Temporal Bias: Time-Encoded Dynamic GPCR Signaling

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Review

Temporal Bias: Time-Encoded Dynamic GPCR Signaling Manuel Grundmann1,2 and Evi Kostenis1,* Evidence suggests that cells can time-encode signals for secure transport and perception of information, and it appears that this dynamic signaling is a common principle of nature to code information in time. G-protein-coupled receptor (GPCR) signaling networks are no exception as their composition and signal transduction appear temporally flexible. In this review, we discuss the potential mechanisms by which GPCRs code biological information in time to create ‘temporal bias.’ We highlight dynamic signaling patterns from the second messenger to the receptor–ligand level and shed light on the dynamics of G-protein cycles, the kinetics of ligand–receptor interaction, and the occurrence of distinct signaling waves within the cell. A dynamic feature such as temporal bias adds to the complexity of GPCR signaling bias and gives rise to the question whether this trait could be exploited to gain control over timeencoded cell physiology. The Temporal Dimension of G-Protein-Coupled Receptor Signaling Communication between and within cells represents one of the most important traits of living matter. Information is usually transported in form of chemical substances (hormones, neurotransmitters, proteins, etc.) through the extracellular space, as well as between intracellular compartments. Reception and transmission of extracellular information into the cell’s interior is often realized by membrane receptors. Of these, G- protein-coupled receptors (GPCRs) constitute the largest group in the human genome and traditionally comprise an important family of drug targets [1]. They process and distribute incoming signals from a vast array of ligands to selected intracellular signaling pathways via adaptor proteins (e.g., G proteins) and thus play an integral part in the orchestration of signal transduction. An immense number of ligands is tapered by around 800 GPCRs to some 21 Ga proteins, encoded by 16 genes, and subsumed into only 4 major groups to then diversify again and allow for a cellular reaction that is perfectly attuned to its environment [2]. Nature apparently has found ways to (i) maintain the information content despite limited number of signal transducers and (ii) encode the biological message so that cells are guided to the intended response. The notion that information is not only encoded by the chemistry of the signaling components but also by their spatial and temporal occurrence, brought about the term of ‘spatiotemporal signaling’ [3]. Signal transduction can thus be categorized into at least three dimensions, (i) quality (which substances take part), (ii) space (where the event takes place) and (iii) time (when the event takes place). The interplay between these dimensions will hereinafter be designated as ‘dynamic signaling’ or ‘signaling dynamics’. Dynamic signaling is deeply entrenched in GPCR signal transduction as an increasing number of reports provide us with a glimpse into spatiotemporal aspects of receptor function [4–10]. Studies employing fluorescence cyclic adenosine monophosphate (cAMP) biosensors, for example, greatly expanded our knowledge about conformational dynamics within GPCR signaling networks [11–18]. For in-depth insight into this rapidly expanding field of research we refer the reader to excellent reviews elsewhere

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Trends Over the [29_TD$IF]past years, the temporal dimension of signaling emerged as a discrete parameter of cell signaling and is referred to as dynamic signaling. Technical developments allowed the assessment of temporal bias[30_TD$IF], such as Single-cell and single-molecule fluorescent biosensors based on resonance energy transfer (FRET/BRET) for intra- and inter-molecular rearrangement and interaction, respectively, or protein-induced fluorescence enhancement (PIFE). Single-molecule tracking and fluorescence correlation spectroscopy (FCS). Optogenetic engineering of the cellular signaling machinery in vitro and in vivo as well as light-controlled chemistry. Electron paramagnetic resonance (EPR) and double electron-electron resonance spectroscopy (DEER). Real-time functional assays such as luminescence/fluorescence second messenger assays. Holistic cellular real-time assays, labelfree optical (DMR) and electrical (CDS) techniques. High-content imaging systems/microscopy with improved temporal and spatial resolution. Computational methods (e.g., molecular dynamics simulations). These developments led to the introduction of several concepts that underlie kinetic aspects of GPCR signaling, such as kinetic scaffolding, ligand residence time, dwell times, frequency filters, oscillatory phenomena, signaling from internalized receptors and structural dynamics as signaling determinant.

https://doi.org/10.1016/j.tips.2017.09.004 © 2017 Elsevier Ltd. All rights reserved.

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This prompted us to consider temporal bias as a kinetic quality beside physical and spatial quality as a category of signaling bias.

[19–24]. For the purpose of this review, we will limit ourselves to molecular mechanisms that underlie dynamic GPCR signaling. We begin at the second-messenger and G-protein level, follow the pathway upstream to the receptor–adaptor interface, and end at the level of ligand– receptor interaction. By highlighting selected research findings, we propose the idea of ‘temporal bias’ that employs time as an additional variable to convey high-fidelity GPCR signaling.

Signaling Dynamics at the Second-Messenger Level Already some decades ago, changes of intracellular calcium concentrations (Ca2+[297_TD$IF]i) following single extracellular stimuli were found to not always occur as transient, monophasic responses but showed a repetitive or oscillating behavior [25]. Ca2+i oscillations have since then been investigated as possible time codes for biological information [26]. Gq-coupled GPCRs are prominent regulators of Ca2+i waves, a discovery underlined by early findings of repetitive calcium spikes in hepatocytes treated with angiotensin II or phenylephrine [25]. The frequency of calcium oscillations was shown to correlate with stimulus concentration [25,27], indicating that frequency functions as a code for agonist efficacy. A plausible mechanism underlying calcium oscillations in nonexcitable cells is provided by the IP3-Ca2+ cross-coupling model [28] (Figure 1). Therein, Gq protein-mediated calcium elevations potentiate phospholipase C (PLC) activity in a positive feedback mechanism, which causes enhanced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and inositol trisphosphate (IP3). Elevated IP3 levels in turn amplify calcium release from internal stores by increased activation of IP3 receptors on the endoplasmic reticulum (ER). High cytosolic calcium concentrations then desensitize IP3 receptors and block further calcium release. Subsequently, depleted calcium stores are replenished by ER-calcium pumps and especially by extracellular calcium influx via activation of membrane calcium channels [store-operated calcium entry (SOCE)]. Once cytosolic calcium concentrations are low enough, IP3 receptors are resensitized to resume another cycle of calcium release [29,30]. Protease-activated receptor PAR1 stimulation in platelets, for instance, evokes calcium spikes that are accurately modeled by this feedback–feedforward system [31] and activation of phospholipase C-b (PLCb) by inducible Gaq proteins also induces cytosolic calcium oscillations in agreement with the IP3-Ca2+ cross-coupling model [32]. Yet, calcium oscillations do not exclusively result from the action of the Ca2+ releasing second messenger IP3 that is produced upon Gq-mediated PLC activity. Repetitive spikes of cytoplasmic Ca2+ also arise when ligand-activated GPCRs engage the G12/13 signaling cascade as previously described for GPR55, a family A GPCR incompetent to elicit Gq activity [33,34]. The concentration of signaling molecules not only can change over time (calcium waves), but also their intracellular location varies dependent on time. Nuclear factors of activated T cells (NFATs) are transcription factors that translocate into the nucleus after activation by an increase of cytosolic calcium to regulate gene expression. Deactivated NFATs then exit the nucleus and are accessible to another cycle [35]. NFATs thus constantly shuttle between cytosol and nucleus with an inherent frequency and, in addition to the calcium waves that activate them, show an oscillating behavior by themselves. 1

In a study designed to understand the rhythmic behavior of this system, a third frequency was introduced when muscarinic M3-receptor-expressing HEK293 cells were stimulated with defined pulses of carbachol using microfluidics [36]. Sumit et al. could show that intermediate rates of agonist pulses (input), which were between the low frequent NFAT shuttling and the high frequent calcium waves, induced maximal NFAT transcriptional activity, whereas both lower and higher frequencies induced less activation (output). The authors proposed a bandpass filter model for this oscillating system. The rate of repetitive calcium signals constitutes the upper limit (low-pass filter) and the NFAT shuttling rate the lower limit (high-pass filter), that is, only input frequencies between these thresholds were effectively transmitted. Thus, the

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Molecular-, Cellular- and Pharmacobiology Section, Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany 2 Kidney Disease Research, Bayer Pharma AG, Aprather Weg 18a, 42113 Wuppertal, Germany

*Correspondence: [email protected] (E. Kostenis).

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Figure 1. Schematic of the IP3-Ca2+ Cross-Coupling Model to Explain Calcium Oscillations. (A) Cytosolic calcium influx is mediated via phospholipase C-b (PLCb) 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 PLCb 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.

biological system defined a specific range of frequency (band-pass) that allows maximal signal transduction [36] (Figure 2). Biological frequency filtering occurs dynamically as cells that are exposed to a periodic stimulus continually adjust the frequency of the oscillating parts within the system depending on the input signal. The synchronization of the temporal pattern of stimulus and response (e.g., pulsatile receptor activation and repetitive calcium signals) is denoted as phase-locking and provides a means for cells to adapt to their momentary environment [37]. The idea of frequency filters to ensure signal fidelity originated from electrical and electronical engineering and was later validated also for biological systems. Box 1 offers a glimpse into parallels of GPCR signaling and digital signal processing.

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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].

Stimulus strength is, however, not only encoded in calcium oscillations but also in oscillations of other signaling molecules, such as protein kinase C (PKC). Conventional PKCs shuttle between cytosol and plasma membrane with different kinase activity depending on the activating calcium waves [38,39]. Parallel imaging of Ca2+i and PKC activation with a Förster-resonance energy transfer (FRET) biosensor revealed that membrane-bound PKC exerts high frequent phosphorylation–dephosphorylation cycles in phase with a high frequent calcium input, whereas cytosolic PKC did not [40]. Hence, PKC activity is sensitive to its micro-environment (intracellular location of PKC determines whether input signals are efficiently transmitted or not) and underlines once again the importance of the interplay between time and space for cellular signal transduction.

Signaling Dynamics at the G-Protein Level Guanine nucleotide-binding proteins (G proteins) are essential transducers in GPCR signaling pathways. G proteins can be classified into monomeric (or small) and heterotrimeric G proteins (consisting of three subunits: Ga, Gb, and Gg) [2]. They are commonly described as molecular switches, which are in an ‘on’ [guanosine triphosphate (GTP)-bound] or an ‘off’ [guanosine

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diphosphate (GDP)-bound] state. G-protein activation is initiated by the dissociation of GDP from and subsequent binding of GTP to the nucleotide binding pocket of the Ga subunit, whereas hydrolysis of GTP to GDP deactivates Ga. Guanine nucleotide exchange factors (GEFs, such as GPCRs in their active conformations) trigger the exchange of GDP for GTP at the inactive G protein and thus lead to G-protein activation. Active, GTP-bound G proteins can then interact with effector proteins to transduce a signal before GTP hydrolysis terminates the signal and allows the G protein to enter another cycle. Transition from the active to the inactive state is mediated by an intrinsic GTPase activity of Ga and by interaction with accessory proteins. In fact, intrinsic GTPase activity, and thus recovery of mammalian G proteins is slow (seconds to minutes). Therefore accelerating proteins called GTPase-activating proteins (GAPs) are needed to efficiently sample an input signal [41] (see also Box 1 for an example of protein-assisted input sampling). Intriguingly, not only the presence of a signal but likewise its absence, that is the exact timing of ‘on’ and ‘off’, is crucial for its interpretation by the cell [42]. It is therefore a biological necessity to precisely control also signal termination, for instance through GAPs. GAP control on G proteins is mostly – although not exclusively (see Box 1) – realized by the family of regulators of G-protein signaling (RGS). Binding of RGS proteins to a G protein lowers the energy barrier for GTP hydrolysis by stabilizing an intermediate state during the GTPase reaction and thereby facilitates the inactivation of the G protein [41,43]. Lack of RGS proteins can cause dysfunction of signal termination as reported for a RGS9 mutation that leads to bradyopsia, a disease that is characterized by an impaired ability to see moving objects under low-contrast conditions [44]. Likewise, reduced abundance of RGS2 in prostate cancer cells is associated with enhanced GPCR signaling and tumor progression in androgen-insensitive forms of prostate cancer [45]. These selected examples underscore the physiological significance of temporal control over G-protein signaling. Beside the negative regulation of G-protein activity, RGS proteins also intensify G-protein signaling [41,46]. This was first detected by RGSmediated acceleration of both onset and termination of G protein-gated inwardly rectifying potassium (GIRK) channel current, an ion channel whose opening is triggered by interaction with Gbg subunits originating from activated heterotrimeric Gai and Gao family proteins [47,48]. Two seemingly contradicting concepts emerged to explain the bilateral behavior of RGS proteins on G-protein activity, namely the kinetic and the physical scaffolding theories (see Box 2). Beside GEFs and GAPs, G proteins are furthermore controlled by G-protein dissociation inhibitors (GDIs). GDIs negatively regulate G-protein signaling through inhibition of the

Box 1. Ensuring Biological Signal Fidelity Signal distortion and artifacts are common problems in digital processing. A major cause of such errors is known as temporal aliasing, which is produced by a mismatch in the sampling rate of analog–digital signal converters, that is, how fast or slow an input signal is scanned and converted into an output signal [88]. If a continuous input signal (analog) is transformed into a binary, on–off-like signal (digital), sampling rate is crucial to ensure high signal fidelity. Although highly simplified, receptors or G proteins can be regarded as biological analog-digital converters or switches that are likewise error-prone and therefore need precise control of their sampling rates. An example of feedback-regulated control over GPCR/G-protein signal sampling rate is given by a study from Waldo et al. PLCb isoforms are main effector proteins of Gaq-mediated signaling. Activated PLCb in turn accelerates GTP hydrolysis (GAP) at the G protein that triggered PLCb activation in a feedback loop [41]. The authors reported the structure of PLCb3 with its activating Gaq protein and described a catch-and-release mechanism to sharpen the dynamics of Gaq signaling [89]. According to their work, GAP activity of PLCb on Gaq ensures signal fidelity by establishing a proper sampling rate corresponding to the input signal. In the absence of PLCb, the active GTP-bound Ga state is relatively long-lived, so there is an increased probability for the G protein to interact with effector proteins and to amplify the signal. However, once bound to PLCb, Gaq is turned off quickly and is thus set again for receiving another activation signal [90] (Figure I). The temporal regulation of Gaq activity by PLCb thus mimics a type of kinetic proofreading: it ensures that PLCb is activated when the receptor is activated but concomitantly turns off the effector when the receptor is turned off again.

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Cells might use this proofreading mechanism to avoid creating artifacts by transducing unintended, random input signals.

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Figure I. Kinetics of G-Protein Dynamics Determine Signal Fidelity. G proteins oscillate between inactive, GDPbound, 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 Gaq pathway stimulates PLCb, which leads to further downstream effects and cell response. Intriguingly, PLCb also functions as a GAP for the Gaq 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 [295_TD$IF](according to [89]).

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Box 2. The Kinetic Scaffolding Theory Explains Positive and Negative Regulation by RGS Proteins RGS proteins not only accelerate GIRK channel deactivation but also speed up the activation step. Notably, they leave steady-state amplitude unaffected [47,48]. This sharpening of GIRK current waveforms is believed to be critical for timing of neuronal communication [47,48]. The discovery of non-GAP domains on RGS proteins was a first attempt to explain this ‘RGS paradox’. Non-GAP domains could potentially scaffold GPCR and G protein so that RGS proteins were thought to sharpen G-protein signaling dynamics by a combination of traditional GAP activity and physical scaffolding with the receptor (GEF) that supports G-protein activation [91]. In contrast, the model of kinetic scaffolding exploited GAP-only activity of RGS proteins to explain both opposite effects [92]. By accelerating GTP hydrolysis and thus providing readily available GDP-bound G protein near the receptor, kinetic scaffolding would permit G proteins to be rapidly recoupled to the receptor. In steady-state, G-protein activation is balanced with its deactivation via receptor-mediated activation and RGS-proteinmediated deactivation, respectively. GAP-stimulated GTP hydrolysis is faster than the dissociation of GTP-bound G protein from the receptor. Consequently, persistent receptor influence directly triggers GDP–GTP exchange [41,93]. After ligand dissociation only GAP-mediated G-protein deactivation remains and thus the signal decreases. Hence, RGS proteins sharpen G-protein responses only near the receptor, but decrease G-protein signaling at distant effector sites [92,93]. Spatial proximity and thus physical scaffolding is a central element in the kinetic scaffolding theory. Therefore, the theories may not be as mutually exclusive as it appears at a first glance. Recently, measurements of GIRK current in Xenopus oocytes indicated that RGS proteins also increased the amplitude of GIRK current [94]. These observations suggest that more Gbg is released by RGS activity, which is inconsistent with the kinetic scaffolding theory that implicates maximal G-protein output at the level without RGS. Therefore, the authors propose an additional explanation for RGS-amplified G-protein signaling by enhancing the nucleotide exchange at the Ga subunit. RGS proteins would thus bind to inactive, GDP-bound G proteins to act as GEF and bind to active, GTPbound G proteins as a GAP [94]. This behavior was furthermore observed to be dependent on the Ga subtype, as Gao but not Gai subunits were affected this way. Hence, RGS proteins mediate signaling bias by selectively acting on one Gprotein isoform but not the other.

GDP–GTP exchange step during G-protein activation. A modeling approach illustrated that increasing GDI concentrations transform the temporal activation pattern of the small G proteins RhoA and Rac1 from a switch-like on/off (that is digital or bistable) to a sustained oscillatory behavior [49]. Although the biological consequence of this transformation remains concealed for the time being, experimental evidence substantiated the functional significance of a temporal modulation of RhoA. RhoA is tightly linked to dynamics of the cytoskeleton and regulates rhythmic cell responses such as protrusion–retraction in the leading edge of migrating cells and tumor metastasis or invasion [50]. Tkachenko et al. reported that RhoA activation and its phosphorylation by protein kinase A (PKA) is kinetically synchronized with the protrusion of PtK1 epithelial kidney cells [51]. PKA-mediated phosphorylation of RhoA here serves as a deactivation signal because phosphorylated RhoA, but not the phosphorylation-incompetent RhoAS188A[301_TD$IF] mutant, permitted the interaction with the GDP dissociation inhibitor of RhoA (RhoGDI) that led to subsequent inhibition of RhoA-effector interaction and signaling shutdown [51]. This kinetic orchestration of RhoA activity by PKA and RhoGDI acts as a pacemaker for PtK1 cell protrusion–retraction cycles and thus finally dictates cell migration. Heterotrimeric G proteins are likewise susceptible to kinetic regulation. A recent study by Furness et al. showed that the conformational rearrangement in the agonist-stimulated, Gscoupled calcitonin (CT) receptor transmits information about signal strength (efficacy) to the coupling G protein by altering its turnover speed [52]. Experiments in COS-7 cells revealed different efficacies of human and salmon calcitonin (hCT vs. sCT) both acting at the CT receptor. hCT and sCT induced different receptor conformations that in turn caused distinct Gs heterotrimer conformations entailing fast or slow cycling speeds and thus high or low efficacy, respectively [52]. This study implicates the induction of distinct G-protein conformations, as has been postulated earlier [53], and emphasizes the possibility that G-protein kinetics can encode agonist efficacy. This contrasts with the traditional understanding where ligandinduced conformational change is confined to the receptor whose conformation has either high or low affinity for G proteins to produce high or low agonist efficacy, respectively. Propagating

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conformational change from the receptor to a downstream level can, however, be broadened to other GPCR adaptor proteins as well, as a recent report found distinct b-arrestin2 conformational states depending on the structure of upstream angiotensin AT1A receptor ligands [54].

Signaling Dynamics Originating from the Receptor–Adaptor Interface An initial receptor impulse may be generated at the plasma membrane by what is generally thought of as ‘canonical’, G-protein-mediated signaling: surface GPCRs activate G proteins at the plasma membrane that then dissociate from the receptor to interact with effector proteins at the membrane or at intracellular locations. Delayed signal impulses emerge either from (or near) the cell membrane or from internalized structures incorporating additional transducers (Figure 3A). The detailed composition of the signaling-competent structures and specifically the function of the constituents thereof is currently still inconclusive but two GPCR adaptor protein families, b-arrestins and G proteins – in cooperation with the endocytic machinery – appear as key components that bias signaling temporally and spatially [55]. b-arrestins were first discovered as mediators of GPCR desensitization [56]. The general assumption is that an activated receptor recruits b-arrestin, which subsequently displaces the G protein and targets the receptor for endocytosis [57]. b-arrestins mediate the interaction between receptor and components of the endocytic machinery, such as clathrin and the AP-2 adaptor complex and thus induce the formation of clathrin-coated pits (CCPs), one pathway to sequester receptors from the cell surface [58]. The scaffolding function of b-arrestins does not merely entail the termination of canonical G-protein signaling and receptor internalization but is also suggested to account for ‘noncanonical’ GPCR signaling [59]. The prevailing tenet of such noncanonical GPCR signaling is that b-arrestins promote receptor concentration in CCPs that subsequently internalize via different endosomal pathways in a dynamin-dependent process [60]. Such desensitized receptors enter endocytosis as GPCR-b-arrestin complexes and serve as the starting point for ‘b-arrestin-mediated’ signaling, most prominently manifested as – but not limited to – activation of the mitogen-activated protein kinase (MAPK) pathway [61]. Recently, this dogma has been challenged by observations that receptor-activated b-arrestin2 forms clathrin-coated structures at the membrane deficient of the receptor and from there induces phosphorylation of extracellular signal-regulated kinases (ERK), a subset of the mammalian MAPK family. Eichel et al. propose an ‘activation-at-a-distance’ mechanism, in which b-arrestin2 gets activated by transient interaction with the b1-adrenergic receptor and organizes in clathrin-coated structures without the receptor as the origin of MAPK signaling [7]. FRET experiments in another study indeed revealed that b-arrestin2 can temporarily maintain its active conformation after dissociation from the activating receptor, reminiscent of a conformational memory effect [62]. Although this effect was relatively short-lived and more evidence for such a memory effect is needed, single molecule spectroscopy experiments revealed first indications that the elementary principle of a conformational memory within single proteins might actually be present [63]. Nevertheless, it is highly likely that arrestin conformational changes that ultimately determine MAPK signaling intensity and duration entirely rely on prior Gprotein activation [64,65]. In addition to b-arrestin’s temporal modulation of G-protein signaling, G proteins themselves can account for delayed GPCR signaling. A nanobody-based biosensor approach provided evidence for the existence of active Gas proteins in complexes with b2-adrenergic receptors at both the cell membrane and intracellular locations as the origin of two spatially and moreover temporally separated Gas-mediated signaling waves [6]. Experiments in primary thyroid follicles previously showed that a temporally-modulated cAMP response from internalized thyroidstimulating hormone (TSH) receptors is important for adequate hormone synthesis and release [66]. Further evidence for intracellular G-protein signaling came from the discovery of a

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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 b-arrestin-coordinated membrane compartments. A third sustained signaling wave finally emerges from receptors at intracellular locations such as endosomes harboring G proteins and/or b-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 m-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 m-opioid receptors are spatially restricted within the cell membrane, which causes sustained G-protein-mediated ERK signaling [294_TD$IF](according to [10]).

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temporal bias of the parathyroid hormone (PTH) receptor, which showed sustained Gasmediated cAMP signaling from internalized agonist–receptor–G-protein complexes but transient cAMP signaling at the plasma membrane [15]. Later, the involvement and molecular mechanism of b-arrestin in the sustained part of the PTH receptor-induced cAMP signaling was studied. Wehbi et al. postulated a mechanism reminiscent of the kinetic scaffolding theory (see Box 2), in which b-arrestin-coupled PTH receptors form a ternary complex with Gbg-dimers to accelerate G-protein turnover and additionally – in contrast to the original kinetic scaffolding theory – increase steady-state G-protein signaling [67]. b-arrestins would here act as scaffold proteins that build signaling complexes consisting of receptors together with G proteins. This step was previously thought to be unlikely because of mutually exclusive binding of G protein or b-arrestin to the receptor (b-arrestin-mediated desensitization of G protein-mediated signaling). Single-particle electron microscopy and bioluminescence resonance energy transfer (BRET) assays, however, revealed megaplexes composed of the chimeric b2V2 GPCR, Gs heterotrimer and b-arrestin2 that may act as a source of sustained G-protein signaling from endosomes [8]. Furthermore, a follow-up study elucidated a Gbg-guided crosstalk mechanism between the b2-adrenergic receptor and signaling downstream of the PTH receptor through formation of even higher ordered super-complexes consisting of Gbg subunits, together with b-arrestin, Gas and PTHR along with the activating ligand PTH and adenylyl cyclase 2 in HEK293 cell endosomes [9]. Studies employing several additional GPCRs, including the vasopressin V2, the luteinizing hormone LH, the glucagon-like peptide GLP-1 and the sphingosine-1-phosphate S1P1 receptor, provide further support for the concept that GPCR ligands de facto create temporal bias at the level of the receptor–adaptor interface [5,68–70].

Signaling Dynamics Originating from the Level of Ligand–Receptor Interaction Recently, the concept of drug residence time for compound selection and optimization in drug discovery came to the fore and has been discussed elsewhere in detail [71,72]. Instead of focusing on the Kd values of the ligands as a measure of affinity, it was proposed to give more attention to their binding kinetics, particularly the koff values, because slowly dissociating ligands have been proven successful as antagonists [73]. The molecular mechanisms governing how such kinetically biased ligands exhibit variable residence times at the receptor are, however, poorly understood. An extended residence time can emerge from slowly dissociating ligands, for instance by induction of receptor conformations that impede ligand unbinding or by trapping the ligand together with the receptor by, for example, cointernalization into endosomes. It was subsequently hypothesized that ligand residence time dictates whether signaling originates only from the plasma membrane or from internalized GPCR–ligand complexes, while rebinding events determine the signal duration, which would have a substantial effect on the signaling dynamics of GPCRs [74] (Figure 3B). Indeed, the difference between the temporally biased ligands PTH and PTH-related protein (PTHrP) to produce a sustained versus a rapid cAMP response was initially explained by either different binding kinetics or different affinities of these two ligands to the G-protein-coupled and -uncoupled PTH receptor [75]. Later, the different temporal signaling pattern could be explained by distinct receptor internalization. PTHrP action was restricted to the cell membrane, whereas PTH induced a cAMP response also from endosomal receptor species, indicating that ligand-binding kinetics shape spatiotemporal receptor signaling [15]. Moreover, ligands that differ in receptor-binding kinetics could theoretically prove useful also as regulators of signal fidelity in the form of a kinetic proofreading (compare with Box 1) [42].

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Threshold-controlled dwell-time duration of GPCR ligands might dictate whether a receptor signal is initiated or not. Long residence times at a receptor could ensure signal initiation while fast ligand dissociation precludes receptor activation. Pharmacological parameters of ligand activity such as efficacy and potency are traditionally determined in experiments under kinetic equilibrium conditions. These setups, however, frequently ignore the fact that cell activation as well as ligand–receptor interaction is a highly dynamic process that has characteristic nonequilibrium features and subsequent consequences for receptor structure and activation [76,77] (see Box 3). Alterations in the ligand-binding behavior may affect the dynamics of GPCR signaling and could thus represent another level to create temporal bias. The impact of the binding kinetics on ligand bias is underlined by two recent publications [78,79]. In the first study, a set of dopamine D2 receptor ligands was inspected for their ability to activate distinct signaling pathways in CHO cells using multiple functional readouts. The extent of signaling bias (quantification to what degree a pathway is preferentially activated) was found to be a variable of the measurement time point of the assay [79]. Aripiprazole and bifeprunox changed their pattern of bias towards certain pathways significantly over time, while other agonists did not or only weakly. Such ‘kinetic bias’ was correlated with different residence times of the ligands at the receptor, that is aripiprazole and bifeprunox dissociated more slowly from the receptor compared to other D2 receptor agonists. However, it remains unanswered here how the different kinetics of the ligand-receptor interaction translate to a temporal bias. One potential explanation would have been that the ligands induce different receptor conformational states over the time and therefore generate different signaling events. The second study investigated the signaling dynamics of the free fatty acid receptor FFA2 in HEK293 cells by detecting two distinct signaling impulses and provided a molecular mechanism for a temporal bias at the level of the ligand–receptor interaction: real-time functional cell phenotyping using label-free biosensors along with molecular dynamics studies unveiled that the allosteric FFA2 agonist 4-CMTB sequentially triggers a first and a second signaling impulse by activating distinct binding sites in a stepwise manner [78]. Thus, interaction with site 1 elicited a first

Box 3. Do Dynamic Conformational Ensembles Determine Signaling Bias? An intriguing question about ligand–receptor interaction is how distinct ligands can bias the signaling of a given receptor. One explanatory approach is that different ligands induce different discrete receptor conformations that in turn trigger the activation of different signaling pathways. However, we now know that receptors do not remain in discrete conformations but constantly oscillate between them. Electron paramagnetic resonance (EPR), especially double electron–electron resonance (DEER) spectroscopy and single molecule fluorescence spectroscopy, revealed that GPCRs actually switch between structural states and show remarkable flexibility [95,96]. Receptor states are therefore better described by an ensemble of different conformations (and different energy states) that the protein samples. Three parameters emerge from these studies that characterize the dynamic behavior of ligand-free and ligand-bound receptor ensembles: (i) retention time of a receptor complex in one conformation, (ii) the frequency of switching between the conformations and (iii) the temporal pattern of long and short retention times in the conformations. The assumption that ligand binding is often accompanied by a shift of the conformational ensemble towards sets of signaling-competent conformations is corroborated by the finding that ligands with different pharmacology (full or partial agonists and inverse agonists) could distinctly modulate the above-mentioned parameters [95,96]. This validates the concept that structural dynamics of the receptor protein determine signaling output. It remains, however, unknown which of the parameters (or if all) are necessary to fully describe the dynamics of ligand-induced GPCR activation, but the reports underscore the need to change our view how receptors are activated: from the perception that receptors undergo a conformational change from one conformation to another (even though with intermediates) towards the recognition of a dynamic switching between whole ensembles of conformations with specific temporal and structural patterns. It is conceivable that these very patterns, that is, the structural dynamics, are the determinants of ligand activity and thus signaling bias. Consequently, linking specific static GPCR structures to distinct signaling outcomes would have to fall short. Future studies will have to confirm this idea and show whether the dynamics instead of individual receptor conformations are the decisive criteria that control subsequent signaling events.

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signaling wave, while subsequent binding to site 2 produced a second signaling wave (Figure 3C). Multistep binding of GPCR ligands with temporary halts at different sites has been documented in several cases – albeit not linked to receptor activation – and represents a captivating concept to gain control over temporally-biased signaling from the ligand-binding level [80–83]. Because standard equilibrium assays generally fail to detect transient cell activation, the recording of cell responses in real time proved an effective way to uncover receptor signaling dynamics. Receptor mobility within the plasma membrane as well as its translocation and clustering by b-arrestins or G-protein-coupled receptor kinases (GRKs) emerge as further determinants of spatiotemporal GPCR signaling. It was shown for the m-opioid receptor (MOP) and the two MOP agonists morphine and DAMGO that DAMGO induced MOP receptor translocation within the membrane and consequently caused transient ERK phosphorylation, whereas morphine restricted MOP receptor redistribution within the membrane by a Gbg- and PKCa-dependent mechanism to induce sustained ERK phosphorylation. Moreover, the morphine signaling fingerprint could be transformed to resemble the DAMGO-induced signaling patterns via inhibition of PKCa and concomitant translocation of the receptor within the membrane corroborating that receptor redistribution within the plasma membrane is as key determinant for spatiotemporally biased MOP signaling [10] (Figure 3D,E). So far, we have presented various examples of fine-tuned signaling mechanisms but there are also contrary findings. Although the concept of fine-tuning cell response with chemically diverse ligands attracts a lot of attention it is not undisputed. A complementary view is offered in a study that measured cell activation in response to b2-adrenergic receptor activation in HEK293 cells with the chemically distinct ligands isoproterenol and salmeterol at early, intermediate, and late time points (determined as second messenger level/receptor endocytosis, protein phosphorylation, and gene transcription, respectively). Although salmeterol showed high affinity/low efficacy and isoproterenol low affinity/high efficacy, both ligands produced an identical integrated response. The authors proposed a modular system to describe the signal transduction cascades, wherein endosomal receptor signaling functions as an ultrasensitive or digital switch that transforms the diversity at the upstream ligand–receptor interface into a stereotyped cell response at the transcriptional level [84]. Such data relativize the tenet of creating subtle cellular responses using chemically refined ligands, and moreover highlight how functional units (modules) within GPCR signal transduction cascades might act as noise filters to ensure biological signal fidelity towards multifarious ligand input (see also Box 1). While plausible, an alternative interpretation is conceivable as well. If low affinity/high efficacy and high affinity/ low efficacy drugs are compared in assays with different sensitivity and amplification (upstream cAMP levels vs. downstream gene transcription) they may differ at low but be equal at high amplification conditions, which is what the authors found and which would be in line with no temporal bias. Regardless, this elegant study emphasizes the need to consider real-time data as an invaluable source of information for pharmacological characterization of drugs.

Concluding Remarks Ligands targeting GPCRs can activate distinct pathways from a multitude of signaling routes, which is also denoted as ligand bias. Apart from subcellular environment (location) and physical composition (quality), GPCR ligands also employ dynamic aspects (time) to convey information from cell to cell. We therefore propose to extend the current view of signaling bias by a kinetic component: temporal bias. Temporal bias is distinct from ‘conformational bias’, where ligands induce different receptor conformations that are linked to distinct signaling outcomes (see Box 3). Its core feature is a time-dependent come and go of signaling events. Temporal bias may coalesce with other types of bias including conformational bias and affect, for example, the magnitude of the latter [52,78,79,84]. To date, conformational bias is both widely accepted and

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Outstanding Questions What are the manifestations of signaling dynamics? How are temporal and spatial aspects interlinked? Can we find a logic behind where and when a signaling event happens? What are the determinants in the dynamics of proteins that define a temporal code? What are the rhythmic patterns of conformational ensembles that dictate the signaling outcome? How are the different time scales connected, from microsecond molecular switches within proteins to seconds, minutes and hours of shuttling molecules, ligand pulses, and transcriptional responses? What are the mechanisms that explain the apparent difference from singlecell dynamics to the temporal behavior of cell populations in tissues and organs? What discerns pathological signaling from physiological signaling temporally? Is it possible to correct aberrant signaling by temporally biased drugs? Can temporal bias be synthetically designed into GPCR-signaling modulators?

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clinically exploited, because researchers have means to design such bias into chemicals. For temporal bias to become pharmacologically accessible, it is still mandatory to establish appropriate experimental setups that allow for capturing even ephemeral signaling events (see Trends). Our understanding of the molecular principles that underlie temporal bias is still incomplete and, furthermore, insufficient for a rational pharmacological intervention with time as the target (see Outstanding Questions). If the molecular mechanisms of signaling dynamics in health and disease conditions were more clearly understood, medicinal chemistry might design temporal bias into compounds to possibly correct disturbed signaling with the compounds’ pharmacodynamic and pharmacokinetic features. This may be further supported by technological advances such as targeted drug delivery systems. Intentional intervention with signal dynamics using temporally-biased drugs would, without doubt, constitute an innovative approach and a hallmark in the cure of diseases, yet it will most likely take time until we are faced with the first drug specifically interfering with the temporal dimension of GPCR signaling. Nevertheless, the idea to target, for example, multiple signaling units dynamically, and the perception of signaling dynamics as a pharmacological target itself, represent quiet harbingers of a (distant) future yet to come [85–87]. [29_TD$IF]Disclaimer Statement The authors declare that there are no conflicts of interest.

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