Commentary Cellscience Reviews Vol.2 No.3 ISSN 1742-8130

Resolving G protein-coupled receptor signaling mechanics in vivo using fluorescent biosensors

Christopher A. Johnston & David P. Siderovski

Introduction

Heterotrimeric G-proteins are molecular switches that relay information intracellularly in response to various extracellular signaling cues (Gilman, 1987; McCudden et al., 2005). This characteristic is achieved by the adoption of two distinct nucleotide-bound states: an inactive, GDP-bound and an active, GTP-bound state. Binding of activating ligands (“agonists”) to G protein-coupled receptors (GPCRs) activates heterotrimeric (Gαβγ) G-proteins by catalyzing the exchange of GTP for GDP on the Gα subunit (Sprang, 1997). Activated Gα and freely liberated Gβγ subunits subsequently modulate specific downstream signaling pathways, including ion channels, adenylyl cyclases, phosphodiesterases, and phospholipases, involved in a vast array of cellular functions (McCudden et al., 2005). Dysregulated G-protein signaling leads to pathophysiologies in numerous organ systems. Furthermore, several important classes of medications currently approved for the treatment of such disorders modify these GPCR signaling pathways either directly or indirectly (Flower, 1999; Rohrer & Kobilka, 1998).

Despite intense research over the past several decades, many key aspects concerning receptor/G-protein interactions and mechanism of action remain incompletely understood. For example, given a lack of structural information with respect to the receptor/G-protein complex, few details are known regarding the sequence of interactions that successfully couple Gαβγ to receptors and the exact molecular details that underlie this functional coupling. Furthermore, how an activated GPCR exerts its exchange-factor activity on Gα is poorly understood, although several models have been proposed (Bourne, 1997; Cherfils & Chabre, 2003; Hamm, 2001; Iiri et al., 1998; Johnston et al., 2005b).

Another unresolved issue regarding the receptor/G-protein complex is whether this interaction is constitutively assembled in an inactive state awaiting activation, or forms only after agonist exposure. The former mechanism is explained by the ‘precoupling’ hypothesis which suggests that G-proteins interact with receptor, including α2-adrenergic receptors, in the absence of agonist and only become activated upon ligand binding to receptor (Burgisser et al., 1982; De Lean et al., 1980; Frances et al., 1990; Shi & Deth, 1994; Tian et al., 1994). This hypothesis is largely favored from a kinetic argument which helps to explain the rapidity of G-protein-mediated signal transduction events, which may not be achieved within a freely diffusional environment (Shea & Linderman, 1997; Zhang et al., 2002b). Ligand/receptor binding assays, along with mathematical modeling, have highlighted the existence of a high- and low-affinity state of the receptor, with the high-affinity state representing the species capable of activating Gα (Burgisser et al., 1982; Leff & Scaramellini, 1998; Leff et al., 1997; Limbird et al., 1980). However, receptor ‘precoupling’ has traditionally been examined within systems containing overexpressed levels of receptors and, therefore, may not accurately reflect a true physiological phenomenon (Tian & Deth, 1993). Moreover, evidence from both experimental and mathematical modeling suggests that receptors have unhindered access to G-proteins, and that receptors do not precouple to G-proteins in the absence of agonist (Azpiazu & Gautam, 2004; Gross & Lohse, 1991; Stickle & Barber, 1993; Stickle & Barber, 1996; Tolkovsky & Levitzki, 1978). This hypothesis, typically referred to as the ‘collision’ model, suggests that G-proteins interact with receptor only after agonist binding and conversion of the receptor to the high-affinity, activated state. One approach to resolve these two competing models of signal transduction mechanics which has recently gained considerable attention is the design of fluorescent biosensors that report both the real-time spatial and temporal aspects of protein activity and protein-protein interactions within living cells (Hahn, 2003; Hahn & Toutchkine, 2002). These technologies could thus be used to help resolve many of the fundamental questions still unanswered in the G-protein signaling field.

FRET imaging

Fluorescence resonance energy transfer (FRET) using chromatic variants of the Aequoria victoria green fluorescent protein involves the non-photonic transfer of energy (Forster, 1948) from a donor fluorophore (cyan fluorescent protein; CFP) to an acceptor fluorophore (yellow fluorescent protein; YFP), an event quantified by the CFP-YFP FRET (CYFRET) ratio. This technique produces high-resolution FRET images showing the location of protein/protein interactions with proximities of less that 50 Å in live cells (Zhang et al., 2002a). Furthermore, the technique allows tracking of protein complex assembly and disassembly in real-time to resolve the spatiotemporal dynamics of protein activation. CFP/YFP-based FRET, and related techniques, have been used previously by a number of groups to study G-protein signaling dynamics in a variety of cellular systems (Azpiazu & Gautam, 2004; Bunemann et al., 2003; Gales et al., 2005; Hoffmann et al., 2005; Janetopoulos et al., 2001; Jin et al., 2000; Lohse et al., 2003; Nobles et al., 2005). For example, FRET techniques have been used to probe the ability of distinct classes of ligands (e.g. agonists, antagonists, partial agonists) to induce distinct conformation changes in GPCRs to explain the difference in pharmacological properties of these receptor ligand classes (Vilardaga et al., 2003). FRET-based techniques have also been used to investigate directly the activation of G-protein heterotrimers in living cells (Bunemann et al., 2003; Janetopoulos et al., 2001). In these cases, care must be taken with respect to the placement of donor and acceptor fluorophores within the signal transduction components, since, for example, attachment of a GFP-variant onto the N- or C-termini of the G-protein alpha subunit leads to steric blockade of Gβγ and/or receptor coupling. The FRET technique is not without other limitations as well (see below and also Toutchkine et al., 2003).

A new report by Hein, et al. now describes the design of a FRET pair created by placing a CFP donor on the N-terminus of the Gγ2 subunit and a YFP acceptor on the extreme C-terminus of a GPCR: the α2A-adrenergic receptor. The authors use this experimental design to monitor real-time interactions between G-protein and receptor within live cells in response to agonist exposure. Outlined below are the critical implications of this work in relation to several paradigms under intense investigation within this field of signal transduction.

Implications of current work

Hein, et al. (Hein et al., 2005) report a FRET-based technique to monitor the interaction of α2A-adrenergic receptors with Gαi1β1γ2 heterotrimers. Their results illustrate visually the formation of receptor/G-protein complexes in real-time in a living cell system and capture the remarkably rapid kinetics of this crucial signal transduction event.

The work presented by Hein and colleagues (Hein et al., 2005) has important implications for several current, and as yet unresolved, topics within the GPCR research field. Most notably, their findings weigh in on the current dispute regarding the timing of receptor/G-protein interactions during agonist-stimulated activation. As stated above, two prevailing models for this interaction are the precoupling model and the collision model. Hein, et al. use a ‘recovery after photobleaching’ technique to show that no significant FRET response between receptor and G-protein exists in the basal state (i.e. in the absence of agonist). These results suggest that α2A-adrenergic receptors and G-protein heterotrimers do not interact in the absence of a stimulus and are thus not precoupled. Furthermore, the authors use data describing the kinetics of the interaction to add support for the collision model of interaction. Specifically, ectopic expression of increasing amounts of G-protein alpha subunit led to decreased time constants for the receptor/G-protein interaction upon agonist activation. These results argue in favor of the collision model in which freely diffusible receptors and G-protein heterotrimers interact upon agonist-induced conversion of the receptor to its high affinity, activated state (Azpiazu & Gautam, 2004; Gross and Lohse, 1991; Tolkovsky & Levitzki, 1978). Moreover, the authors show that increasing G-protein expression levels leads to a rate constant for receptor/G-protein interaction that approximates previously determined rates of receptor activation (conversion to the high affinity state, Vilardaga et al., 2005), demonstrating a key assumption of the collision model of coupling (Stickle & Barber, 1993; Stickle & Barber, 1996). These data together add support to the collision model of interaction; however, they do not necessarily refute recent results suggesting that G-protein signaling components may segregate to specified regions within the plasma membrane and are not at least somewhat restricted in their diffusional properties (Ostrom & Insel, 2004). It must be noted that while these results are in agreement with a previous study using a similar FRET-based approach with M2- and M3-muscarinic receptors (Azpiazu & Gautam, 2004), a recent study also employing a FRET-based assay strikingly similar to that used by Hein, et al. indeed demonstrated an apparent precoupled state between Gαβγ and various GPCRs (including the α2A-adrenergic receptor, Nobles et al., 2005). The basis for these discrepancies is unclear; however, they may in part reflect the inherent limitations to FRET-based techniques (see below).

Another classical paradigm in GPCR signaling addressed by the FRET approach of Hein, et al. is the issue of ‘spare receptors’ (or ‘receptor reserve’, Kenakin, 2004). In an elegantly designed experiment, the authors simultaneously image receptor/G-protein interactions and perform patch-clamp electrophysiological recordings of GIRK channel activation (via the freed Gβγ subunit) following G-protein activation (Hein et al., 2005; see Figure 5B therein). Application of a ‘low’ concentration of agonist elicited a fraction of the FRET response as compared to a ‘high’ agonist concentration (indicating fewer receptor/G-protein interactions at low agonist concentration), whereas a maximal GIRK current was elicited by both agonist concentrations. Thus, at an agonist concentration that activates a submaximal number of receptors to engage G-protein, a full effector response is obtained. These results illustrate the non-uniform stoichiometry of the system, and highlight the ability of a single receptor to activate multiple G-protein heterotrimers leading to sufficient signal amplification to elicit maximal effector responses. While studies of GPCR signal transduction have traditionally been able to show the kinetics of ligand binding and subsequent downstream second messenger signaling, the studies by Hein, et al. represent the first kinetic demonstration of the most proximal event in this ubiquitous signaling pathway, that is, interaction of heterotrimeric G-proteins with an activated receptor.

Using a Gα mutation that more strongly associates with activated receptor (Gαi1-N270D; first identified by Dohlman and colleagues in the yeast Gα subunit Gpa1, see (Wu et al., 2004), Hein et al. illustrate that the steady-state rate of receptor/G-protein interaction is rather fast (reflected in an increased magnitude of FRET response with the N270D mutation compared to wild-type Gα) and that G-proteins, once activated, fully dissociate from the receptor during a significant part of the guanine nucleotide cycle (Figure 1). Current models of heterotrimeric G-protein signaling suggest that the pathway components (i.e. receptor; G-protein; effector; accessory proteins) are properly ‘sorted’ and spatially constrained either by plasma membrane compartmentalization (Ostrom & Insel, 2004) or by specialized scaffolding proteins (Hall & Lefkowitz, 2002; Kimple et al., 2001). Kinetic analyses have also suggested the existence of a ternary complex of activated receptor/G-protein/effector that, under steady-state conditions, regulates signal initiation and termination as well as the magnitude of effector signaling responses (Biddlecome et al., 1996; Mukhopadhyay & Ross, 1999). These models help to rationalize the observed efficiency and spatiotemporal dynamics of signaling and are common among other signal transduction pathways (Kim & Sheng, 2004; Kolch, 2005). Results by Hein, et al., while certainly not refuting these models, suggest that activated G-proteins do have at least a limited capacity for diffusion away from their activating receptor in order to participate in effector activation.

Figure 1. Experimental approach used by Hein and colleagues to visualize the agonist-induced interaction between the α2A-adrenergic receptor and Gαi1β1γ2 heterotrimer. (Left) In the absence of agonist (norepinephrine), the α2A-adrenergic receptor fused to YFP via its intracellular C-terminus exists in the inactive, low-affinity state (R) without an associated G-protein heterotrimer. Excitation (using light at 436 nm) of CFP fused to the C-terminus of the Gγ subunit results in a minimal excitation of YFP and low FRET ratio (EmYFP/EmCFP; emission of YFP [at 535 nm] over emission of CFP [at 480 nm]). (Middle) Upon norepinephrine binding, the receptor undergoes conformational changes to adopt the active, high-affinity state (R*). Once activated, the receptor ‘collides’ with and binds an inactive, GDP-bound Gαβγ heterotrimer. Upon binding, energy is transferred in a non-radiative fashion from the excited, Gγ-fused CFP molecule to the receptor-fused YFP molecule, yielding an increased FRET ratio. (Right) Following receptor-catalyzed guanine nucleotide exchange (or “GEF” activity), Gα-GTP and Gβγ freely diffuse to interact with downstream effectors, such as GIRK channels analyzed by Hein, et al.

Finally, the authors measure FRET responses following treatment of cells with pertussis toxin (PTX). This bacterial toxin catalyzes the ADP-ribosylation of a cysteine residue in the C-terminal helix conserved within Gαi family G-proteins – a modification thought to inhibit proper interaction of heterotrimer with receptor (Katada et al., 1984). However, the exact mechanism of PTX-mediated inhibition of Gi-coupled receptor signaling remains incompletely understood. Hein, et al. show that cells treated with PTX still yield a considerable FRET response upon agonist application, despite complete abolition of GIRK channel activation. These results suggest that PTX-‘inactivated’ Gαiβγ heterotrimers may still interact with activated receptors while not undergoing nucleotide exchange. This finding impacts upon our notions as to how G-proteins interact with activated receptors – a mechanism that remains elusive (Bourne, 1997; Hamm, 2001). The data presented by Hein, et al. support a hypothesis that heterotrimeric G-proteins first interact with receptor via the Gβγ subunit followed by a secondary interaction with the Gα subunit (Herrmann et al., 2004). Given that a FRET response was still seen within PTX-treated cells upon agonist application suggests that this ‘secondary’ Gα interaction with activated receptor is not necessary for Gαβγ/receptor coupling but is required for G-protein activation (i.e., nucleotide exchange). These results support a prevailing model of receptor-mediated G-protein activation requiring simultaneous interactions of the receptor with the Gβγ subunit and with the C-terminus of Gα (Bourne, 1997; Onrust et al., 1997).

Concluding remarks

Despite the advancing insights provided by Hein, et al., the conclusions discussed therein are not without limitations. The FRET-based techniques used are limited in that they rely on i) GFP-modified signal transduction components, ii) overexpression systems, and iii) cell systems not necessarily representative of in vivo environments. Additionally, the experiments presented were limited to the study of a single GPCR that may not be representative of other rhodopsin-family receptors, let alone other subfamilies of GPCRs (Pierce et al., 2002). Indeed, a recent FRET-based study examining receptor/G-protein interactions argues in favor of a precoupled state for several different GPCRs (Nobles et al., 2005). However, results from experiments using a similar approach with the M2-muscarinic receptor (Azpiazu & Gautam, 2004) appear to agree with data presented by Hein, et al. and the conclusions of this independent study also support the collision model of receptor/G-protein interaction. Alternatives fluorescent imaging techniques to FRET-based sensors may help resolve these apparent contradictions (see below).

G-proteins represent a crucial family of signal transduction molecules that govern a variety of physiological functions. Moreover, GPCRs have traditionally been (and continue to be) a major exploitable drug target, giving rise to a plethora of clinically relevant molecules (Flower, 1999). Thus, a more complete understanding of the fundamental properties of GPCRs and how they interact with, and activate, their target G-proteins is of utmost importance to future drug discovery. The use of fluorescence-based imaging techniques has been illuminating to our understanding of protein activity in living cells and the spatiotemporal dynamics of several signal transduction pathways (Hahn, 2003; Hahn & Toutchkine, 2002). Hein, et al. have employed such technologies to provide new insights into the dynamics of GPCR/G-protein interactions. Their work, all within a living cell environment, provides evidence for a collision model of interaction, directly illustrates the concept of ‘spare receptors’, demonstrates the rapid kinetics of receptor-mediated signaling, and also provides new insight into the mechanism of action of pertussis toxin as a tool with which to interrogate Gi-coupled signaling.

Future approaches should now be designed to investigate these and other issues related to G-protein signaling dynamics under more ‘physiological’ conditions. Biosensors capable of reporting the activity of endogenous receptor and G-protein activity would obviate the need for overexpression and the potential pitfalls which lie therein. For example, we and others have recently used phage display screening to identify small peptides sequences capable of interacting with Gα subunits in nucleotide-dependent fashions (Ja & Roberts, 2004; Johnston et al., 2005a; Johnston et al., 2005b, and unpublished observations). These molecules, once properly derivitized with appropriate fluorescent molecules, could potentially be used within a living cell system to visualize the activity of endogenous Gα activity.

Acknowledgements

We thank Dr. T. Kendall Harden for critical review of this commentary. Work in the Siderovski laboratory is supported by NIH grants R01 GM062338 and P01 GM065533. C.A.J. is supported by a National Institute of Mental Health postdoctoral training award (1-F32-GM076944-01).

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