18]  S. Snyder,et al.  Inositol 1,4,5-trisphosphate receptors as signal integrators. , 2004, Annual review of biochemistry.

[19]  T. Kendall Harden,et al.  Protein Kinase C Phosphorylates RGS2 and Modulates Its Capacity for Negative Regulation of Gα11 Signaling* , 2001, The Journal of Biological Chemistry.

[20]  Lubert Stryer,et al.  Biochemistry 5th ed , 2002 .

[21]  J. Keizer,et al.  Two roles of Ca2+ in agonist stimulated Ca2+ oscillations. , 1992, Biophysical journal.

[22]  L. Runnels,et al.  Determination of the affinities between heterotrimeric G protein subunits and their phospholipase C-beta effectors. , 1999, Biochemistry.

[23]  R. Tsien.  Fluorescent indicators of ion concentrations. , 1989, Methods in cell biology.

[24]  J. Kehrl,et al.  Heterotrimeric G protein signaling: roles in immune function and fine-tuning by RGS proteins. , 1998, Immunity.

[25]  Huiping Jiang,et al.  Pertussis Toxin-sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors* , 1996, The Journal of Biological Chemistry.

[26]  Gary B Willars,et al.  Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+. , 2003, The Biochemical journal.

[27]  D. Cantrell,et al.  Protein kinase C and beyond , 2004, Nature Immunology.

[28]  F. Colotta,et al.  Targeting C5a: recent advances in drug discovery. , 2005, Current medicinal chemistry.

[29]  Hoon Kim,et al.  Monte Carlo Statistical Methods , 2000, Technometrics.

[30]  M. Simon,et al.  Molecular Mechanism of the Inhibition of Phospholipase C β3 by Protein Kinase C* , 2000, The Journal of Biological Chemistry.

[31]  S. Soltoff,et al.  P2Y6 Nucleotide Receptor Mediates Monocyte Interleukin-8 Production in Response to UDP or Lipopolysaccharide* , 2001, The Journal of Biological Chemistry.

[32]  S. Rhee,et al.  Regulation of phospholipase C-beta 4 by ribonucleotides and the alpha subunit of Gq. , 1994, The Journal of biological chemistry.

[33]  Irene Litosch,et al.  Novel Mechanisms for Feedback Regulation of Phospholipase C‐β Activity , 2002 .

[34]  L. Stryer,et al.  Highly cooperative opening of calcium channels by inositol 1,4,5-trisphosphate. , 1988, Science.

[35]  H. Nakata,et al.  Heteromeric association creates a P2Y-like adenosine receptor , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Simon,et al.  Activation of phospholipase C beta 4 by heterotrimeric GTP-binding proteins. , 1994, The Journal of biological chemistry.

[37]  Mano Ram Maurya,et al.  A kinetic model for calcium dynamics in RAW 264.7 cells: 2. Knockdown response and long-term response. , 2007, Biophysical journal.

[38]  Mano Ram Maurya,et al.  A kinetic model for calcium dynamics in RAW 264.7 cells: 1. Mechanisms, parameters, and subpopulational variability. , 2007, Biophysical journal.

[39]  Julie A. Pitcher,et al.  Pleckstrin Homology Domain-mediated Membrane Association and Activation of the -Adrenergic Receptor Kinase Requires Coordinate Interaction with G Subunits and Lipid(*) , 1995, The Journal of Biological Chemistry.

[40]  M. Simon,et al.  A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated transgene expression , 2006, Proceedings of the National Academy of Sciences.

[41]  P C Sternweis,et al.  Regulation of purified subtypes of phosphatidylinositol-specific phospholipase C beta by G protein alpha and beta gamma subunits. , 1993, The Journal of biological chemistry.

[42]  Bryan L Roth,et al.  G-protein-coupled receptors at a glance , 2003, Journal of Cell Science.

[43]  Madhusudan Natarajan,et al.  A global analysis of cross-talk in a mammalian cellular signalling network , 2006, Nature Cell Biology.

[44]  E M Ross,et al.  GTPase-activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS-like proteins. , 2000, Annual review of biochemistry.

[45]  L. Birnbaumer,et al.  G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[46]  S. Rhee,et al.  Activation of phospholipase C isozymes by G protein beta gamma subunits. , 1993, The Journal of biological chemistry.

[47]  M. Simon,et al.  Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein. , 1993, Proceedings of the National Academy of Sciences of the United States of America.