Temporal Bias: Time-Encoded Dynamic GPCR Signaling Manuel Grundmann, Evi Kostenis  Trends in Pharmacological Sciences  Volume 38, Issue 12, Pages 1110-1124 (December 2017) DOI: 10.1016/j.tips.2017.09.004 Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Schematic of the IP3-Ca2+ Cross-Coupling Model to Explain Calcium Oscillations. (A) Cytosolic calcium influx is mediated via phospholipase C-β (PLCβ) activation downstream of G-protein-coupled receptors (GPCRs) that increase intracellular inositol trisphosphate (IP3) levels. Calcium release is triggered by binding of IP3 to IP3 receptors at the endoplasmic reticulum (ER). Elevated calcium concentrations further promote PLCβ activation. (B) Enhanced PLC activity leads to further increase of cytosolic calcium concentrations. (C) Maximal intracellular calcium levels desensitize IP3 receptors to stop calcium release from the ER. Cytosolic calcium concentration decreases while depleted calcium stores are refilled. (D) Low basal cytosolic calcium levels resensitize IP3 receptors to resume a new cycle of calcium release from the ER. Note that, just as for calcium, IP3 levels also undergo oscillations in this model. Trends in Pharmacological Sciences 2017 38, 1110-1124DOI: (10.1016/j.tips.2017.09.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 GPCR Signaling as Band-Pass Filter. Pulsed G-protein-coupled receptor (GPCR) stimulation leads to high-frequency oscillations of intracellular calcium release via canonical Gq-protein-mediated signaling. Translocation of the transcription factor NFAT is guided by phosphorylation and dephosphorylation cycles that are under control of the intracellular calcium oscillations. Inactive NFAT resides in phosphorylated form in the cytosol. Calcium-mediated dephosphorylation leads to NFAT translocation into the nucleus to regulate gene transcription. NFAT shuttling shows significantly slower frequencies than calcium flux. Maximal cell response upon GPCR activation is achieved if ligand pulses are matched to the cell's inherent band-bass filter property, wherein calcium oscillations determine the upper limit and NFAT translocation the lower limit of the filter. Reproduced, with permission, from [36]. Trends in Pharmacological Sciences 2017 38, 1110-1124DOI: (10.1016/j.tips.2017.09.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Temporal Bias Emerging from the Receptor or Ligand–Receptor Level. (A) G-protein-coupled receptors (GPCRs) can induce distinct but coalescing signaling waves. Canonical G protein-mediated signaling from the plasma membrane triggers the first transient wave, while a second impulse is generated by noncanonical signaling from internalizing structures such as β-arrestin-coordinated membrane compartments. A third sustained signaling wave finally emerges from receptors at intracellular locations such as endosomes harboring G proteins and/or β-arrestins. Ligands targeting the receptor can control which signaling pattern prevails. (B) Ligand-binding kinetics might dictate whether a GPCR cointernalizes with its ligand or not. Ligands with balanced on and off rates do not induce receptor–ligand cointernalization, while ligands with substantially longer residence times trigger receptor endocytosis along with the agonist. Thus, an initial cell response originates from plasma-membrane-situated receptors. After internalization, endosomal enrichment of ligand and rebinding phenomena generate sustained signaling from internalized structures (adapted from [74]). (C) Transient occupancy of receptor epitopes by stepwise-binding ligands can generate a sequential receptor activation with temporally distinct signaling waves (according to [78]). (D,E) The membrane redistribution of receptors controls temporal GPCR signaling. (D) DAMGO (diamond) stimulation of μ-opioid receptors leads to translocation across the cell membrane and thus determines transient G protein-mediated extracellular signal-regulated kinase (ERK) signaling. (E) Morphine (oval)-stimulated μ-opioid receptors are spatially restricted within the cell membrane, which causes sustained G-protein-mediated ERK signaling (according to [10]). Trends in Pharmacological Sciences 2017 38, 1110-1124DOI: (10.1016/j.tips.2017.09.004) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure I Kinetics of G-Protein Dynamics Determine Signal Fidelity. G proteins oscillate between inactive, GDP-bound, and active, GTP-bound, forms. GDP–GTP exchange is triggered by guanine nucleotide exchange factors (GEFs) such as GPCRs. Hydrolysis of GTP to GDP is substantially accelerated by GTPase-activating proteins (GAPs). Activation of the Gαq pathway stimulates PLCβ, which leads to further downstream effects and cell response. Intriguingly, PLCβ also functions as a GAP for the Gαq protein that activates it. The resulting accelerated G-protein cycling ensures higher sampling rates to match upstream GPCR activation cycles, which finally leads to higher signal fidelity (according to [89]). Trends in Pharmacological Sciences 2017 38, 1110-1124DOI: (10.1016/j.tips.2017.09.004) Copyright © 2017 Elsevier Ltd Terms and Conditions