Investigating RGS proteins in yeast

Seminars in Cell & Developmental Biology 17 (2006) 352–362

Review

Investigating RGS proteins in yeast

Claire Hill a,∗, Alan Goddard a, John Davey a, Graham Ladds b

a Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UKb Division of Clinical Sciences, Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK

Available online 5 April 2006

Abstract

Regulator of G protein signalling (RGS) proteins are vital in the adaptation of cells to stimulation via G protein-coupled receptors. Yeast havebeen integral in elucidating the important role that RGS proteins play within cellular processes. In addition to extensive characterisation of theendogenous RGS proteins, these organisms have enabled the identification and analysis of numerous mammalian homologues. The simplicity andplasticity of the yeast pheromone-response pathway has facilitated studies which would have been impossible in mammalian systems and it iscertain that yeast will continue to have a great impact on this field of research in the future.© 2006 Elsevier Ltd. All rights reserved.

Keywords: RGS; Yeast; SST2; GAP; Review

Contents

1. GPCR signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3532. RGS proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3533. Yeast as a model system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

3.1. Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3533.2. Saccharomyces cerevisiae SST2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3543.3. SST2 GAP activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3543.4. Regulation of SST2 activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3553.5. The role of the N-terminus of SST2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3563.6. Mathematical modelling of SST2 action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

4. The study of mammalian RGS proteins in Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3564.1. Yeast as a bioassay for putative RGS proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3564.2. The role of the RGS N-terminal domain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3564.3. Selectivity of RGS action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3574.4. Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3574.5. Schizosaccharomyces pombe Rgs1p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

5. The study of heterologous RGS proteins in Schizosaccharomyces pombe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3586. RGS proteins in other yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3587. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

The discovery of the regulator of G protein signalling (RGS)family of proteins has greatly enhanced the understanding of Gprotein-coupled receptor (GPCR) signalling pathways. The RGS

∗ Corresponding author. Tel.: +44 2476 528361; fax: +44 2476 523701.E-mail address: [email protected] (C. Hill).

family, consisting of approximately 30 known members, plays apivotal role in determining the strength and duration of signallingvia G proteins and provides an important opportunity to influ-ence signalling. The study of RGS proteins in mammalian cellsis hampered by the sheer number of different, cross-interactingGPCR-G�-RGS-cascades, but great progress has been madethrough the use of simpler systems such as yeast. Through yeast-

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C. Hill et al. / Seminars in Cell & Developmental Biology 17 (2006) 352–362 353

based studies, a wide range of mysteries concerning the action,molecular targets and regulation of both endogenous and mam-malian RGS proteins have been unravelled. This knowledge canthen be applied to higher eukaryotic systems.

1. GPCR signalling

GPCRs transmit their signals through heterotrimeric G pro-teins consisting of G�, G� and G� subunits. Activation of theseG proteins is achieved through exchange of GDP for GTP onthe G�-subunit resulting in the dissociation of the G� and G��components, which both progress to exert various effects withinthe cell. G protein signalling ceases upon inactivation of theG�-subunit, as a result of GTP hydrolysis, and reformation ofthe G�� heterotrimer. Although the G�-subunit itself containsan intrinsic GTPase, this activity is not sufficient to account forthe observed rate of GTP hydrolysis in vivo (reviewed [1,2]).The higher rate of hydrolysis is achieved through the action ofGTPase activating proteins (GAPs), which include of membersof the RGS family. These proteins exert their activity by stabil-ising the transition state of GTP hydrolysis in the G�-subunitthereby increasing the reaction rate by as much as 1000-fold[3–7].

2. RGS proteins

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ing it difficult to glean insights into the actions and interactionsof these proteins. Yeast provides an ideal simple model organ-ism for the study of GPCR signalling though the use of thepheromone-response pathway. Yeast are single-celled eukary-otes that provide a GPCR-based signalling pathway with ahigh degree of structural and functional similarity to mam-malian hormone and neurotransmitter pathways. Indeed, someof the components of these pathways share such a high degreeof evolutionary conservation that they are functionally inter-changeable. Knock-out mutants for each of the yeast pathwaycomponents are also available. These highly robust organismsare fast growing and have no requirement for elaborate steriletechnique or expensive media. Yeast therefore represent a rel-atively low-cost investment and the ability to couple a varietyof reporter genes to pheromone-responsive promoters allowsboth qualitative and quantitative assays to be performed. Takentogether, these attributes make yeast highly suitable for use inHTS and detailed investigation of GPCR-based signalling cas-cades (reviewed [9]).

3.1. Saccharomyces cerevisiae

The budding yeast Sc. cerevisiae can exist as haploid cells oftwo mating types, MAT� and MATa, which prepare for matingvia the reciprocal exchange of diffusible mating pheromones,�-factor and a-factor (Fig. 1) [15,16]. These small peptidesbtCuestctccMS2oco

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The RGS family prototype, SST2, was identified in the bud-ing yeast Saccharomyces cerevisiae and was determined toegulate the pheromone response [8]. The yeast pheromone-esponse pathway is one of the best characterised examplesf eukaryotic signal transduction and provides a simple andenetically tractable environment which lends itself to high-hroughput screening (HTS) (reviewed [9]). SST2 was identifieds a result of one such screen [8], and study of this proteinncreased understanding of this important family. RGS family

embers were subsequently identified in a number of higherukaryotes [10–12] with over 30 mammalian RGS proteinsnown at present.

RGS proteins are defined by a highly conserved 130-residuetructural domain known as the RGS-fold. Determination ofhe crystal structure of human RGS4 in a complex with G�i113] demonstrated that the globular RGS-fold forms nine �-elices in two sub-domains, a four-helix bundle (helices 4–7)nd a domain composed of the N- and C-termini (helices 1–3nd 8–9, respectively). The G�-subunit contains three switchegions in which major conformational changes occur duringhe GDP-GTP cycle [14]. The RGS-fold contacts these regionsia interhelix loops 3/4, 5/6 and 7/8 but forms no direct contactith the GTP molecule. In addition to the RGS-fold, a number ofGS proteins contain domains and motifs which may play roles

n the cell in addition to the well-characterised GAP activity.

. Yeast as a model system

GPCR signalling cascades within mammalian cells are highlyomplex. For example, human heart cells express at least 13PCRs, 9 different G�-subunits and 13 RGS proteins [9], mak-

ind to receptors of the GPCR family, STE2 and STE3, respec-ively [17], resulting in activation of a G protein heterotrimer.onsequently, the G�- (GPA1), and G��- (STE4-STE18) sub-nits dissociate allowing signal transduction through the positiveffector, the G��-subunit [18]. Cellular responses to pheromonetimulation include polarised growth, induction of new generanscription, G1 cell-cycle arrest and fusion to form a diploidell [19]. G�� acts to induce these responses through the activa-ion of a mitogen-activated protein kinase (MAPK) cascade withlose homology to its mammalian counterparts. This pathway,omposed of a MAPKKK (STE11), a MAPKK (STE7) and twoAPKs (FUS3 and KSS1), is activated via the scaffold protein

TE5 and ultimately results in the increased expression of over00 genes via the transcription factor STE12 [20]. G�� also actsn a FAR1-CDC24 complex allowing rearrangement of the actinytoskeleton required for polarised cell growth in the directionf a potential mating partner [21,22].

An important feature of the pheromone pathway in yeast ishe attenuated response during prolonged stimulation. Cells fail-ng to mate are able to recover from the pheromone-inducedell-cycle arrest and resume normal haploid growth [23]. Thisrocess of desensitisation is an absolute requirement to allowells to return to their unstimulated state. Much of the focus oftudies in this field has concentrated on the receptor. Desensiti-ation in yeast bears a striking resemblance to hormone desensi-isation in animal cells, with both the yeast pheromone receptornd the mammalian �-adrenergic receptors undergoing phos-horylation and internalisation following stimulation [24–27].east therefore presents itself as a simple organism in which to

nvestigate the mechanisms involved in desensitisation and thetudies described in this review demonstrate that, in addition to

354 C. Hill et al. / Seminars in Cell & Developmental Biology 17 (2006) 352–362

Fig. 1. The mating pheromone-response pathway in Sc. cerevisiae. Binding of�-factor to the STE2 GPCR expressed on the surface of a-cells, or the recip-rocal binding of a-factor to STE3 on �-cells, results in GDP-GTP exchangeon the GPA1 G�-subunit and the release of the STE4-STE18 G�� dimer. Thisheterodimer activates a MAP kinase cascade consisting of MAPKKK (STE11),MAPKK (STE7) and MAPK (FUS3), mediated by the STE5 scaffold protein.The STE12 transcription factor is subsequently phosphorylated and acts uponpheromone-responsive elements (PREs) to drive expression of genes requiredfor mating. Attenuation of the response occurs via the intrinsic GTPase activityof GPA1, enhanced by the SST2 RGS protein.

receptor desensitisation, G proteins are also subject to adaptationprocesses.

3.2. Saccharomyces cerevisiae SST2

After prolonged exposure to pheromone the pheromonereceptors are down-regulated and internalised along withthe bound pheromone. Following this down-regulation anddegradation of pheromone, the receptors re-accumulate atthe cell surface [27]. Although at this stage the receptors areable to bind pheromone, they are no longer able to initiatea response [23,26]. This observation led to interest in thenewly discovered SST2 protein which was identified in 1982in a screen for mutants that demonstrated supersensitivity to�-factor-induced G1 arrest [8]. The screen yielded two groupsof mutants, SST1 and SST2. The effects of the SST1 mutationwere a-cell specific and the result of the action of a secretedprotease which inactivates pheromone [28]. The SST2 mutantsresponded to low levels of pheromone and had a defect inrecovery from cell-cycle arrest. This effect was demonstratedin both a-cells and �-cells and highlighted SST2 as a factorpotentially involved in the desensitisation process.

SST2 was cloned in 1987 and its expression was demon-strated to increase approximately 50-fold upon stimulation withpheromone [29]. It was therefore proposed that SST2 acts tointerrupt the activity of a component of the pheromone-response

pathway. In 1992, Weiner et al. demonstrated a functional rela-tionship between STE2 and SST2, reporting that substitutionsin the third intracellular loop of the �-factor receptor STE2increased the rate at which cells were able to recover frompheromone-induced cell-cycle arrest [30]. This increased rateof adaptation was observed only in cells carrying a functionalSST2. Subsequent studies also placed STE2 and SST2 in arelated pathway but did not determine the mechanism of SST2action [31,32]. It was later observed that overexpression ofGPA1 can partially overcome the supersensitivity of �SST2mutants, thus implicating a direct interaction between GPA1and SST2 [33]. The direct intracellular target of SST2 wasconfirmed in 1995 through the use of a gain-of-functionmutant [33]. This mutant, SST2 (P20L), was able to blockreceptor-directed signalling along with pheromone-inducedgene transcription and cell-cycle arrest. However, these effectswere overcome by expression of a constitutively active mutantof GPA1. This study demonstrated that SST2 acts as a negativeregulator of GPA1 activity and provided the first clue to themechanism of SST2 action.

3.3. SST2 GAP activity

SST2 was not recognised as the RGS prototype until 1996[34]. At this time, several groups described a 130-residue domainin a number of proteins, GOS8 (now RGS2) [10], GAIP [11] andEfarrish[w[csAbmsttowGsh[GG

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GL-10 [12], leading to the identification of a distinct proteinamily. Dohlman et al. demonstrated that SST2 acts as a GAPgainst GPA1 [35]. In the absence of GPA1 the pheromone-esponse pathway is constitutively active and SST2 is unable toeverse the resulting growth arrest [35], demonstrating that SST2s unable to act in the absence of GPA1. This finding was con-istent with other studies which demonstrate that RGS proteinsave GAP activity toward members of the G�i and G�q families3–6]. SST2 was also unable to act against endogenous GPA1hilst in the presence of a GTPase-defective mutant of GPA1

35] (GPA1 (R297H), a mutation which alters an arginine residueonserved in all G� subunits [36]) indicating a role for SST2 intimulation of GTP hydrolysis. This was confirmed in 1998 bypanovitch et al. in a study demonstrating that SST2 does notind GTP but rather binds preferentially to the transition stateimic of GPA1, GPA1·GDP-AlF4

− [37]. Similar results wereeen with human RGS4 by Berman et al. [38], indicating thathese proteins act to promote GTP hydrolysis through stabilisa-ion of the transition state thus lowering the activation energyf the reaction. Further evidence for the GAP activity of SST2as provided through a screen which isolated the GPA1 mutant,PA1 (G302S) [39]. Glycine 302 is located in one of the three

witch regions which undergo a conformation change upon GTPydrolysis and is conserved amongst the G� family of subunits40–46]. The mutant protein was able to bind and hydrolyseTP via its endogenous GTPase activity but was resistant to theTPase enhancement normally provided by SST2.At this time SST2 was the only RGS protein whose cognate

� protein had been identified. This made yeast invaluable forhe study of G�–RGS interactions since it was the only organ-sm in which knock-out mutants of both of these proteins were


available. Siekhaus and Drubin demonstrated that, in the absenceof the pheromone receptor, SST2 is required to prevent sponta-neous G protein signalling [47]. Inactivating mutations in SST2allowed spontaneous activation of the pheromone pathway tolevels of activity normally seen in the presence of pheromone.Such studies are impossible to perform in mammalian cells dueto the complex interplay between the myriad of GPCR signallingcomponents within these cells.

The demonstration of SST2 GAP activity, in parallel witha mechanism of SST2 action proposed by Dietzel and Kurjan[29], allows a more complete model to be constructed (Fig. 2).SST2 prevents spontaneous GPA1 activation in the absenceof stimulation. ∆SST2 cells are supersensitive to pheromonesince these cells lack the pheromone-independent quantity ofSST2 that would normally act to modulate spontaneous GPA1activity. Following stimulation, the receptors are recycled andbound pheromone is degraded. Upon re-accumulation at thecell surface, the receptors are able to bind ligand. However,since pheromone stimulation results in an up-regulation of SST2expression, there is sufficient SST2 to prevent a second response.In a ∆SST2 strain, a further response is permitted since SST2 isnot produced following pheromone stimulation [29].

3.4. Regulation of SST2 activity

SST2 clearly plays an indispensable role in the attenuationo

process needs to be strictly regulated. SST2 has a short half-lifeand is degraded rapidly [35,48], however this process appears tobe regulated through phosphorylation of Serine 539 [49]. Phos-phorylation of this residue occurs in response to pheromoneand requires the MAPK genes FUS3 and KSS1, in additionto components of the upstream MAPK cascade [49,50]. S539

lies adjacent to a region containing two PEST motifs (contain-ing high local concentrations of proline, glutamic acid, serineand threonine) [35] which are often found in proteins that, likeSST2, are degraded rapidly [51]. The location of S539, betweenhelices found away from the G�-binding region, indicates thatphosphorylation could occur in the presence of bound GPA1and would have no direct effect on the interaction between thetwo proteins [49]. Pheromone stimulation results in a 50-foldincreased in SST2 mRNA and protein levels [29]; phospho-rylation and stabilisation of SST2 acts to enhance this effectand allows sustained expression of SST2 [49]. The phospho-rylation event is regulated by an elegant feedback-loop. In theabsence of pheromone, degradation of SST2 is allowed to occur.However, upon pheromone stimulation, phosphorylation by thepheromone-induced MAPK activation results in a reduction ofSST2 degradation. This, in turn, limits the amount of free G��,which ultimately decreases the levels of active MAPK availableto phosphorylate SST2 [52].

SST2 also undergoes a pheromone-independent cleavageevent resulting in separate N- and C-terminal fragments [53].T

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f pheromone signalling and the activity of such an influential

ig. 2. The SST2-mediated adaptation response. Upon initial stimulation of thubunit, resulting in dissociation from the receptor and the G�� subunits. ThMAP kinase cascade (not shown) resulting in the expression of a number o

resent in unstimulated cells is insufficient to prevent this response. However, uponeceptors are phosphorylated, before internalisation and ligand degradation. The GPCDP/GTP exchange on GPA1 once again. However, in wild-type cells (red arrow), tTP hydrolysis on GPA1, and an immediate attenuation of the response. Thereforeheromone. In contrast, if SST2 is deleted (blue arrow), no enhancement of GTP hynterpretation of the references to colour in this figure legend, the reader is referred to

he cleavage sites, S414 and S416, lie just preceding the RGS-

ropriate GPCR by pheromone, GDP-GTP exchange occurs on the GPA1 G�-erodimer then stimulates the activation of the STE12 transcription factor viaes required for the mating response, including SST2. The low level of SST2
stimulation, SST2 is markedly up-regulated. To aid in the adaptation process,Rs are then returned to the cell surface and can be re-stimulated, causing the
he accumulation of SST2 post-stimulation results in an increase in the rate of, cells have a period post-stimulation in which they are unable to respond todrolysis occurs and cells are able to respond to stimulation immediately. (Forthe web version of the article.)


fold, although the C-terminal fragment displays no GAP activityin vivo and neither fragment is able to rescue the ∆SST2 phe-notype. However, co-expression of the two fragments enablespartial restoration of the pheromone-induced growth arrest [53].Cleavage results in a redistribution of the N- and C-terminalfragments to the microsome and soluble fractions, respectively[53]. Full length SST2 is localised to microsomes and theplasma membrane via N-terminal DEP (Dishevelled, Egl-10 andPleckstrin homology) domains [54]. SST2, like many large RGSproteins, contains two DEP-domains implicated in the recruit-ment of proteins to the plasma membrane. As described above,the C-terminal fragment alone is unable to regulate the action ofGPA1 indicating that, in the full length protein, the N-terminusmay be required to direct the RGS domain to its point of actionat the plasma membrane. Although the C-terminal RGS domainplays no further part in the signalling process, the N-terminaldomain may retain additional, as yet uncharacterised, roleswhich will be discussed in the next section.

3.5. The role of the N-terminus of SST2

A number of RGS proteins contain large N-terminal exten-sions and the study of SST2 has shed some light on the potentialroles of these domains. Findings have indicated that the DEP-domain-containing N-terminus may play a role in modulatingthe stress response leading to transcription of stress responseeitPpacaapitv

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occurs after residues S414 and S416 thus producing an N-terminalregion containing the entire MPI domain [58]. The interactionbetween SST2 and MPT5 occurs independently of the RGSdomain and evidence suggests that this domain may inhibit theinteraction between the MPI domain and MPT5 [58]. Stud-ies have suggested that MPT5 does not act to specificallyregulate the action of SST2 but may in fact be required forefficient packaging or nucleocytoplasmic transport of mRNA-containing ribonucleoprotein particles [54]. MPT5 also interactswith CDC28 and the protein kinases KSS1 and FUS3, indicatinga role downstream of GPA1 [57].

3.6. Mathematical modelling of SST2 action

A relatively new development in the study of the action ofRGS proteins has been the utilisation of mathematical mod-elling. The simplicity of the yeast system lends itself to sucha process and to date three studies have utilised computationmodelling to gain an insight into how the yeast pheromone-response pathway is regulated [59–61]. Such in silico modelswill no doubt enable a much greater understanding of GPCRsignalling pathways and, as with the in vivo yeast studies, infor-mation gained from these models may be applied to the muchmore complex mammalian systems.

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lement (STRE)-containing genes [55]. Seventeen potentialnteractants were identified in a yeast two-hybrid screen usinghe N-terminus of SST2 as bait [55]. Three of these proteins,EP12, TLG2 and VPS36, are known to be required for theroper sorting of proteases, normal vacuolar morphology, andre implicated in the modulation of transcription of a STRE-ontaining gene ([55] and references therein). These proteinsre also necessary for full activation of both the stress-responsend pheromone-response pathways [55]. The role of these com-onents in protein sorting implicated the N-terminus of SST2n a similar role, consistent with a number of other RGS pro-eins, including RGS GAIP and RGS-PX1, which also regulateesicle-mediated trafficking processes [56].

Studies of the potential interactants have concentrated onPS36, a deletion of which results in both a diminishedheromone response and reduced signalling by downstreamomponents such as STE12 [55]. Overexpression of the N-erminus of SST2 resulted in a further reduction of signallingn the ∆VPS36 strain. Conversely, overexpression of VPS36 in∆SST2 strain resulted in an enhanced pheromone response.he pheromone-response pathway is inhibited by the SST2-terminal fragment and activated by VPS36. The opposite sce-ario occurs in the stress-response pathway. Such studies suggesto-operative regulation of the pheromone- and stress-responseathways via SST2 and VPS36 [55].

Another SST2-interactant is the RNA-binding protein MPT557]. Studies of this interaction provided further evidence to sup-ort suggestions that SST2 contains two functional domains,C-terminal RGS domain and an N-terminal MPI (MPT5-

nteracting) domain [58]. The MPI domain consists of residues–135 and 333–401. Interestingly, proteolytic cleavage of SST2

. The study of mammalian RGS proteins inaccharomyces cerevisiae

.1. Yeast as a bioassay for putative RGS proteins

The study of SST2 in yeast unveiled the significance of RGSroteins in cellular signalling processes and researchers becameeen to apply this newfound knowledge to mammalian systems.s discussed previously, a number of mammalian homologuesf SST2 were known, including RGS1, RGS2 and RGS3, butheir importance had not previously been realised. Yeast enabledhe identification of the fourth member of the family, RGS4,hrough a screen of rat brain cDNAs which allowed ∆SST2

utants to grow on plates containing pheromone [62]. The real-sation that mammalian RGS proteins could complement the

SST2 phenotype provided the first evidence of the functionalignificance of the sequence similarity between the mammalianGS family members and SST2, and led to the utilisation ofeast as a bioassay for putative RGS proteins. A number of otherammalian RGS proteins have since been shown to complement

he ∆SST2 mutant including the human proteins RGS1, RGS2,GS3, a truncated version of RGS3 [62], RGS8 [63], RGS16

64,65] and RGS18 [66], and the murine proteins RGS2, RGS5,GS16 [67] and RGS4 [68].

.2. The role of the RGS N-terminal domain

The study of mammalian RGS proteins within yeast hasnabled a greater understanding of the action of these proteinsith much research focusing on plasma membrane localisationf the R4 family of RGS proteins. The RGS family is divided


into a number of subfamilies based on sequence similarity andthe additional domains and motifs they contain [69]. The R4family is considered the simplest of the groups since membersconsist of little more than the RGS-fold. The short N-terminusof this family is important in the recruitment of these proteinsto the plasma membrane [70]. In the absence of the N-terminal33 residues, both plasma membrane localisation and function ofhuman RGS4 are lost. These 33 residues are sufficient to recruitthe soluble GFP protein to the plasma membrane implicatingthis region as a membrane targeting domain [70]. Plasma mem-brane localisation is required for RGS4 function since functionis restored by addition of a heterologous C-terminal membraneanchor.

The N-terminal 30-residues of RGS4 are predicted to forman amphipathic �-helix and share sequence similarity with theequivalent region in a number of R4 family members, includinghuman RGS2, RGS5, RGS8 and RGS16 [71]. Plasma membranelocalisation and biological function of RGS2 [72], RGS4 [71],RGS8 [63] and RGS16 [64,73] were lost through disruptionof this region, indicating that the �-helix may be sufficient forplasma membrane association of these proteins. The action ofthe N-terminal membrane targeting domain in yeast led Ajit andYoung to utilise this region to target RGS proteins such as RGS7and RGS10S, which are non-functional in yeast, to the plasmamembrane, thus allowing them to function [74]. This extendedthe use of the yeast-based bioassay for a greater range of RGSpsc

ttlt[urts

4

pmyetwirkpgtty

The identification of this second yeast RGS protein led toquestions regarding the specificity of the two RGS proteins forthe two G� subunits. A key observation in the action of theendogenous RGS proteins in Sc. cerevisiae is that SST2 andRGS2 exert specific, non-overlapping functions, thus demon-strating the selective action of RGS proteins for different sig-nalling pathways [79]. Additionally, deletion mutants of theSST2 and RGS2 genes are complemented in some cases by dif-ferent mammalian RGS proteins. For example, in a comparisonof the actions of murine RGS2, RGS5 and RGS16, only RGS16was able to complement the ∆SST2 mutant. In contrast bothmuRGS5 and muRGS16 could complement the RGS2 deletionmutant. Murine RGS2 exerted only minor inhibitory effects inboth cases [79]. This observation provides potential insight intoone of the key unanswered questions in the study of RGS pro-teins. As previously eluded to, many different RGS proteins arefound in a single type of cell. How then do these RGS proteinsknow upon which receptors they should exert their effects? Thepresence of so many different RGS proteins in mammalian cellshas hampered the study of RGS specificity and it now seemsthat yeast do not only provide a simple bioassay for the detec-tion of RGS activity of proteins, but also allow the study of thespecificity of these proteins.

The yeast pheromone-response pathway provides a sensi-tive, reproducible assay in which to detect quantitative differ-ences in RGS potency toward GPA1. Heximer et al. utilisedyhosaprscdt5cdic

kmsbR

4

aee(s

roteins. Additionally, this system should allow high-throughputcreens for small molecule RGS inhibitors to be performed toomplement those already performed for RGS4 [75].

Studies in yeast have also extended to the N-terminal G pro-ein �-like (GGL) domain present in a number of RGS proteins inhe R7 family. This domain is known to specifically bind to G�5eading to the suggestion that the RGS/G�5 complex may poten-ially substitute for the G��-subunit in some signalling systems76,77]. This effect was investigated in the yeast ∆SST2 strainsing RGS7 and RGS9 [78]. Co-expression of RGS9 and G�5esulted in an enhanced cell-cycle arrest and provided evidencehat RGS9/G�5 may indeed function as a G��-subunit underome conditions.

.3. Selectivity of RGS action

As discussed above, the study of RGS proteins in yeast hasrovided insights into the role of the N-terminal domains andotifs that these proteins contain. Further studies utilising the

east model have alluded to the method in which RGS proteinsxert their specificity for the numerous receptors and G pro-eins contained within mammalian cells. Such a use for yeastas first considered through studies of the second RGS protein

n Sc. cerevisiae, RGS2 [79]. This protein acts as a negativeegulator of glucose-induced cAMP signalling which plays aey role in the control of metabolism, stress-resistance and cell-roliferation. RGS2 was isolated as a multi-copy suppressor oflucose-induced loss of heat resistance and was shown to func-ion by directly inhibiting the G� protein in this pathway, GPA2,hrough binding strongly to the transition state of GTP hydrol-sis [79].

east to demonstrate a difference in potency of inhibition ofuman RGS2 and RGS4 towards G�i proteins through the usef GPA1 as a G�i class protein [80]. Kong et al. also demon-trated that four mammalian RGS proteins, RGS1, RGS2, RGS5nd RGS16, act to differentially inhibit the activation of aheromone-responsive reporter by the STE2 receptor [81], aesult which correlates with studies in vitro that have demon-trated that some RGS proteins display differential activity forertain classes of G� proteins [4,5,82]. Furthermore, it wasemonstrated that all four of these RGS proteins are equally ableo inhibit the signalling from the human somatostatin receptor(SST5) when functionally expressed in yeast [81]. This result

learly indicates that the GPCR itself may play a key role inetermining the extent of inhibition and the selectivity of RGSnteractions with the G� protein and suggests a potential trimericomplex between GPCRs, G� proteins and RGS proteins [81].

Studies utilising Sc. cerevisiae have provided a wealth ofnowledge about RGS action against GPA1, however, mam-alian G� subunits couple poorly to the G��-subunit in this

ystem [83]. Alternative yeast-based systems have thereforeeen considered to allow the interaction between mammalianGS proteins and mammalian G� subunits to be studied.

.4. Schizosaccharomyces pombe

The fission yeast Schizosaccharomyces pombe provides anlternative system in which to study RGS proteins and otherlements of G protein signalling. Like Sc. cerevisiae, Sz. pombexists as haploid cells of two mating types, M (Minus) and PPlus). Mating in Sz. pombe is initiated in response to nitrogentarvation by the exchange of mating pheromones M-factor and


P-factor [84]. These pheromones bind to the GPCRs Map3p(M-factor) and Mam2p (P-factor) respectively, and activate aMAPK cascade consisting of Byr2p, Byr1p and Spk1p, homol-ogous to the cascade found in Sc. cerevisiae. In contrast to Sc.cerevisiae, where the signal is propagated by the G��-subunit,in Sz. pombe the G�-subunit Gpa1p transmits the pheromonesignal [84]. In this respect Sz. pombe allows easier investigationof G�-subunit activity. Loss-of-function mutations in Sc. cere-visiae GPA1 result in cell death [85]. In contrast such mutationsare tolerated by Sz. pombe [86] thus allowing greater versatilityin the studies permitted in this system. Pheromone signalling inSz. pombe results in increased cellular agglutination, formationof elongated cells known as shmoos, G1 cell-cycle arrest, fusionto form a diploid, and induction of new gene transcription as aresult of activation of the transcription factor Ste11p [84].

4.5. Schizosaccharomyces pombe Rgs1p

The Sz. pombe rgs1 gene was identified as a result of the Sz.pombe genome sequencing project due to the similarity it dis-played to other members of the RGS family [87]. It was laterisolated in a screen for novel Ste11p target genes [87,88]. TheRGS-fold of the Rgs1p protein is most closely related to theequivalent domains in Sc. cerevisiae SST2 and Aspergillus nidu-lans FlbA, and this RGS-fold can be fitted within the resolvedstructure of RGS4 through the introduction of additional inser-ttptsf[lssmt

gilRRtNdt[wpstDRoo

Sz. pombe and data indicate that Rgs1p is unable to exert anyeffects against Gpa2p, the G protein involved in this pathway[88]. It is likely that an RGS protein for this pathway remains tobe discovered.

5. The study of heterologous RGS proteins inSchizosaccharomyces pombe

The hypersensitive phenotype of the ∆rgs1 strain can bepartially rescued by the overexpression of Sc. cerevisiae SST2,although this protein is unable to overcome the mating defectcaused by the mutation [87]. It is interesting to note that SST2was only able to partially rescue the mutant phenotype, hav-ing no effect on the mating defect. Like many RGS proteins,Rgs1p contains a long N-terminal extension which may playadditional roles within the cell. It is possible that regions withinRgs1p, perhaps the N-terminus, play roles in the mating processthat cannot be restored by SST2. Additionally, loss of SST2 inSc. cerevisiae had no effect on the mating efficiency. The studyof heterologous RGS protein in Sz. pombe has been extended toinclude a number of human RGS proteins ([93], Hill, Davey andLadds, unpublished results; Fig. 3) [94]. These results indicatethat human RGS proteins display differential activity towards theSz. pombe Gpa1p G�-subunit with human RGS1 able to abol-ish signalling at all concentrations of pheromone tested, humanRGS4 able to reduce pheromone signalling at low concentra-tRodtSs

Sliet

6

fdramsSSNSeGoa

ions which are not thought to affect the folding of this pro-ein [87]. ∆rgs1 cells demonstrated the ability to initiate theheromone-response pathway in a normal way through agglu-ination, G1 cell-cycle arrest and pheromone-dependent tran-cription, however, these cells are hypersensitive to pheromone,ailed to mate and formed longer shmoos than normally observed87,88]. Such results indicate that Rgs1p acts at a relativelyate stage in the pheromone-signalling process. Studies alsouggested that rgs1 expression is up-regulated by pheromonetimulation [88], a result that correlates with the presence ofultiple TR-box motifs (Ste11p-binding sites [89,90]) close to

he start of this gene [87].Rgs1p localisation was determined through tagging with

reen fluorescent protein (GFP) and was predominantly presentn the nucleus, a pattern unaltered following pheromone stimu-ation [88]. This finding correlates with studies of mammalianGS2 and RGS10 [91]. Removal of the C-terminal RGS-fold ofgs1p resulted in a complete loss of function, although the dis-

ribution was not affected [88], reflecting a possible role for the-terminus in sub-cellular localisation. Two other domains wereetermined to be essential for activity, a DEP domain and an N-erminal fungal differentiation regulator (Fungal-DR) domain88]. As previously discussed, SST2 contains two DEP domainshich are thought to play a role in recruitment of proteins to thelasma membrane. The DEP domain within Rgs1p may play aimilar role perhaps in shuttling the protein from the nucleuso its point of action at the plasma membrane. The Fungal-R domain only appears to be present in three fungal proteins,gs1p, FlbA and SST2 but appears to be critical in the functionf these proteins [35,88,92]. To date there have been no reportsf an RGS protein regulating the glucose-sensing pathway in

ions of pheromone but not high concentrations, and humanGS2 appearing to elicit no effect on the response. As previ-usly discussed, human RGS proteins expressed in Sc. cerevisiaeemonstrate differential activity towards two different recep-ors. If these observations are considered in the context of thez. pombe system it could also be postulated that these proteinshow different specificities towards the Mam2p receptor.

Although still in its infancy, the study of RGS proteins withinz. pombe demonstrates great potential. A number of unpub-ished observations within this system correlate with those seenn Sc. cerevisiae and the role of the G�-subunit as the positiveffector provides an important opportunity to study these pro-eins.

. RGS proteins in other yeast

As a final consideration, the very recent discovery of an RGSamily member, SST2, in the yeast Candida albicans must beiscussed [95]. C. albicans demonstrates a mating pheromone-esponse pathway comparable with that found in Sc. cerevisiaend Sz. pombe. Two genes have been identified as encoding forembers of the RGS family; however, only one of these demon-

trates similarities to other fungal RGS proteins. C. albicansST2 demonstrates a very similar construction to Sc. cerevisiaeST2 with a C-terminal RGS-fold, central DEP domain and an-terminal Fungal-DR domain. C. albicans cells lacking theST2 gene demonstrate hypersensitivity to pheromone and areven responsive to a relatively inactive form of the pheromone.PCR signalling cascades have been identified in a number ofther yeast, including pathways regulating asexual developmentnd germination in Penicillium mameffei [96,97], adenylyl cyl-


Fig. 3. Analysis of human RGS proteins in Sz. pombe. Sz. pombe cells were engineered such that production the enzyme Ura4p was coupled to the pheromone-responsive sxa2 promoter [94]. Ura4p is essential in the biosynthesis of uracil and allows growth on uracil-deficient medium. Strains with increased signalling activityrequire less agonist to produce sufficient Ura4p to support growth. By contrast, strains with reduced signalling require more agonist to produce the same effect.The wild-type strain is capable of growth only when stimulated with at least 10−8 M pheromone. Removal of Rgs1p from this strain (vector) increases spontaneoussignalling through the pheromone-response pathway and allows growth even in the absence of pheromone. This effect can be overcome by expression of Rgs1pfrom a plasmid. Similarly, expression of human RGS1 reduces signalling and allows growth only when stimulated with 10−7 M pheromone. Conversely, RGS2 isunable to attenuate signalling and cell growth is observed even in the absence of stimulation. RGS4 is most similar to the endogenous Rgs1p, allowing growth onlyat 10−8 M stimulation or higher.

case activity in Kluyveromyces lactis [98], and nutrient sensingin Cryptococcus neoformans [99,100]. Although RGS proteinswithin these pathways remain undiscovered, it is likely that theyare present and may in future supplement knowledge in this field.

7. Concluding remarks

Due to the complex interplay between GPCR signallingcascades in mammalian cells, model eukaryotes have oftenbeen employed to analyse such systems. The yeast pheromone-response pathway is one of the best characterised examples ofGPCR-mediated signal transduction with close homology to itsmammalian counterparts. Mutants of components within thispathway are readily generated and can often be complementedby heterologous proteins. Such studies led to the identification ofthe RGS family prototype, SST2, in Sc. cerevisiae. It was sub-sequently demonstrated that mammalian RGS proteins couldfunctionally complement the ∆SST2 mutation. Investigation of

these important proteins in isolation has provided a plethora ofinformation regarding their functions and mechanism of action.Indeed, a number of the studies undertaken in yeast would havebeen impossible to perform in the context of a mammalian cell.It is clear that this model system will continue to contribute tothe understanding of the activity of these essential and complexsignalling regulators.

Acknowledgements

This work was supported by the Biotechnology and Biologi-cal Sciences Research Council UK (CH and AG) and the Univer-sity Hospitals of Coventry and Warwickshire NHS Trust (GL).

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Investigating RGS proteins in yeast

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