p Nar Review

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Biology and Functionsof the RGS9 Isoforms

Kirill A. Martemyanov *

and Vadim Y. Arshavsky {

*Department of Pharmacology,University of Minnesota, Minneapolis,Minnesota 55455{Departments of Ophthalmology andPharmacology, Duke University,Durham, North Carolina 27710

I. Introductory Remarks...................................................................... 206II. RGS9 Exists as Two Splice Isoforms with Distinct Nonoverlapping

Expression Patterns......................................................................... 206III. RGS9 Isoforms are Modular Multidomain Proteins... .... .... .... ... .... ... .... ... . 207IV. Gb5 is an Obligatory Subunit of RGS9....... ....... ....... ....... ...... ....... ........ 208 V. The DEP Domain Mediates RGS9 Association with a Novel Class of

Membrane Anchors......................................................................... 209 VI. The PGL Domain is the Unique Structural Feature

of the RGS92 Isoform..................................................................... 209

VII. Spatial Organization of the RGS9Gb5 Complex .. . . . .. . . . .. . . . .. . . . . .. . . . . .. . . . .. . . 210 VIII. RGS92 Gb5S R7BP Regulates G Protein Signaling in the Striatum .. .. .. ... .. 211IX. RGS91 Gb5L R9AP Regulates Visual Signal

Transduction in Vertebrate Photoreceptors ... ... .... ... .... .... .... ... .... ... .... .... 213 X. The Role of the Effector Enzyme in Regulating Transducin GTPase

and the Concept of Afnity Adapters............ ....... ....... ....... ....... ....... .... 216 XI. Comparing the Functional Properties of RGS9 Isoforms Expressed in the

Same Cell Type Suggests a Hypothesis on the Evolutionary Origin of Phototransduction........................................................................... 218

XII. Mechanisms Regulating the Ga Recognition Selectivity and CatalyticActivity of RGS9............................................................................. 219

References.................................................................................... 221

Two splice isoforms of the ninth member of the regulator of G proteinsignaling (RGS) protein family are expressed in the nervous system, wherethey are engaged in such diverse functions as vision and behavior. RGS91regulates phototransduction in rods and cones, while RGS92 regulates dopa-mine and opioid signaling in the basal ganglia. The main goal of this review is toillustrate how these functions are fulfilled through the interplay between theintrinsic molecular properties of RGS9 isoforms and their interactions with

several key protein partners in the cells in which they are expressed.

Progress in Molecular Biology Copyright 2009, Elsevier Inc.and Translational Science, Vol. 86 205 All rights reserved.DOI: 10.1016/S1877-1173(09)86007-9 1877-1173/09 $35.00


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I. Introductory Remarks

The family of regulator of G protein signaling (RGS) proteins is comprisedof a large number of members with highly variable amino acid sequences andsignicant variation in the overall domain architecture. A vast body of studiesconducted over the past decade revealed that RGS proteins are expressed in virtually every eukaryotic cell and are engaged in the regulation of highly diverse cellular processes, including but not limited to cell division,1 neuronalexcitability,2 sensory reception,3 angiogenesis,4 and vasoconstriction.5 Thesingle unifying characteristic of all conventional RGS proteins is their ability to negatively regulate G protein signaling. They accomplish this function by accelerating the rate of the GTP hydrolysis on G proteinasubunits and, there-fore, serve as GTPase activating proteins, or GAPs. However, recent studiesindicated that certain RGS proteins can also regulate G proteins without poten-tiating their GTP hydrolysis68 and that some of the RGS protein family mem-bers do not regulate G protein GTPase activity at all.9 Furthermore, most RGSproteins are equipped with a variety of domains in addition to their catalytic RGShomology domain. These domains appear to both modulate the GAP functionof RGS proteins and engage RGS proteins in controlling other signaling events(see Ref. 10 for review). Therefore, understanding the roles of individualRGS proteins in regulating discrete signaling events requires elucidation of themechanisms coordinating their functional dynamics and association with bindingpartners in the context of the signaling cascades which they regulate.

Progress in this direction is aided by studies of representative members whose functions are well dened. An outstanding example is RGS9, which isarguably the best understood RGS protein in regards to its physiological role.Early studies of RGS9 were summarized over 8 years ago in a review by Wenseland colleagues.11 However, many exciting developments have been reported inrecent years. The goal of this review is to integrate our current understandingof the roles of RGS9 in regulating such diverse processes as vision and behaviorin the mammalian nervous system.

II. RGS9 Exists as Two Splice Isoforms with Distinct Nonoverlapping Expression Patterns

One of the most interesting early observations about RGS9 was that it hastwo splice isoforms expressed in the nervous system with nonoverlapping,cellspecic proles.1214 The RGS9 gene is composed of 19 exons that can

206 MARTEMYANOV AND ARSHAVSKY


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be spliced in two different ways. Splicing of the rst 17 exons yields a shortisoform, called RGS91, encoding a 481 residue polypeptide. Alternatively,exon 17 may be skipped and substituted by exons 18 and 19, resulting in alonger protein product of 671 amino acids, called RGS92. Accordingly, theseisoforms differ only at their Ctermini where the 18 amino acid stretchfromRGS91 is replaced by the 209 amino acidlong domain in RGS92.1214

This pattern of splicing provides RGS9 with an alternative conguration of its Cterminal domains while keeping the rest of the molecule identical.Distinct functional consequences of such structural exibility are detailed inthe following sections. This differential splicing of the RGS9 gene occurs in astrict tissuespecic manner. RGS91 is found exclusively in the photoreceptorcells of the retina, whereas RGS92 is expressed only in the central nervoussystem, with a dramatic enrichment in the striatum. Interestingly, the mRNAsof the two splice isoforms also differ considerably in size, with the RGS91 variant being signicantly longer (9.5 kb) than the RGS92 isoform (2.4 kb),suggesting that alternativ ely spliced mRNAs may contain different untranslatedregulatory elements.1214

III. RGS9 Isoforms are Modular Multidomain Proteins

RGS9 is a multidomain protein sharing a common organization with threeother mammalian proteins: RGS6, RGS7, and RGS11, which along with RGS9are dened as the R7 subfamily RGS proteins.1517 Sharing about 50% homology,these proteins are remarkably conserved in evolution, with their orthologuespresent in all animals ranging fromCaenorhabditis elegans to humans.17

Like other R7 RGS proteins, RGS9 contains ve distinct domains (Fig. 1).The domain directly responsible for interacting with G proteinasubunits andstimulating their GTPase activity is the catalytic RGS homology domain,present in all RGS proteins.18,19 Directly upstream from the RGS homology domain is the G protein gsubunit like domain (GGL). Originally identied by Siderovski and colleagues,20 this domain is structurally homologous to conven-tional G protein gsubunits. The two Nterminal domains of RGS9 are termedDEP (Disheveled, Egl10, Pleckstrin)21 and R7H (R7 Homology).15 The latteris also known as DHEX (DEP helical extension).22 While the DEP domain isfound in over 30 unrelated signaling molecules,21 the R7H domain is foundonly in R7 RGS proteins.15 Recent evidence suggests that in RGS9 boththe DEP and R7H domains are organized as a single structural unit.22,23

The fth structural element of the RGS9 isoforms is represented by their variable Ctermini.

REGULATOR OF G PROTEIN SIGNALING 9 207


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V. The DEP Domain Mediates RGS9 Association witha Novel Class of Membrane Anchors

DEP domains of several proteins have been described as membranetargeting elements that either directly associate with membranes36,37 or bindto membrane proteins, such as GPCRs.3840 The same functional role is playedby the DEP domain of RGS9. Examination of the membraneassociationmechanisms of RGS9 isoforms has led to the discovery of two membraneassociated proteins that bind to the DEP/R7H domains of RGS9. RGS91 inphotoreceptors forms a tight complex with the RGS9 anchor protein(R9AP),41,42 whereas RGS92 in the striatum interacts with the R7 binding

protein (R7BP).43,44

Although the similarity between R9AP and R7BP isrelatively low (18% identity), both proteins share signicant homology withmembers of the SNARE protein family, which are involved in vesicular trans-port and exocytosis and contain characteristic Cterminal coiled coil motifs.45,46

Like SNARE proteins, R7BP and R9AP are resident membrane proteinsanchored either via a transmembrane segment (R9AP)41 or via lipid modica-tion by palmitoylation (R7BP).43,47

Studies of R9AP and R7BP knockout mice revealed another importantfunction of these proteins. Elimination of R9AP48 or R7BP49 resulted in severe

downregulation of RGS91 and RGS9

2, respectively. The DEP/R7H domainsof RGS9 contain specic instability determinants that target it for proteolytic

degradation b y cellular cysteine proteases upon dissociation from its mem-brane anchor.49 These determinants are shielded by R7BP or R9AP within thecorresponding complexes, but any RGS9 produced in excess of the anchorproteins undergoes rapid degradation. These observations indicate thatin vivoRGS9 exists as an obligate heterotrimer built from three constitutive subunits:RGS9, Gb5, and either R7BP or R9AP. Finally, the association of each RGS9isoform with its respective anchor has been shown to mediate its targeting to

specic subcellular compartments, the outer segments of photoreceptors(in the case of RGS91) and the postsynaptic density of striatal neurons(in the case of RGS92).39,46,49

VI. The PGL Domain is the Unique Structural Featureof the RGS92 Isoform

In contrast to the rather unremarkable short Cterminus of RGS91, theCterminus of RGS92 is represented by a welldened domain containingseveral predicted proteinprotein interaction sites, such as polyprolinerichsequences that are usually recognized by proteins containing SH3 motifs.50



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Yet, no specic interactions involving the Cterminal domain of RGS92 havebeen reported so far. Perhaps the most dening feature of this domain is thesequence located at the extreme Cterminus which bears signicant similarity to the gsubunit of PDE6 (PDEg), an effector enzyme in phototransductioncascade. Owing to this similarity, we propose to call this domain PDE gammalike (PGL). Consistent with this apparent homology to PDEg, the PGL domain wasshown to contribute to high afnity binding of RGS92 to activated Ga t andGao,50 a hallmark feature of PDEg discussed in a separate section below.

VII. Spatial Organization of the RGS9G b 5 Complex

The recently solved crystal structure of the RGS9Gb5 complex22 provideda major insight into understanding the arrangement of its individual domains(Fig. 2). Multiple domains of RGS9 are tightly integrated with Gb5 via a seriesof intermolecular interactions. The GGL domain of RGS9 forms the primary contact between these subunits, and the structural organization of the GGLGb5 module is very similar to the organization of conventional G proteinbg subunits. The GGLGb5 module is sandwiched between the DEP/R7Hand RGS domains, both forming additional contacts with Gb5. Remarkably, the

surface of Gb5 forming contacts with DEP/R7H shows a conservation of many residues present in other G protein bsubunits, which directly interact withGa subunits and effectors51 (see alsoRef. 52for review). This conservation ledtwo groups of authors to hypothesize that this surface may become accessible to

RGS

GGL

DEP

R7H Gb 5

FIG. 2. Structural organization of the RGS91Gb5S complex. Crystal structure22 revealed that

Gb5 is tightly bound to the GGL domain of RGS9 in a fashion resembling conventional G proteinbgsubunit interactions. In addition, the DEP/R7H and RGS domains make direct contacts withopposite interfaces of Gb5. Distinct protein domains are coded by different colors. Image wasrendered using the pdb coordinates (2PBI) and the DS ViewPro software.



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Ga subunits upon specic intermolecular rearrangements of RGS9Gb5 that would displace the DEP/R7H domain from its association with Gb5.22,53

However, several documented failures to reconstitute R7 RGS Gb5 proteins with various Ga subunits in vitro20,54,55 suggest that such rearrangements are very unlikely and that the shielding by the DEP/R7H domain is strong enoughto preclude this Gb5 surface from binding other proteins. Perhaps the mem-brane anchors, R9AP and R7BP, which also bind to the DEP/R7H domain,could serve as additional gatekeepers of such inexibility.

VIII. RGS92 G b 5SR7BP Regulates G ProteinSignaling in the Striatum

Several early studies revealed that the long splice isoform of RGS9 is selec-tivelyenriched in the striatum.12,14,56 More recentstudies also detectedRGS92 inseveral other regions of the central nervous system, such as the periaquiducal gray matter, the dorsal horns of the spinal cord, and the cortex, albeit at much lowerlevels.5760 As described above, RGS92 exists as an obligate heterotrimer with Gb5S and R7BP.34,44 Genetic knockout of either Gb5 or R7BP destabilizesRGS92 and results in its proteolytic degradation.34,49 The predominant expres-sion of RGS92 in the striatum, a region associated with the processing of rewardstimuli and providing motor coordination, has placed an emphasis on examiningthe role of RGS92 in mediating the behavioral responses to administration of addictive drugs and to manipulations disrupting motor performance.

Behavioral studies conducted with the RGS9 knockout mouse yielded thefollowing phenotypic observations: (1) Enhanced locomotion inresponse to thestimulation of dopamine signaling by apomorphine and cocaine.61 (2) Increasedsensitivity to the rewarding and analgesic effects of morphine.58 (3) Increasedphysical dependence on and delayed tolerance to morphine.58 This observationis also supported by an earlier nding that the reduction of RGS92 expressionlevels by antisense oligonucleotides results in reduced tolerance to opioids.57

(4) An accelerated development of tardive diskinesia, a motor discoordinationcondition associated with hypersensitivity of the dopamine receptors in thestriatum.39 (5)Signicant deteriorationof basic motor coordination.62 In contrast, virusmediatedhyperexpressionofRGS92inmousestriatumledtotheinhibitionof locomotor responses upon stimulation of dopamine signaling, the opposite of what was observed in the knockout animals.61 Taken together, these data suggestthat RGS92 negatively regulates G proteinmediated signals triggered by thedrugs of abuse: the elimination of RGS92 potentiates drug effects whereasRGS92 hyperexpression leads to less pronounced effects.



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Consistent with the role of RGS92 in regulating the effects of the addictivedrugs, several studies have pointed out potential feedback mechanisms by whichchanges in signaling via both dopamine and opioid receptors modulate theexpressionofRGS92. The le v el of RGS92 in the striatum increase upon chr oniccocaine61 or acute morphine58 administration, but decrease when morphine58 oramphetamine63 are administered chronicall y.Likewise, RGS92 levels were alsofound to be elevated in Parkinsons patients.64

Despite the wealthof behavioral data implicatingRGS92 instriatal functions,the molecular and cellular mechanisms of its action are not so well understood.RGS92 is known to beexpressed in all medium spiny neurons as well as incholinergic interneurons.39,61,65 Pharmacological studies indicate that RGS92regulates signaling downstream from the D2 dopamine receptors. Striatalneurons lacking RGS92 exhibited greater inhibition of thepostsynaptic NMDAreceptor signaling in response to D2 receptor stimulation.39 Likewise, infusionof recombinant RGS domain of RGS92 into acutely dissociated cholinergicinterneuronsdecreasedmodulation ofcalcium channelactivitybyD2receptors.65

In addition to its effects on dopamine signaling, RGS92 has also beenreported to regulate the sensitivity of the responses elicited by themopioidreceptor. In melanophores, transfection of RGS92 was shown to inhibitpigment aggregation,12 while in PC12 cells, expression of RGS92 resultedin decreased ERK1/2 activation38 in response to stimulation of mopioid recep-tors. Furthermore, two groups reported that RGS92 can be coimmunopreci-pitated with antibodies against the mopioid receptor, suggesting that bothproteins may exist in a complex.38,66

Despite clear evidence that RGS92 in the striatum is involved in regulationof D2 and mopioid signaling, it remains unclear whether these two receptors arethe only ones regulated byRGS92. Striatal neurons receive extensive inputs fromneurons that in addition to dopamine and opioids, include GABA, glutamate, andacetylcholine.67 Most of these neurotransmitters signal via receptors that activatethe Gi/o subclass of G proteins, which serve as primary targets for RGS92. Thisopens the possibility that the spectrum of receptors regulated by RGS92 is even wider. For example, studies with transfected cells and reconstituted liposomesdemonstrated that the RGS92 Gb5 complexes can effectively control G proteinsignaling driven by the M2 muscarinic acetylcholine receptor.33,68 Thus, furtherstudies are required to establish the complete spectrum of RGS92 specicity beyond its action on D2 andmopioid receptors.

Overall, we can envision three potential modes of RGS92 functioning in thestriatum. First, RGS92 may serve as the universal coordinator of G proteinsignaling originating from several receptors. In this scenario, RGS92 wouldintegrate the duration of cellular responses to various neurotransmitters. Second,RGS92 action may be limited to the regulation of G proteins originating fromspecic type(s) of GPCR, such as dopamine D2 receptor, whereas other



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neurotransmitters or hormones, such as opioids, act presynaptically to modulatethe release of the transmitter, which postsynaptic receptors are specically regu-lated by RGS92. Finally, there is a possibility that RGS92 may serve as a bridgebetween GPCR activation and the regulation of cellular processes other thanG proteinmediated neurotransmission. For instance, several recent reportsindicated that RGS92 and other members of the R7 family can translocateinto the nucleus where they presumably regulate transcription and/or othernucleusspecic processes.43,6971 Clearly, these scenarios are not mutually exclu-sive, and discriminating among them remains at the center of current studies.

IX. RGS91 G b 5LR9AP Regulates Visual SignalTransduction in Vertebrate Photoreceptors

In contrast to poorly understood molecular mechanisms of RGS92 actionin striatal neurons, the role of RGS91 in regulating G protein signaling inphotoreceptors is well established. In fact, the very idea that G proteinmediated signals can be regulated through acceleration of their GTPase activity rst surfaced in studies of vertebrate visual signal transduction (or photo-transduction). Phototransduction begins when a molecule of the visual pig-ment rhodopsin, located in the membranes of the outer segment compartmentof rods or cones, captures a photon of light. This brings rhodopsin into itsphotoexcited conformation (metarhodopsin II or R*) which activates theG protein transducin by triggering a rapid exchange of bound GDP for GTPon transducins asubunit (Ga t). At the next step, Ga tGTP stimulates theactivity of its effector enzyme, cGMP phosphodiesterase (PDE, also knownas PDE6). PDE activation occurs via Ga tGTP binding to one of the two smallgsubunits (PDEg) and releasing the inhibitory constraint that PDEg otherwiseimposes on the catalytic sites located within the PDEa or b subunit. PDEactivation leads to the hydrolysis of cGMP in photoreceptor cytoplasm and theclosure of the cGMPgated channels located in the plasma membrane. Chan-nel closure results in membrane hyperpolarization and decreased release of theneurotransmitter glutamate at the photoreceptor synaptic terminal (see Refs.7274 for detailed reviews on phototransduction and75 for the most recentupdate). A schematic representation of this cascade and an example of a rodlight response are shown inFig. 3.

As exemplied inFig. 3, both the rising and recovery phases of the photo-response take place on the rapid subsecond timescale. This requires that allcomponents of the phototransduction cascade are both activated and inactivated during this short time interval. It was understood by the late 1980s thatrhodopsin inactivation is accomplished by a twostep mechanism involving its



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phosphorylation by a specic kinase and subsequent arrestin binding. This typeof receptor inactivation mechanism is ubiquitously utilized in many GPCRsignaling pathways (reviewed inRef. 76). However, the mechanism responsiblefor the termination of PDE activation by transducin remained unclear. It wasthought that, like in any other G protein pathway, transducin signaling persistsuntil the GTP molecule bound to Ga t is hydrolyzed to GDP and Pi, whichallows Ga t dissociation of from PDEg and the return of PDE in its inactivestate. However, biochemical measurements showed that the rate of GTPhydrolysis measured with puried transducin was nearly 100fold slower thanthe photoresponse recovery rate (e.g., Refs. 7779). This was particularly puzzling because the current dogma in the eld was that heterotrimericG proteins did not have their own GAPs.80

The rst hints to the solution of this problem were obtained when trans-ducin GTPase was analyzed in concentrated suspensions of photoreceptormembranes or in rod outer segments with highly preserved disk stack structure.8183 These experiments revealed that the transducin GTPase ratemeasured under such conditions is signicantly higher than in cellfree systems

GTP

P i

PDE

Rho

RGS9-1

Single-photonresponse

0 0.2 0.4 s

Ga t Ga t

Gb Gb

hn

Gg Gg

b

b

a

a

g g

g g

cGMP GMP

FIG. 3. The role of RGS91 in regulation of vertebrate phototransduction. Absorption of aphoton by a molecule of rhodopsin (Rho) located in the disk membranes of the photoreceptor outersegment stimulates the GDP/GTP exchange on the G protein transducin (Ga t) leading to itsactivation. Activated GTPbound Ga t interacts with the gsubunit of the effector enzyme, PDE,leading to the hydrolysis of the second messenger, cGMP. PDE activation persists until RGS91binds to Ga t and stimulates its GTPase activity, which returns all of these proteins to their inactivestate. A transient reduction in the cGMP concentration in the photoreceptor cytoplasm evokes anelectrical response of the cell. A typical response to a single photon activation recorded from amouse rod is shown in the right panel (courtesy of M. E. Burns, UCDavis). Rapid recognition of

Ga t by RGS91 requires a contribution from PDEg, which dramatically enhances the afnity of RGS91 for Ga t and thus serves as an afnity adapter. The gure is modied fromRef. 50 andreproduced with permission.



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reconstituted from puried proteins. They further suggested that photorecep-tors may contain GTPase activating proteins, or other factors playing a similarrole in activating transducin GTPase.

It was shown several years later that the addition of PDEg, the immediatepartner of Ga tGTP, to photoreceptor membranes depleted of endogenous PDEbut retaining endogenous transducin resulted in a severalfold increase in thetransducin GTPase rate.84 In the same year it was found that another G proteineffector, PLCb1, stimulates the GTPase activity of the corresponding G proteins,Gq/11 .85 Together, these observations raised the hypothesis that G protein effec-torsin general may serve as GTPase activating proteins.86 However, a subsequentstudy 87 argued that PDE g cannot activate transducin GTPase without anothermembranebound protein present in the photoreceptor membranes, the conclu-sion solidied in three subsequentstudies.8890 The identity of this protein wasestablished 5 years later as RGS91.3 As discussed above, RGS91 in photorecep-tors exists as a constitutive dimer with Gb5L,24,34 and the entire RGS91 Gb5Lcomplex is tethered on the membrane surface by the transmembrane anchorR9AP.41,46

The nal conrmation of the role of RGS91 in transducin inactivation camefrom studies of RGS9 knockout mice, which are characterized by slow rates of photoresponse recovery in both rods35 and cones.91 More recently, it was shownthat the lack of RGS91 function in humans lead to the visual disorder bradyop-sia (slow vision).9294 The chief complaints of bradyopsia patients are difculty in adjusting to changes in luminance and photophobia. For example, they experience temporary blindness for 510 s after walking out of a relatively poorly lit space into the sunlight. A similar problem is experienced when they move back from a bright to a dark environment. Another signature characteris-tic of bradyopsia is patients inability to see rapidly moving objects, particularly in a low contrast visual environment. This condition is stationary and does notget better or worse with age, indicating that bradyopsia is not accompanied by progressive retinal degeneration (unlike disorders produced by mutations inseveral other phototransduction proteins; reviewed inRef s. 9597), matchingthe nondegenerative phenotype of RGS9 knockout mice.35

A recent study by Krispel and colleagues98 (see also Ref. 99 for a brief review), demonstrated that the rate at which transducin is inactivated by RGS91 serves as the single most important parameter determining the overallkinetics of photoresponse recovery in rods. Earlier studies argued that thetimely recovery of the photoresponse requires efcient deactivation of bothrhodopsin and transducin, with the slower of these two steps determining theoverall rate of response recovery (reviewed inRef. 100). However, the nature of this ratelimiting step remained elusive. Krispel and colleagues overexpressedRGS91 Gb5L R9AP in rods (this was achieved by overexpression of the R9APgene) and found that this leads to accelerated photoresponse recovery.



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Consistently, underexpression of RGS91 Gb5L R9AP in R9AP hemizygousmice was accompanied by a reduced photoresponse recovery rate. In contrast,overexpression of rhodopsin kinase, which is responsible for rhodopsin inactivation, did not cause a marked effect on the rod response recovery rate,reinforcing the authors conclusion that this rate is set by transducin GTPasecontrolled by RGS91. Interestingly, cones, which recover from light excitationsignicantly faster than rods, also have signicantly higher RGS91 levels.101

Therefore, the expression level of RGS91 is likely to serve as a key determinantof the temporal characteristics of individual types of photoreceptor cells.

X. The Role of the Effector Enzyme in Regulating TransducinGTPase and the Concept of Affinity Adapters

The discovery that the primary role in activating transducin GTPase in rodsand cones belongs to RGS91, raised the question of the role of PDEg, theoriginal protein implicated in transducin GTPase regulation. A study utilizing acombination of kinetic and pulldown assays55 established that PDEg acts by increasing the afnity of RGS91 Gb5L for Ga t. When measured under optimalconditions, the afnity of RGS91 Gb5L for Ga tGTP PDEg was $ 22 timeshigher than for Ga tGTP alone. This effect is observed with both free PDEgand PDEg within the PDEabg 2 holoenzyme, although the latter is about twiceless efcient in potentiating RGS91 activity.84,88 Interestingly, this effect of PDEg is much more prominent when measured with the RGS91 Gb5L com-plex (native or recombinant) than with the recombinant RGS9 homology do-main, in which case it never exceeds$ 23fold.18,19,102104 This argues that thenoncatalytic domains of RGS91 Gb5L play a signicant role in setting itsinteraction specicity, a concept discussed in detail in one of the sections below.

Importantly, normal interactions between RGS91 and PDEg are criticalfor timely photoresponse recovery in intact photoreceptors. This was estab-lished in the study of a transgenic mouse in which wildtype PDEg wassubstituted by its W70A mutant.105 This mutation was previously showed toimpair the ability of PDEg to affect transducin GTPase106 (see also Ref. 107).Photoresponses in W70A mice recovered much slower than in wildtype mice,revealing the essential role of PDEg in this process, yet about twice fasterthan in RGS9 knockout mice, indicating that RGS91 retains some ability toinactivate transducin even without cooperating with PDEg.

One way to conceptualize the role of PDEg in regulating transducinGTPase is to call it an afnity adapter between an RGS protein, RGS91,and its G protein target, transducin. Such an afnity adapter may be needed toassure an orderly sequence of proteinprotein interactions during the



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propagation of a photoresponse.50,105,108 A very low afnity of RGS91 Gb5Lfor free Ga tGTP may serve to prevent Ga t from hydrolyzing bound GTPbefore it has a chance to activate its effector, PDE. However, a much higherRGS91 Gb5L afnity for Ga tGTP PDE is essential for performing GTPhydrolysis on the physiologically rapid timescale of photoresponse recovery.

Realizing the signicance of PDEgs role serving an afnity adapterbetween RGS91 and transducin, we sought to investigate whether RGS92in the striatum may also utilize a similar mechanism to interact efciently withits biological target(s). We noted that the unique Cterminal PGL domain of RGS92 shares a signicant degree of homology with PDEg (see above) andused recombinant proteins to test whether this domain enhances the afnity between RGS92 and partner G protein asubunits.50 Following previousreports that the most likely physiological targets of RGS92 are G proteins of the Gi/o subfamily,12,14 we tested its ability to activate the GTPase activity of Ga o, Ga i, and Ga t and found that RGS92 displays the highest GAP activity toward Gao and Ga t. Given that transducin is absent from the striatum, weproposed that the most likely biological target of RGS92 is Gao. We nextcompared the ability of RGS92 to activate the Gao GTPase with that of RGS91, the isoform naturally lacking the PGL domain. Remarkably, theactivity of RGS92 alone was high and very similar to that of RGS91 in thepresence of PDEg. Just as in the case of transducin, RGS91 alone was not very active, but the addition of a recombinant Cterminus of RGS92 potentiated itsactivity, just as did PDEg. Complementary pulldown assays with immobilizedRGS9 isoforms showed that RGS91 was able to efciently bind activated Gaoonly when PDEg was present, whereas RGS92 bound Gao with the same highefciency whether or not PDEg was present.

These experiments have led us to conclude that the Cterminal PGLdomain of RGS92 is not just a structural but also a functional homolog of PDE g and that it serves as an afnity adapter between RGS92 and itsbiological target, Gao. However, unlike the case of RGS91, the afnity adapterfor RGS92 is an integral part of this protein. We speculate that the utility of thelatter arrangement may reect the fact that in the Go pathways effectorsare typically activated by itsbgsubunits (reviewed in Ref. 109), whereas theprimary function of activated Ga o is to interact with RGS proteins to achieveinactivation of the entire pathway on a physiologically rapid timescale. There-fore, it is not surprising that an afnity adapter in this case is a part of the RGSprotein molecule, which is a benecial arrangement for efcient G proteinasubunit binding. An example of such a putative pathway is illustrated inFig. 4. Here, Go activation by the D2 dopamine receptor is followed by thedissociation of thebgsubunits from GaoGTP. Gbg then activates the inwardly rectifying K channel (GIRK) whereas Gao interacts with RGS92 to ensurethe appropriate duration of this channel opening event.



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XI. Comparing the Functional Properties of RGS9 IsoformsExpressed in the Same Cell Type Suggests a Hypothesis

on the Evolutionary Origin of Phototransduction

In our most recent study, we compared the function of each RGS9 spliceisoform in the physiological context of the same cell.110 This was achieved by transgenic substitution of RGS91 in mouse rods with RGS92. A somewhatunexpected result was that in dim to moderate light rods expressing RGS92displayed the same rate of the photoresponse recovery as wildtype miceexpressing RGS91, but in bright light RGS92 rods showed a more efcientphotoresponse recovery than wildtype rods. Key to understanding thisresult isthe fact that rods contain about 10times more transducin than PDE.74 Giventhat RGS91 is an efcient GAP for the Ga tGTP PDE g complex but not freeGa tGTP, rapid transducin inactivation by RGS91 in wildtype rods can besustained only as far as the amount of activated transducin does not exceedthe cellular content of PDEg. In contrast, RGS92 has its own builtin afnity adapter, the PGL domain, and therefore RGS92 can serve as an efcient GAPfor both Ga tGTP bound to PDEg andGa tGTP produced in excess of PDEg inbright light.

Despite this plausible mechanistic explanation, this result inevitably bringsthe question why rods normally employ an apparently inferior RGS9 isoform.Although we realize that in biology the question why is not nearly as

GTP

PGL

RGS9-2

P i

K+ Ga o Ga oGb Gb

Gg Gg

D2R

FIG. 4. Putative role ofRGS92 in regulating G proteinsignaling pathways of the striatal neurons.In this example, the D2 dopamine receptor activates G protein, Go. The bgsubunit of Go modulatesthe conductivity of the inwardly rectied K channel, whereas Gao interacts with RGS92 to regulatethe duration of this signaling event. The PGL domain of RGS92 serves as a builtin afnity adapter which endows RGS92 with high afnity for free GTP bound Ga subunits and allows their rapidinactivation. The gure is modied fromRef. 50and reproduced with permission.



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legitimate as the question how, searching for the answer provided us anopportunity to think about the evolutionary origin of the visual transductionpathway. RGS proteins, including the members of the R7 subfamily, exist in alleukaryotic organisms, whereas no PDEg genes have yet been discovered inspecies more ancient than the lamprey.111 This suggests that when PDEgemerged in evolution to allow PDE activation by transducin, high afnity interactions of the transducin prototype with RGS protein(s) already existed.This necessitated that PDEg effectively compete with RGS proteins for bind-ing to activated transducin in order to maintain high visual sensitivity. There-fore, we speculate that the evolution of the entire Ga t PDE g RGS9 molecularmodule involved both the improvement of the afnity between transducin andPDE g and the selection for an RGS protein isoform that would not inactivatetransducin before it binds and activates PDE. RGS91 is the only knownprotein that follows this rule.

Furthermore, the similarities in sequence and function between theCterminus of RGS92 and PDEg suggest that one may have evolved fromanother in evolution. Indeed, the RGS9 and PDEg genes are located on thesame region of chromosome 17 (within loci 17q2417q25 in the humangenome). One possibility is that the PDEg gene may have originated fromthe duplication of a portion of the RGS9 gene coding the PDEglike domain.Alternatively, the Cterminal domain of RGS92 may have originated fromthe duplication of the PDEg gene. Unfortunately, the current gaps in thegenome sequences of the lamprey and its immediate predecessor, the hagsh(see Ref. 112 for a review on evolution of the eye), require us to wait beforeconcluding which of these alternatives is more plausible.

XII. Mechanisms Regulating the Ga RecognitionSelectivity and Catalytic Activity of RGS9

As discussed in the preceding sections, efcient interactions of the RGS9isoforms with their Ga t or Gao targets requires the presence of afnity adap-ters: PDEg for RGS91 or the PGL domain of RGS92. Detailed mutagenesisstudies of RGS91 revealed that the key role in discriminating between freeactivated G protein asubunit and activated Ga in a complex with PDEg isplayed by the noncatalytic domains of the RGS9Gb5 complex.

As noted above, the RGS9 homology domain itself shows only a modest,less than threefold substrate discrimination in favor of the transducinPDE gcomplex.18,19,102104 Still, this is the only known RGS homology domain whoseability to activate transducin GTPase is potentiated by PDEg. Every other RGSdomain tested so far is inhibited by PDEg.113116 Examination of the crystalstructure of the triple protein complex containing the RGS9 homology domain,



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Ga t and PDE g117 and evolutionary trace analysis116 identied several aminoacids at the Ga interaction interface of RGS9 in close proximity to the PDEgbinding region that are present in RGS9 but not in the closely related domainof RGS7. The substitution of just two of these residues in the RGS7 homology domain for corresponding residues from RGS9 (E387L and P394R) reversedthe effect of PDEg from inhibition of RGS7 domain activity to stimulation.116

The reciprocal substitution of these two residues in RGS9 for those from RGS7(L353E and R360P) also reversed its specicity by mak ing the Ga tPDE gcomplex a less preferred substrate than free activated Ga t.102 These observa-tions indicate that the RGS domain from RGS9 has unique properties allowingit to positively cooperate with PDEg.

However, the ability of the entire RGS91 Gb5 complex to discriminatebetween free Ga t and Ga tPDE g is much higher than that of the RGS9 domainalone and exceeds 20fold when measured under optimized condi-tions.18,19,55,102 Interestingly, this overall effect originates from opposite con-tributions from the individual molecular modules of RGS91 Gb5.18,19 TheGGLGb5 module imposes a very strong reduction in the afnity of theRGS9 domain for transducin without affecting the ability of this domain tobind slightly better to Ga tPDE g than to free Ga t. All other structures withinRGS91 Gb5L (the Nterminus of Gb5L, the DEP/R7H domain, and the shortCterminus of RGS91) reverse the inhibition imposed by GGLGb5S onbinding Ga tPDE g, but not on binding free Ga t. This ultimately results in thephysiologically high difference in the RGS9Gb5L afnity for free activatedtransducin and its PDEg complex.

In the subsequent study, 102 we investigated whether this large contributionof the noncatalytic domains of RGS91 Gb5L consists of enhancing the smallintrinsic specicity of the RGS9 homology domain or whether the noncatalyticdomains set the ability of RGS91 Gb5L to preferentially recognize Ga tPDEgregardless of the properties of RGS9 homology domain. This was accomplishedby introducing the double L353E/R360P mutation into the sequence of fulllength recombinant RGS91. As described above, this mutation reverses thespecicity of the recombinant catalytic domain toward favoring free activatedGa t. However, the same mutation introduced in the fulllength RGS91 did notreverse its ability to favor Ga tPDEg over free Ga t. Yet, the degree of thisdiscrimination was reduced from $ 23fold in wildtype RGS91 Gb5L to $ 6fold in the mutant. Overall, this study rmly established that the noncatalyticdomains of RGS91 Gb5L play a decisive role in establishing its substratespecicity, although the high degree of this specicity observed under physiolog-ical conditions requires an additional contribution from the catalytic domain.

Another mechanism controlling the GAP activity of RGS9 is its potentia-tion by R9AP.42,118,119 The effect of R9APis very large and achieves$ 70foldpotentiation in the case of the native R9AP42 and up to $ 20fold in the case of



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recombinant R9AP.46,118 Consequentially, the physiologically rapid inactivationof transducin in rods and cones can be achieved only by the entireR9APRGS91 Gb5L complex.46,48,92

R9AP can potentiate the GAP activity of RGS91 Gb5L, only when it isattached to the membrane (whether in photoreceptors or heterologous systems)because the soluble truncation mutant of R9AP containing all parts necessary forRGS91 Gb5L binding but lacking the transmembrane segment cannot stimulatethe GAP activity of RGS91 Gb5L.119 A formal kinetic analysis119 revealed thatthis stimulation primarily consists of the allosteric RGS91 Gb5L activation, which ultimately results in over an order of magnitude increase in the rate of transducin GTPase. An additional smaller contribution results from an appro-ximately twofold enhancement of the afnity between RGS91 Gb5L and trans-ducin. Whether these effects originate from direct proteinprotein interactionor fromthe consequencesofplacing RGS91 Gb5L next to the membranesurfaceis presently unknown.

Recent studies with transgenic mice expressing RGS92 in their rodsindicated that the activity of RGS92 could be likewise stimulated by R9AP,110 although these proteins are not known to be expressed in the samecell types. By analogy, it is plausible to think that the RGS92specic anchor,R7BP, is also capable of potentiating the GAP activity of RGS92 (or bothisoforms), but this remains to be elucidated.

In conclusion, we would like to emphasize that studies of the RGS9 isoformshave provided a valuable framework for addressing many questions regardingthe functional roles and regulation of regulators of G protein signaling in general. We hope that consolidation of the current state of understanding of this key signaling protein will help to facilitate investigations of the less understoodmembers of the RGS family, provide insight into the principles of G proteinregulation in retina and striatum, and spur investigations on the outstandingquestions regarding the functional regulation of RGS9 splice isoforms.

Acknowledgments

We would like to thank Sid Gospe for critical reading of the manuscript. The authors weresupported by NIH grants DA021743 and EY018139 to K.A.M. and EY10336 and EY12859 to V.Y.A.

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