Abstract
Guanine nucleotide-binding proteins (G-proteins) act as transducers of external stimuli for intracellular signaling, and control various cellular processes in cooperation with seven transmembrane G-protein-coupled receptors (GPCRs). Because GPCRs constitute the largest family of eukaryotic membrane proteins and enable the selective recognition of a diverse range of molecules (ligands), they are the major molecular targets in pharmaceutical and medicinal fields. In addition, GPCRs have been known to form heteromers as well as homomers, which may result in vast physiological diversity and provide opportunities for drug discovery. G-proteins and their signal transduction machinery are universally conserved in eukaryotes; thereby, the yeast Saccharomyces cerevisiae has been used to construct artificial in vivo GPCR biosensors. In this chapter, we focus on the yeast-based GPCR biosensors that can detect ligand stimulation and oligomer formation, and summarize their techniques using the G-protein signaling and protein-protein interaction assays.
Keywords
- yeast
- G-protein
- G-protein-coupled receptor
- signal transduction
- oligomer formation
- reporter gene assay
- protein-protein interaction
1. Introduction
Guanine nucleotide-binding proteins (G-proteins) are highly conserved among various eukaryotes, and act as signal transduction molecules [1, 2]. In cooperation with seven transmembrane G-protein-coupled receptors (GPCRs), G-proteins transduce external stimuli to intracellular signaling and control a wide variety of cellular processes. GPCRs, which represent the largest family of integral membrane proteins and present more than 800 genes in the human genome [3], engage a wide range of ligands. GPCR ligands range from small molecules to large proteins, such as hormones, neurotransmitters, ions, tastants, odor molecules and even light [4]. Thus, GPCRs are involved in various physiological processes, and are the targets of several prescribed drugs [5, 6, 7, 8].
Agonist ligand binding to a GPCR causes ligand-specific active conformational changes, and allows the receptor to couple to G-proteins that are composed of Gα, Gβ and Gγ subunits [9]. Subsequently, heterotrimeric G-proteins dissociate from the receptor, and then G-protein signaling generates second messengers such as cyclic adenosine monophosphate (cAMP), inositol phosphates, and intracellular Ca2+. These second messengers trigger different cellular and ultimately physiological responses [10]. During these processes, G-proteins switch from an inactive state to an active state by exchanging a guanosine diphosphate (GDP) molecule from the Gα subunit for guanosine triphosphate (GTP). To resume an inactive state, G-proteins hydrolyze GTP to GDP [11].
Historically, GPCRs transduce signals only as single monomeric entities (homomers) [12]. However, in the past two decades, several studies have shown that GPCRs also transduce signals as heteromers [13, 14, 15, 16, 17, 18]. Heteromerization is involved in both the regulation and modulation of GPCR signaling, consequently increasing the potentially large functional and physiological diversity of various GPCR-mediated processes (e.g., ligand binding, receptor biosynthesis, cellular trafficking, maturation, G-protein activation, and internalization) [19, 20, 21, 22, 23, 24]. Therefore, heteromerization among GPCRs may provide new opportunities for drug discovery [25, 26]. For example, GPCR heteromers may be new molecular targets for therapeutic treatments, or for developing more potent and selective compounds, such as bispecific or bivalent ligands, with reduced side effects [27, 28, 29]. The mechanism of GPCR heteromerization has been under debate, because the identification of individual heteromer pairs is ongoing and the
2. G-protein signaling
Heterotrimeric G-proteins, as peripheral membrane proteins, interact with the plasma membrane on the cytoplasmic side. G-proteins consist of three subunits, Gα, Gβ, and Gγ, which are widely conserved in eukaryotic species, and there are various subfamilies within each subunit, especially the Gα subunit. The heterotrimeric G-proteins transduce messages from GPCRs, which regulate important functions such as vision, taste, smell, heart rate, blood pressure, neurotransmission, cell growth, and numerous other processes [10, 35]. When, in response to extracellular stimuli, GPCRs transduce ligand-specific intracellular signaling cascades, they activate a GDP to GTP exchange on the Gα subunit, resulting in Gα dissociation from the Gβγ complex. Free Gα or Gβγ interacts with several downstream effectors including phospholipases, adenylyl cyclases, phosphodiesterases, tyrosine kinases, ion channels, and ion transporters in human and other mammalian cells [36, 37].
2.1. Heterotrimeric G-protein signaling in yeast
The yeast
2.2. Improvement of the sensitivity of the yeast G-protein signaling
To increase the sensitivity of human GPCR expressing yeast cells, several modifications of yeast-based biosensors have been reported. The yeast’s single GPCR (yeast pheromone receptor Ste2p) is often deleted to avoid competitive expression with heterologous GPCRs [30]; therefore, expressing human GPCR on the plasma membrane of
Yeast Gpa1p is equivalent to mammalian Gα. Gpa1p shares particularly high homology with the human Gαi classes, and GPCRs from a variety of species, including human, are able to both interact with Gpa1p and activate yeast pheromone signaling [32, 64, 65]. Various genetic modifications allow many other human GPCRs to function as yeast signaling modulators. In one such modification, a chimeric Gpa1p system, referred to ‘as “transplants”, has’ been employed to substitute only five Gpa1p C-terminus amino acids for those of human Gα subunits, of which there are three key families: Gαi/o, Gαs, and Gαq [66]. Indeed, these transplants have allowed functional coupling of various GPCRs (including serotonin, purinergic, muscarinic, and many other receptors) to the yeast pheromone pathway with greater coupling efficiency [32, 66, 67, 68].
The use of fluorescent reporter genes can provide the most simple and convenient procedure for comparative quantification of signaling levels, as this removed the need for laborious operations such as sample preparations and enzyme reactions. GFP is commonly chosen as the fluorescent reporter and enhanced green fluorescent protein (EGFP) is often utilized as the GFP. However, the
2.3. Detection of GPCR agonists by utilizing yeast G-protein signaling
Many heterologous GPCRs (including muscarinic, neurotensin, serotonin, somatosta-tin, adrenergic, olfactory, and many other receptors) have been functionally expressed in yeast, successfully demonstrating the feasibility of yeast-based GPCR biosensors [31, 32, 63, 64, 65, 66, 67, 71, 72, 73].
For example, the cyclic neuropeptide somatostatin, known to inhibit growth hormone release, regulates the human endocrine system through somatostatin receptor (SSTR) binding. There are five identified SSTR subtypes (SSTR1 − SSTR5) [74, 75]. SSTR2 and SSTR5 are known to regulate acromegaly patient growth hormone secretion, and are also expressed in most growth hormone secreting tumors [76]. Several researchers demonstrated functional expression of human SSTR2 and SSTR5 in yeasts, and SSTR5 has been often used for constructing yeast-based somatostatin-specific biosensors. To modify the functional expression of human SSTR5 and somatostatin-specific signaling functions in yeasts, addition of signal sequences derived from secretion or membrane proteins (e.g., prepro- and pre-regions of α-factor, and a N-terminal 20 amino acids of yeast Ste2p; Ste2N) to the N-terminus of the receptor, and implementation of the chimeric Gpa1/Gαi3 transplant (see Section 2.2) were tested [77]. Additionally, the
Neurotensin receptor type-1 (NTSR1), a member of the GPCR family, is another example of site-directed mutagenesis of human SSTR5. Neurotensin is the natural ligand of NTSR1, as well as a central nervous system neuromodulator [80]. As neurotensin is also involved in many oncogenic events [81], NTSR1 is a significant therapeutic target. To monitor the activation of human NTSR1 signaling responding to its agonist, a fluorescence-based microbial
Angiotensin II (Ang II) type 1 receptor (AGTR1) is also a GPCR and its natural ligand, Ang II, is an important effector molecule for the renin-angiotensin system. Thus, AGTR1 controls blood pressure and volume in the cardiovascular system [84, 85]. Interaction of Asn295 with Asn111 may play a role in determining the ligand peptide binding selectivity of AGTR1 receptors [86, 87]. Therefore, a single alanine or serine mutation was introduced at Asn295 of human AGTR1, and the Asn295-mutated (N295A and N295S) AGTR1 was expressed in the IMFD-72ZsD yeast strain [88]. When exposed to Ang II and Ang II peptidic analogs, which differ in affinity toward AGTR1, these cells resulted in successful signal transmissions inside the yeast cells. Additionally, the secretory expression plasmids for angiotensin peptides (Ang II, Ang III, and Ang IV) were transformed into the yeasts expressing AGTR1-N295A or AGTR1-N295S, showing the ZsGreen fluorescence with different intensities according to the respective agonistic activities. In contrast, the monoamine neurotransmitter serotonin (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family [89]. Nakamura et al. expressed the human HTR1A in the IMFD-72ZsD strain to enable improved detection of HTR1A signaling in response to the 5-HT [90]. The authors further validated the capability of this improved yeast biosensor for antagonistic ligand characterization and site-directed mutants of human HTR1A.
The rat M3 muscarinic acetylcholine receptor (M3R) has been used for rapid identification of functionally critical amino acids with random mutagenesis [67]. In this system, the
Human formyl peptide receptor-like 1, which was originally identified as an orphan GPCR, has been used to isolate agonists for functionally unknown GPCRs [93]. Both a library of secreted random tridecapeptides and a mammalian/yeast hybrid Gα subunit were employed for histidine prototrophic selection via the
In the case of olfactory receptors (ORs), Minic et al. optimized a yeast system for functional expression of rat I7 OR and subsequent characterization. In engineered yeasts lacking endogenous Gpa1p, the olfactory-specific Gα subunit (Gαolf) was co-expressed. When the receptor was activated by its ligands, MAPK signaling was switched on and luciferase (as a functional reporter) synthesis was induced [71]. Marrakchi et al. successfully expressed human olfactory receptor OR17-40 in yeast based on Minic’s biosensor system to detect the conductometric changes [94]. Fukutani et al. improved the firefly luciferase-based biomimetic odor-sensing system [60], and replaced the N-terminal region of mOR226 with the corresponding domain of the rat I7 receptor [95]. They further improved some ORs by the coexpression of either odorant accessory binding proteins or the receptor transporting protein 1 short (RTP1S) [96]. Tehseen et al. demonstrated that the
2.4. Yeast cell-surface display technology for single-cell signaling assay of GPCR peptides
Yeast cell-surface display technology is a platform to tether functional proteins and peptides expressed in yeast to the cell surface [99, 100, 101, 102]. Cell-surface display of peptides can be used as a powerful ligand screening based on the yeast GPCR signaling assay systems [70, 103]. Displaying peptidic ligands by fusing them to an anchor protein in the yeast can enable a series of biological processes within a single cell, from peptide synthesis to agonist detection against an already expressing cognate GPCR. In such a system, a library of peptides is individually tethered to the plasma membrane on GPCR-producing yeast cells via attachment to a glycosyl-phosphatidylinositol (GPI) anchor. Upon phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI, the peptides, which are fused to the anchor protein, are released from the membrane and trapped in the cell wall [103]. In principle, the host cells unconsciously detect the binding of peptidic ligands to relevant receptors on the membrane and report the peptides resulting agonistic activation. Thus, this technique facilitates concomitant library synthesis and identification of peptide ligands at the single-cell level [104, 105].
Ishii et al. have developed a system for cell wall trapping of autocrine peptides (CWTrAP), which activates human SSTR5 signaling using short anchor proteins (e.g., 42 a.a. of Flo1p; Flo42) [103]. The engineered yeast strain concomitantly expressing human SSTR5 and somatostatin peptide successfully induced
3. Oligomerization among GPCRs
Many GPCRs have the capacity to form homomers or heteromers that show unique functional and biochemical characteristics including receptor pharmacology, regulation, and signaling [14, 107, 108]. Therefore, GPCR oligomers could be potential molecular targets for the development of new therapeutic agents. Yeast is a potential host for making cell-based biosensors for eukaryotic proteins and biological processes of interest [109], because varied reporting systems are available that can facilitate assays in yeast cells [110, 111, 112]. Notably, the “gold standard” for testing protein-protein interactions
3.1. Biophysical RET technologies to study GPCR oligomers in yeast cells
Varieties of resonance energy transfer (RET)-based techniques have promoted the visualization of GPCR oligomers in living cells. Fluorescence resonance energy transfer (FRET) is a strictly distance-dependent energy transfer technique using a cyan fluorescent protein (CFP) as energy donor and a yellow fluorescent protein (YFP) as energy acceptor, but other pairings are also possible [117]. Highly sensitive, bioluminescence resonance energy transfer (BRET) is based on the distance-dependent transfer of energy between a bioluminescent energy donor and a fluorescent acceptor molecule [118, 119].
Overton and Blumer [120] used subcellular fractionation and CFP/YFP FRET to demonstrate that oligomerization of the endogenous mating pheromone Ste2p receptors occurs via a stable association between protomers in yeast. Subsequently, the authors employed FRET in live yeast cells for detection of Ste2p oligomerization with its transmembrane domains [121, 122, 123, 124]. Furthermore, FRET experiments with yeast cells demonstrated the oligomer formation of functional human complement factor 5a (C5a) receptors [125].
BRET was later used to increase the detection sensitivity for Ste2p oligomerization. Increased sensitivity was needed, because the C-terminal regions of full length Ste2p protomers did not reach a proximity sufficient for effective energy transfer [126]. With the BRET system, Gehret et al. [126] demonstrated that mutations previously reported as blocking Ste2p receptor oligomerization decreased but did not completely eliminate oligomerization. Previously, BRET has been employed in yeast to analyze the protein interactions involved with heterogeneous olfactory receptors [127, 128].
3.2. Membrane Y2H technology to study GPCR oligomers in yeast cells
In contrast to FRET and BRET technologies (see Section 3.1), mY2H method is based on transcription-dependent reporter gene assays, permitting colorimetric evaluations with
In the split-ubiquitin mY2H system, the N- and C-terminal halves (Nub
Historically, the split-ubiquitin mY2H system was employed to screen interacting membrane-associated proteins (not GPCRs) for GPCRs, such as the μ-opioid receptor (MOR) [131, 132] and the M3 muscarinic acetylcholine receptor (M3R) [133]. Jin et al. identified GPR177, the mammalian ortholog of
The split-ubiquitin mY2H system has also been applied to identify GPCR heteromers [34]. Nakamura et al. developed a specialized method to screen candidate heteromer partners for target human GPCRs based on the split-ubiquitin mY2H method [34]. The authors noted that mating-associated induction of cell-cycle arrest, which causes robust growth inhibition in yeast, might impair the assessment of reporter gene activity. Therefore, the authors constructed a MAPK signal-defective yeast strain. This modified host permitted the rapid and facile detection, not only of target human GPCR heteromerization, but also of ligand-mediated conformational changes in living yeast cells [34]. Thus, the modified mY2H would be available to identify GPCR heteromer components and potential therapeutic targets for regulating physiological activities.
Furthermore, the authors subsequently designed a reporter switching system that can switch the expressions between two reporter genes (one from ON to OFF and the other from OFF to ON) in response to the Y2H readout (Figure 2C and D) [134]. Cre/
Using a modified split-ubiquitin mY2H approach, Sokolina et al. reported the systematic interactome analysis of 48 clinically important human GPCRs in their ligand-unoccupied state [135]. The authors also carried out additional in-depth functional validation on selected GPCR protein-protein interactions using biochemical and cell-based assays as well as knockout and knock-in animals. The authors found that a G-protein-regulated inducer of neurite outgrowth 2 (GPRIN2) and the GPR37 receptor, both physically and functionally, interact with the serotonin 5-HT4d receptor, a promising target for Alzheimer’s disease [135].
3.3. GPCR oligomerization and G-protein signaling
GPCR oligomerization can increase the potential for diversity in the regulation and modulation of GPCR signaling, and thus the specific evaluation of signaling properties among various receptor oligomer pairs. This work has important implications, not only for the development of new drugs, but also for the understanding of signaling networks [22]. This unique system was developed for simultaneous detection of oligomer formation and GPCR signaling activation. This new methodology uses a combination of the split-ubiquitin mY2H assay and a G-protein signaling assay, and is expected to facilitate the identification of physiologically relevant GPCR oligomers [136]. Using this system, Nakamura et al. monitored the physiological relevance of yeast pheromone receptor Ste2p, in both native and mutated forms. In addition, the authors demonstrated the simultaneous detection of homo- and heteromerization, and somatostatin-induced signaling of the human SSTR5 somatostatin receptor [136]. In the future, this system will be useful for identifying agonists that bind to the heteromer, promising to serve as a powerful platform for uncovering the novel functions, modes of action, and potential molecular targets of GPCR heteromerization for the development of new therapeutic agents.
4. Conclusion
In summary, we focused on yeast-based biosensors employed for the detection of GPCR ligand stimulation and oligomer formation, and described yeast-based techniques using the G-protein signaling and protein-protein interaction assays. Due to their involvement in signal transduction machinery, GPCRs are excellent therapeutic targets for various diseases and clinical indications [137]. The identification of new physiologically relevant GPCR oligomers provides a promising opportunity for drug discovery, based on the effect of allosteric communication between GPCR protomers (each subunit within the oligomer complex) on known pharmacological properties. Thus, approaches for investigating the relationship between oligomerization and GPCR signaling are necessary for creating oligomer-specific bivalent ligands. Additionally, there is great potential for identifying previously undiscovered physiological diversities and therapeutic targets through the generation of comprehensive and interactive GPCR oligomer maps. It is also important to expand our knowledge of the molecular details of GPCR-mediated signal transduction, including the identification of all proteins that interact with clinically relevant GPCRs. Further development of various methods, including yeast-based approaches and the investigation of GPCR oligomers, are expected to facilitate these outcomes in the near future.
Acknowledgments
This work was supported in part by a Grant-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (JSPS), and a Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe; iBioK) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
Conflict of interest
The authors declare no commercial or financial conflict of interest.
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