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Heterotrimeric G protein signaling in polycystic kidney disease

Taketsugu Hama and Frank ParkDepartment of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis,Tennessee

Hama T, Park F. Heterotrimeric G protein signaling in polycystic kidneydisease. Physiol Genomics 48: 429–445, 2016. First published May 13, 2016;doi:10.1152/physiolgenomics.00027.2016.—Autosomal dominant polycystic kid-ney disease (ADPKD) is a signalopathy of renal tubular epithelial cells caused bynaturally occurring mutations in two distinct genes, polycystic kidney disease 1(PKD1) and 2 (PKD2). Genetic variants in PKD1, which encodes the polycystin-1(PC-1) protein, remain the predominant factor associated with the pathogenesis ofnearly two-thirds of all patients diagnosed with PKD. Although the relationshipbetween defective PC-1 with renal cystic disease initiation and progression remainsto be fully elucidated, there are numerous clinical studies that have focused uponthe control of effector systems involving heterotrimeric G protein regulation. Amajor regulator in the activation state of heterotrimeric G proteins are G protein-coupled receptors (GPCRs), which are defined by their seven transmembrane-spanning regions. PC-1 has been considered to function as an unconventionalGPCR, but the mechanisms by which PC-1 controls signal processing, magnitude,or trafficking through heterotrimeric G proteins remains to be fully known. Thediversity of heterotrimeric G protein signaling in PKD is further complicated by thepresence of non-GPCR proteins in the membrane or cytoplasm that also modulatethe functional state of heterotrimeric G proteins within the cell. Moreover, PC-1abnormalities promote changes in hormonal systems that ultimately interact withdistinct GPCRs in the kidney to potentially amplify or antagonize signaling outputfrom PC-1. This review will focus upon the canonical and noncanonical signalingpathways that have been described in PKD with specific emphasis on whichheterotrimeric G proteins are involved in the pathological reorganization of thetubular epithelial cell architecture to exacerbate renal cystogenic pathways.

heterotrimeric G proteins; kidney; signal transduction; polycystic kidney disease;accessory proteins

POLYCYSTIC KIDNEY DISEASE (PKD) remains one of the mostcommon inherited disorders, which can lead to a progressiveloss in renal function (52–54). Under normal conditions, thekidney possesses a multitude of functions, including its pri-mary roles as a filtration system to eliminate waste productsfrom the blood, regulation in long-term blood pressure bycontrolling fluid and electrolyte balance, acid-base regulation,and production of endocrine hormones, such as erythropoietin,renin and active forms of vitamin D (148). In PKD, however,numerous variants in either the polycystic kidney disease 1(PKD1) or polycystic kidney disease 2 (PKD2) gene, which arethe two primary genes associated with causing the autosomaldominant form of PKD (or ADPKD), can negatively impactthe normal function of the kidney by pathologically altering thearchitecture of the kidney through a transformative process thatchanges the tubular epithelial cells, largely in the collectingducts, into fluid-filled sacs known as cysts (52, 54). Themechanisms by which cyst initiation and formation occur dueto the malfunctioning gene products of PKD1 and PKD2,

known as polycystin-1 (PC-1) and polycystin-2 (PC-2), respec-tively, remain to be fully described (60).

There is considerable evidence that this pathological switchin the tubular epithelial cell to form cysts is quite complex andinvolves a cascade of diverse changes, including missorting ofproteins to incorrect cellular locations (5), misalignment of cellorientation (42), hypersecretion of fluids (53), reorganizationof the extracellular matrix (190), apoptosis (49), and abnormalinduction in cell proliferation (90). Numerous orthologous andnonorthologous rodent models of PKD have been generated tofurther elucidate the importance of these above described andpossible other novel modes of action in vivo (55, 60, 116). Atthe present time, however, a major focus of many preclinicaland numerous clinical trials is the importance of neutralizingheterotrimeric G protein-dependent signalopathies by eitheractivating or inactivating cell surface G protein-coupled recep-tors (GPCRs) to control cystic disease progression (53, 59, 60,174, 190).

Heterotrimeric G proteins consist of three distinct subunits,�, �, and �, which are regulated through canonical interactionswith cell surface GPCRs (189) or noncanonical associationswith membrane tyrosine kinase receptors or cytoplasmic ac-cessory proteins (127). In the classic model of GPCR signaling,ligand binding induces a conformational change in the guaninenucleotide exchange factor domain of the receptor facilitating

Address for reprint requests and other correspondence: F. Park, Univ. ofTennessee Health Science Center, College of Pharmacy, Dept. of Pharmaceu-tical Sciences, 881 Madison Ave., Rm. 442, Memphis, TN 38104 (e-mail:[email protected]).

Physiol Genomics 48: 429–445, 2016.First published May 13, 2016; doi:10.1152/physiolgenomics.00027.2016. Review

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the transfer of GTP onto the inactive G�-GDP subunit. Thisexchange promoted the activation of G�-GTP, which is themain signaling complex evaluated during cyst formation andprogression in PKD. Although unbound G�� has been largelyobserved as an ancillary byproduct to G�-subunit activation,its role as a regulator of signaling output, particularly on ionchannel activity, cannot be completely ignored (31, 32, 86). Inthe end, G protein signaling is terminated by the reassociationof unbound G�� with inactivated G�-GDP subunits.

A growing body of evidence has demonstrated that theregulation of heterotrimeric G protein activation is more di-verse than previously anticipated. This was attributed to thedistinct renal localization pattern for each G protein subunitexpression, the combination of G��-complexes that actuallyexist, and the GPCR subtypes that are expressed within eachcell type (127, 128). Moreover, intracellular proteins have beenidentified to intercede at the level of the receptor-ligand com-plex through unique modes of action to compete for either G�or G�� binding and modulate the termination of second mes-senger-mediated signaling. In addition, these accessory oradaptor proteins could also perturb the magnitude of thecellular response, protein trafficking, cell orientation, andmany other biological processes that would be relevant to thepathogenesis of cystic disease (127, 128). Alternatively, het-erotrimeric G proteins could also be involved in an integratedcross-talk network by functioning as intermediary signal pro-cessors between traditional GPCR and other unitary transmem-brane tyrosine kinase receptors (105).

In the majority of the studies describing GPCR-based sig-nalopathies in PKD, the involvement of heterotrimeric Gproteins is not generally discussed. A major reason may be thatmany investigators assume that the specific heterotrimeric Gprotein ��-complexes with their cognate GPCR have alreadybeen well documented. However, as new data continue toemerge regarding unexpected interactions by heterotrimeric Gproteins or the identification of novel signaling pathwaysoutside of the canonical ligand-GPCR systems, further inves-tigations into the role of heterotrimeric G proteins are war-ranted to confirm their role as key signal modulators in path-ways associated with cystic disease progression. With this inmind, we will provide a comprehensive overview by whichheterotrimeric G proteins play a pivotal role in canonicalGPCR pathways, as well as unconventional modes of action,including those regulated by PC-1, during the cystogenicprocess in PKD. In some cases, we will ascribe some modes ofaction by which heterotrimeric G protein subunits have beendescribed in other biological systems to potentially provideimportant new insight into heterotrimeric G protein signalmodulation in PKD.

POLYCYSTIN-1

PC-1 has been categorized as an atypical GPCR and hasshown the capability to transmit physical cues from the exter-nal milieu into the cell by converting this information into abiochemical signaling output (118, 126, 192). The mecha-nosensation mediated by PC-1 has been postulated to beassociated with its cellular localization within or at the base ofthe cilia (118, 119). Unlike the standard GPCR, which has aheptahelical transmembrane configuration, PC-1 has been pre-dicted to have 11 transmembrane-spanning regions (68). Sim-

ilar to all GPCRs, however, PC-1 could selectively bind mul-tiple classes of G� subunits at a specific stretch of 20 aminoacids in the COOH terminus, known as a G protein-bindingdomain (GBD) (129, 130, 204). In addition, PC-1 has a highlyconserved autoproteolytic site, GPCR proteolysis site (GPS),within a GPCR-Autoproteolytic INducing (GAIN) domainlocated on the NH2-terminal side of the first transmembranespan. The GAIN domain was found in cell adhesion GPCRs tomaintain tubular epithelial cell morphology (3). In fact, loss ofthe GPS in PC-1 could structurally reorganize the cell towarda highly aggressive form of renal cystogenesis (202), but theimportance of PC-1 targeting to the cilia remains to be resolved(19, 159). These findings provide some insight that PC-1 hasthe properties of a ligand-independent GPCR, yet the physio-logical implication to directly regulate heterotrimeric G proteinsignaling to maintain normal tubular architecture and functionremains to be fully understood.

G Protein Subunit Binding to PC-1 COOH Terminus

As mentioned above, a number of G�-subunits, most nota-bly G�12 and G�i/o, have been shown to interact with PC1and PC1-like proteins (31, 32, 129, 130, 203–205) (Fig. 1).Initially, Parnell et al. (129, 130) demonstrated that heterotri-meric G proteins with select G�-subunits, namely G�i/o, G�s,and G�12/13, interact with the COOH-terminal tail of PC-1.The efficiency of the G�-subunit binding to PC-1 was depen-dent upon a stretch of 74 amino acids, which contained theGBD flanked by conserved tyrosine and serine phosphorylationmotifs (129). Complete abolition of G protein binding was onlyobserved when the serine phosphorylation motif was alsoremoved in conjunction with the GBD (129). Subsequently, Yuet al. (203, 204) confirmed a selective interaction betweenPC-1 and G�12, but not its closely related G�13 (203), andthat other components of the COOH-terminal region werepartially required to maintain wild-type binding of G�12 toPC-1 (204).

In addition to binding heterotrimeric G protein subunits, theGBD catalyzes the degradation of GTP to GDP nucleotidesbound on G�i-subunits (129). Although these data were sug-gestive that PC-1 may act similar to conventional GPCRs, itwas not known whether the inactivated G�-GDP remainedbound to PC-1. A subsequent study, however, demonstratedthat mutant G�12-subunits bound to either GTP or GDP arecapable of binding to PC-1 (203, 204). From these studies,PC-1 may be predicted not only to activate heterotrimeric Gproteins, but also to behave as a scaffold to form proteincomplexes to control downstream signaling. Additional studiesare clearly needed to determine whether the activation status(i.e., GTP/GDP bound form) of each distinct G�-subunit playsa crucial role in the efficient binding to PC-1. Regardless, theCOOH-terminal tail of PC-1 has shown flexibility in its abilityto interact with multiple classes of G�-subtypes, which leads tocomplementary or differing modes of signal processing and/oreffect. To date, heterotrimeric G protein interaction with PC-1has been described to control downstream signal pathwayactivation or directly modulate ion channel activity (Fig. 1).

JNK Signaling: Role of G�i- and G�12-Subunits

Initial experiments by Parnell et al. (130) demonstrated thatc-Jun NH2-terminal kinase (JNK) activation is mediated by

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interaction of G�i-subunits with the COOH-terminal tail ofPC-1, but that the effector was unbound G��. Subsequentexperiments showed that the activation of JNK cannot besolely attributed to G�i-subunits but involves other classes ofG�-subunits, since blockade with pertussis toxin inhibited only�25% of the PC-1-mediated JNK signaling (130). In tubularepithelial cells, G protein �12-subunits promote apoptosis byactivating JNK and enhance Bcl-2 degradation. PC-1 interfereswith the activation of apoptotic signaling by directly binding toG�12 (204). Even under conditions of reduced by G�12binding to PC-1, the inhibition of apoptotic signaling is main-tained. Moreover, constitutively active G�12, QL�12, is ableto bind to PC-1, and that mutant G�12 can uncouple theinteractions between QL�12 and PC-1 to prevent apoptotic

signaling (204). These results suggest that G�12 can bind toPC-1 in either the active GTP-bound or inactive GDP-boundform.

G�i-Subunits: Ion Channels and Other Signaling Pathways

Antiapoptotic signaling was also observed following G�i-subunit interaction with PC-1, which activates Akt through aG��-PI-3K pathway (11). In addition, G�i-subunits are capa-ble of activating AP-1 activity, but this activation is at leasttwofold less potent than G�q- or G�12/13-subunits (130). Theintermediary steps between G�-subunit activation to the induc-tion of AP-1 is not known, but there may be a link with JNKactivation (130).

To date, however, the role of G�i/o subunits with PC-1 hasbeen described as a key regulator of ion channels through aG��-dependent mechanism. Under basal conditions, overex-pression of PC-1 exerts an inhibitory effect on N-type Ca2�

channels, while promoting G protein-coupled inward rectifyingpotassium channel activity (31, 32). Genetic and pharmacolog-ical approaches have demonstrated that the ion channel regu-lation can be attributed to the COOH-terminal region of PC-1to bind G�i/o-subunits to liberate unbound G�� (31, 32, 46).However, the role of PC-1 to control ion channel activity byG�� is not universal and is dependent upon heterologousproteins that bind to the coiled coil domain in the COOH-terminal tail of PC-1, which is located near the GBD (129).PC-2, which is also known as the transient receptor protein P2ion channel (176), has been shown to have a direct relationshipwith PC-1 at the coiled coil domain (57, 135, 195). The role ofPC-2 within the heteromeric PC-1/PC-2 complex was found toblock G protein activation by PC-1, and the loss of theCOOH-terminal region of PC-2 is responsible for restoring theability of PC-1 to modulate ion channels by activating G��(31, 32). The positive influence by which PC-1 activates PC-2ion channel activity is not dependent upon G protein signaling,

A

BFig. 1. Heterotrimeric G protein regulation by polycystin-1. Polycystin-1(PC-1; in blue) is embedded within the plasma membrane due to its 11transmembrane-spanning regions. G protein binding and activation are regu-lated by a region known as GBD in the COOH terminus end of PC-1 in thecytoplasm and could regulate G� (A) or G�� signaling (B). A: the GBD couldinteract with multiple classes of GTP-bound G�-subunits (G�12, G�, G�s,and G�) activated by heterologous GPCRs (such as thrombin receptors) orinteracting with inactivated forms of G�-GDP. PC-1 could interact with G�12to block apoptosis, possibly through the activation of AP-1. G�q signalingcould activate calcineurin to promote NFAT translocation to the nucleus. OtherG�-subunits could activate AP-1 activity. The role of G�s activation by PC-1is not fully described but has been shown to interact directly with PC-1. PC-1could also interact directly with other proteins, PC-2 and RGS7, at the coiledcoil domain. The COOH-terminal cytoplasmic tail has 2 cleavage sites (de-noted by the dotted line in the COOH-terminal tail of PC-1) to generate eithera small or larger fragment that can be translocated to the nucleus to eitheractivate STAT 3/6 or �-catenin. B: other accessory proteins in the vicinity ofPC-1 may intercept G protein subunits, such as AGS3, to promote PC-2 ionchannel regulation through a G��-dependent mechanism. G�i-subunit inter-action with PC-1 could prevent apoptosis by activating Akt or activateJNK/AP-1 signaling. G��-signaling mediated by PC-1 could negatively con-trol N-type calcium channel and activate GIRK channels. AGS3, activator ofG protein signaling 3; GBD, G protein-binding domain; GPCR, G protein-coupled receptor; GPS, G protein-coupled receptor proteolytic site; NFAT,nuclear factor of activated T cells; PC1, polycystin 1; PC2, polycystin 2;RGS7, regulator of G protein signaling 7; SAPK/JNK, stress activated proteinkinase/Jun amino terminal kinase.

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but rather upon the physical association between the COOH-terminal tails of PC-1 and PC-2 (31, 46, 57, 195).

G�q-Signaling

In neurons, the role of G�q-signaling in the presence orabsence of murine PC-1 did not appear to play a role in ionchannel activity, since there was no marked change in theendogenous activity of the M-type K� channel (32). In asubsequent study, the role of the PC-1/G�q-complex wasshown to be involved in the activation of other signalingpathways unrelated to ion channel regulation. Puri et al. (134)demonstrated a synergistic role for G�q to enhance the PC-1activation of calcineurin and nuclear accumulation of nuclearfactor of activated T cells in HEK293T cells. G�q-subunits arealso involved with promoting AP-1 activity in the presence ofPC-1, similar in magnitude with G�12/13-subunits (130).Since transgenic overexpression of PC-1 can also perturb itsnormal physiological function to promote renal cyst formation(168), the contrasting results regarding the role of G�q-acti-vation with respect to PC-1 is likely due to the differences inthe experimental design (endogenous vs. transfected overex-pression of G�q), cell lines studied (sympathetic neurons vs.HEK293T), and experimental readout (electrophysiologicalmeasurement of ion channels vs. signal transduction pathwayanalysis). Further studies using Pkd1-deficient or hypomorphcells are needed to confirm whether G�q signaling is actuallyregulated by PC-1 in renal tubular epithelial cells.

Accessory Proteins

The regulation of G protein subunit binding and downstreamsignaling by PC-1 may be modulated by the presence ofheterologous proteins. Similar to PC-2, proteins could directlybind to specific regions near the GBD in PC-1 to interfere withG protein signaling, possibly by affecting G protein interaction.Alternatively, proteins may localize near PC-1 and interferewith G protein signaling elicited by PC-1 by binding to Gprotein subunits during the activation/inactivation cycle. Un-like PC-2, not all proteins that bind to the COOH-terminal endof PC-1 can alter G protein signaling. Most notably, regulatorof G protein signaling 7 (RGS7), which is a member of a largefamily of accessory proteins that directly binds to G�-subunits,exhibits a physical protein-protein interaction in the coiled coildomain of PC-1 (76). Other than slowing the degradation ofRGS7 (76), the RGS7/PC-1 complex was not determined toalter the ability by PC-1 to control ion channel activity (32).Mutant forms of G�i/o-subunits [i.e., G�oA (G184S) or G�i1

(G183S)], which were designed to prevent binding to the RGSdomain of RGS7, did not change PC-1-dependent regulation inCa2� channel activity (32). Because of these findings, theauthors concluded that RGS7 is not directly involved withdisrupting the PC-1/heterotrimeric G protein signaling axis(32), but further studies may be warranted to substantiate thisconclusion. Several RGS proteins, including RGS7, have beenshown to preferentially bind to G�5 rather than G�o-GDPcomplexes (92). Regardless of the presence of pertussis toxin,an inhibitor of G�i/o protein activity, RGS7 could interact withG�5 and the deactivation of the mutant G�i/o could be basedentirely upon its intrinsic GAP function. Therefore, the lack ofregulation on ion channel activity may be masked by thepreference of G protein subunits binding to RGS7 within a

particular cell type. It is also important to note that the absenceof the whole or even a partial fragment of the coiled coildomain is sufficient to markedly reduce G protein �-subunitbinding to PC-1 (129, 204). This could suggest that heterolo-gous protein binding at the coiled coil domain does not nega-tively impact, but may positively recruit, G proteins to PC-1 foractivation. To date, however, there have been no publisheddata demonstrating that protein interaction in the COOH ter-minus of PC-1 could modify G�-subunit binding to regulate Gprotein signaling. Further studies are needed to clearly eluci-date the role of G protein subunit binding and activation usingmutant PC-1 with only mutations in the GBD, and not dele-tions in the entire PC-1 COOH-terminal region.

As mentioned above, other accessory proteins may indi-rectly control PC-1-dependent G protein signaling by interact-ing with the activated G protein subunits, and not with PC-1itself. One example is activator of G protein signaling 3(AGS3), which could bind multiple inactive G�i/o-GDP-sub-units and function as a guanine dinucleotide dissociation in-hibitor. The liberation of free G��-dimers was shown toinduce heteromeric PC-1/PC-2 ion channel activity, but notwith PC-2 expression alone (86). No definitive study hasshown a direct protein-protein interaction between AGS3 andPC-1, but a recent study by Yeh et al. (200) clearly localizedAGS3 to the basal body/centrosome region of the cilia, acommon site of PC-1 expression. If this spatial localization forAGS3 is similarly observed in renal epithelial cells, then AGS3could be in close proximity to the same pool of G�i/o-subunitsthat interact with PC-1 and would be available to modulatedownstream effector signaling, including the direct control ofPC-2 ion channel activity through unbound G��-dimers (86).

Cleavage and Nuclear Localization of PC-1 COOH-terminalTail

In addition, the importance of G protein binding on PC-1and its effects on the efficiency of posttranslation cleavage ofits COOH-terminal tail to produce two distinct smaller proteinfragments, �35 kDa product containing the entire intracellularCOOH-terminal tail (24, 87) or �16 kDa product (p112) (99),remain to be determined. These truncated COOH-terminalfragments could translocate to the nucleus to promote geneexpression by interacting with either �-catenin or STAT-3/-6,respectively (99, 186, 187). The extent of the cleavage of theCOOH-terminal tail depends upon the expression of PC-2 andchanges in intracellular calcium (11). Of these two PC-1fragments, the larger COOH-terminal tail portion may likelycontain the GBD, which could be involved in the process ofcleavage efficiency, nuclear translocation, and possibly tran-scriptional activation.

For these reasons, to fully appreciate the physiologicalimportance of G protein interaction with PC-1 to controlnormal tubular epithelial cell function, genetic constructs ex-pressing mutant versions of PC-1 in which the GBD has beenmodified or removed to prevent G protein binding need to beevaluated. These additional studies would provide direct evi-dence regarding the functional consequences of PC-1 to bindand initiate G protein signaling to promote cardiovascularabnormalities and cystic disease development.

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DYSREGULATION OF HETEROTRIMERIC G PROTEIN-DEPENDENT SIGNAL TRANSDUCTION PATHWAYS IN PKD

Loss of polycystin activity could dysregulate numeroushormonal systems that are dependent upon heterotrimeric G

protein regulation in both vascular and tubular epithelial cellsin the kidney (Fig. 2). To date, the gradual temporal changes inthe activation or repression of signaling pathways during theearly and late phases of cystic disease progression remain to be

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fully described. In nearly all cases, heterotrimeric G proteinsignaling following ligand binding to a specific GPCR hasfocused upon the role of the G�-subunit, with less informationknown about G��, in tubular epithelial and vascular cellfunction. In this section, we provide an overview for some ofthe candidate GPCR pathways that have been described invascular and tubular epithelial cells in PKD and elucidate thecellular and/or functional changes in cardiovascular and renalfunction using data from animal models and humans withPKD.

cAMP Regulation by Adenylyl Cyclase and Calcium: Role ofG�i-, G�s-, and G�q-Subunits

Renal tubular epithelial cells from animal models and hu-mans with PKD1 or PKD2 mutations exhibit elevated produc-tion of cAMP (102); molecular strategies targeting the controlof adenylyl cyclase activity have been a major focus in drugdevelopment for PKD treatment. G�s (stimulatory) and G�i(inhibitory) were shown to be positive and negative regulatorsof adenylyl cyclase activity, respectively. In the nephron,numerous isoforms of adenylyl cyclase are present, particularlyadenylyl cyclase isoforms 3, 4, and 6 (AC6), but to date, onlyAC6 has been shown to play a crucial role in cystic diseaseprogression (137). Under normal conditions, increased cAMPdecreases the rate of tubular epithelial cell proliferation byactivating the protein kinase A/c-Raf pathway (197). In cysticepithelial cells, however, increased cAMP (197) or decreasedintracellular calcium (198) could switch kinase activation fromc-Raf (Raf-1) to B-Raf kinase and lead to abnormal activationof the MEK/ERK1/2 signaling (Fig. 3). This would result inincreased tubular epithelial cell proliferation, increased fluidsecretion, and/or decreased tubulogenesis.

At present, there are several GPCR systems directly associ-ated with controlling cAMP levels through either G�i or G�s,including arginine vasopressin receptors, somatostatin receptor(SSTR) subtypes, �-adrenergic receptors, adenosine receptors,

and E-prostanoid receptor subtypes. In addition, strategies toreduce cAMP levels by increasing intracellular calcium levelsthrough G�q-activation has been investigated (Figs. 2 and 3).Increased G�q-activation by GPCR stimulation could promoteeither accelerated cAMP catabolism through phosphodiester-ase (PDE) activation or inhibition of adenylyl cyclases (60).

Arginine vasopressin receptors. In the kidney, arginine va-sopressin (AVP) was shown to have a dual role in modulatingrenal blood flow distribution and control the urine-concentrat-ing mechanism through interactions with two distinct types ofreceptors, vasopressin V1a (V1aR) and V2 receptors (V2R)(98, 124, 125). In the context of PKD, the primary therapeutictarget of the AVP system has been to prevent or disrupt V2Ractivation. Mechanistically, V2R activation promotes G�s dis-sociation from G�� to stimulate adenylyl cyclase activity andsubsequently increase cAMP production. Recent data wouldsuggest that AVP stimulation activates AC6 as the primaryeffector system to produce cAMP (137), and not AC3 or AC4(77, 78). The V2R is primarily expressed in the distal tubularepithelial cells (98), and its localization is consistent with itsfunctional role to control the urine concentration process.Changes in the cAMP levels directly alter the function of theNa�/K�/2 Cl� cotransporter in the thick ascending limb ofHenle and also control the number of aquaporin-2 (AQP2)water channels and urea transporters inserted into the apicalmembrane in the principal cells of the collecting duct (109,208). In cystic epithelial cells, the increased levels of cAMPare primarily through V2R activation in the basolateral mem-brane, but there was evidence that the V2R could also bepartially misrouted to the apical membrane (144). On the basisof these findings, selective V2R antagonists, such as Tolvap-tan, have been developed as a treatment strategy to attenuatecystic disease progression by preventing the activation of theG�s subunits (61, 171, 173).

As described earlier, alterations in intracellular calciumlevels could also play a role in regulating cAMP levels. In

Fig. 2. Schematic overview of heterotrimeric G protein signalopathy in renal tubular epithelial and vascular cells in polycystic kidney disease. HeterotrimericG proteins play diverse physiological roles either canonically to transmit extracellular stimuli into the cell following GPCR stimulation or noncanonically throughrecently identified accessory proteins. There are 4 distinct classes of G� subunits, G�s, G�i, G�q/11, and G�12/13, which are depicted in this schematic, exceptfor G�12 (described in Fig. 1). 1) Cystic tubular epithelial cells. G�s activation by multiple GPCR, including V2R, EP2/EP4, and �1/�2, could increase cAMPproduction and activate downstream proliferative pathways, which include Raf/MEK/ERK1/2, or other cellular functions, such as water and electrolyte balance.ANG II stimulation of the AT1R could also increase cAMP, but the mechanism of action is not known. Conversely, G�i functions to antagonize cAMPproduction by activating SSTR or EP4 but could also regulate pathways mediated by Smo or FzR that control cytoskeleton remodeling, cell migration, and ionchannel activity (shown in Fig. 1). G�q activation by B2R, ETB, and AT1R could activate PLC-dependent signaling cascades, which could increase intracellularcalcium or activate DAG to promote PKC function. In some cases, B2R and AT1R could also promote ERK 1/2 signaling by cross talk with EGFR. ETB inhibitscAMP production through increased production of PGE2, which is mediated by PKC. 2) Vascular cells. In smooth muscle cells, G�q activation by the AT1Ror ETA/ETB could promote vasoconstriction, which could be counteracted by opposing AT2R-G�q vasodilation signaling pathways in the same smooth musclecells. Alternatively, B2R or ETB stimulation of G�q signaling in endothelial cells or neighboring tubular epithelial cells could produce paracrine factors, suchas nitric oxide or prostaglandins, which could traverse to the smooth muscle cells and modulate tone. 3) Polarity and mitotic spindles. In some situations,G�i-subunits could be involved in the process of epithelial cell polarity and mitotic spindle orientation. There is evidence that accessory proteins from theactivator of G protein signaling family, namely AGS3 and AGS5/LGN, could be involved in this process. ACE, angiotensin converting enzyme; AC6, adenylatecyclase 6; AGS3, activator of G protein signaling 3; ANG I, angiotensin I; aPKC, atypical protein kinase C; AQP2, aquaporin 2; ANG II, angiotensin II; AT1R,angiotensin II receptor type 1; AT2R, angiotensin II receptor type 2; AVP, arginine vasopressin; A3AR, A3 adenosine receptor; B2R, bradykinin B2 receptor;CaMKII, calcium/calmodulin-dependent protein kinase II; CFTR, cystic fibrosis transmembrane conductance regulator; cGMP, cyclic guanosine monophosphate;COX1/2, cyclooxygenase 1/2; DAAM-1, dishevelled associated activator of morphogenesis 1; DAG, diacylglycerol; Dvl, disheveled; EGFR, epidermal growthfactor receptor; Epac, exchange protein directly activated by cAMP; EP2, prostaglandin E2 receptor 2; EP4, prostaglandin E2 receptor 4; ER, endoplasmicreticulum; Erk1/2, extracellular signal regulated kinase 1/2; ET-1, endothelin-1, ETA, endothelin type A receptor; ETB, endothelin type B receptor, FzR, frizzledreceptor; Gli2/3, glioma-associated oncogene 2/3; GSK-3, glycogen synthase kinase 3; IP3, inositol triphosphate; JNK, c-jun N-terminal kinase; LGN,leucine-glycine-asparagine repeat protein; MEK, MAPK/ERK kinase; mInsc, mammalian homolog of Inscuteable; NHE, Na/H exchanger; NO, nitric oxide;NuMA, nuclear mitotic apparatus; PC1, polycystin-1; PC2, polycystin-2; PGE2, prostaglandin E2; PI3-K, phosphoinositide 3 kinase; PKA, protein kinase A;PKC, protein kinase C; PLA2, phospholipase A2; PLC, phospholipase C; PTCH, Patched; Rock, Rho kinase; SST, somatostatin; SSTR, somatostatin receptor;UT, urea transporter; SMO, Smoothened; V2R, vasopressin type 2 receptor.

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terms of the AVP system, the V1aR has been shown to activateCa2�-dependent pathways through G�q-subunits, and thischange in calcium modulates the V2R-dependent cAMP pro-duction in renal tubular epithelial cells. Moreover, the V1aRcould selectively reduce renal medullary blood flow by vaso-constriction of the renal vasculature. This would minimizesolute wash-out in the renal medulla and, thereby, maximizeurine concentration (43, 44). Since polydipsia has been ob-served as a side effect due to chronic V2R blockade in patientswith ADPKD (61, 171), there may be some potential benefit ifa partial antagonist to the V2R is employed to block cAMPproduction through the G�s pathway, which also has modestV1aR agonist ability to activate Ca2� pathways in both thetubules and blood vessels. However, there are caveats to thisproposed logic, since the vascular sensitivity to AVP in PKDkidneys remains largely unknown and selective V1aR stimu-lation has been shown to promote persistent elevations insystemic blood pressure (28, 82) or lead to inappropriateactivation of procystic hormones, such as PGE2 (see nextsection). It is also important to note that intracellular calciumlevels in PKD epithelial cells are lower than in noncysticepithelial cells, so the counterregulatory systems that would beinvolved in reducing cAMP through V1aR are likely inferior tothe AVP/V2R/G�s axis. To date, however, the role of thisV1aR pathway in PKD remains to be investigated, and the

primary mode of action by AVP is through the actions of theV2R.

Prostaglandin E2 receptors. Prostaglandin E2 (PGE2) hasbeen implicated as a causative factor in cystic disease by anumber of observations, including its accumulated levels incyst fluid, ability to promote cAMP production and chloridesecretion, and induction of cyst growth in Madin-Darby caninekidney (MDCK) epithelial cells (199). PGE2 exerts its biolog-ical effects by binding to one of four distinct GPCR subtypesknown as the E-prostanoid receptors (EP1-4) (201), but theprimary receptors are likely EP4 (40, 95) and possibly EP2(41). PGE2 binding to EP4 activates G�s-subunits to inducecAMP production in murine IMCD3 and also PC-1-deficientcollecting duct cell lines (40, 95). There is also evidence inhuman cystic epithelial cells that EP2 may also play a role inthe cAMP response (41). In some cells, PGE2 could alsoactivate G�i-subunits to inhibit adenylyl cyclase through EP4stimulation (201). Alternatively, there is evidence that PGE2synthesis could be positively regulated by AVP through theV1aR in either the interstitial medullary cells or cortical col-lecting ducts (15), and this could synergize the production ofcAMP. These latter findings may partially explain why themagnitude of cAMP induction by PGE2 in cultured cells fromADPKD and ARPKD cysts is markedly less robust comparedwith AVP (8). Consistent with other hormones activatingcAMP/PKA in cystic epithelial cells, PGE2 activates the con-ventional B-Raf/MEK/ERK1/2 pathway. In addition, PGE2could mediate cAMP-dependent activation of membrane-asso-ciated exchange protein 1/2 (Epac1/2) (201). Although the roleof Epac1/2 in the cystic kidney needs further study, there isevidence that the cAMP-Epac pathway promotes cyst growthby hyperproliferation in liver cells (6).

These studies demonstrate that there may be a synergisticrole to synthesize PGE2 by other procystic hormones and thatPGE2 could subsequently promote increased cAMP-dependentprocystic signaling through the activation of distinct G proteinsubunits.

Adrenergic receptors. In general, adrenergic receptor acti-vation controls long-term blood pressure through a number ofdistinct modes, including fluid and electrolyte balance, renalblood flow distribution, and renin production (27, 35). Hyper-activity of the sympathetic nervous system has been shown tobe associated with high blood pressure in humans withADPKD (79). Selective loss of renal nerve activity reduces thekidney-to-body weight ratio, an index of cystic disease pro-gression, in the Han:Sprague-Dawley (SPRD) rat, a nonor-thologous model of ADPKD (45). The adrenergic receptorsubtypes associated with the sympathetic nervous system arecategorized as � and � and are distributed in various vascularand tubular epithelial cells. Blockade of the �1/�2-receptorsubtypes, which are involved in G�s-activation of adenylylcyclase, does not improve renal function as measured bymicroalbuminuria and glomerular filtration rate in ADPKDpatients (180, 206). Renal tubular epithelial cell expression of�1- and �2-receptor subtypes are localized throughout thenephron from the proximal tubules to the collecting ducts (161,196). Incubation with isoproterenol, a nonselective �1/�2-receptor agonist, could induce cAMP production in microdis-sected distal tubules and cortical collecting ducts (111, 149)and correlate with mitogenesis in renal tubular epithelial cells(193). The adrenergic �2-receptor has been shown to inhibit

Fig. 3. Overview of cAMP regulation by distinct heterotrimeric G proteinsubunits in renal tubular epithelial cells. Intracellular levels of cAMP havebeen shown to be regulated by an integration of numerous GPCR pathways inrenal tubular epithelial cells. Adenylyl cyclase type 6 (AC6) could be eitherdirectly activated by GPCR/G�s or inhibited by GPCR/G�i. In addition,GPCR/G�q signaling could stimulate intracellular calcium, which could beinvolved in reducing AC6 activity or accelerating cAMP catalysis by phos-phodiesterase (PDE) activation. Upon changes in cAMP levels, protein kinaseA (PKA)-dependent phosphorylation of Raf kinase has been shown to beinvolved in controlling distinct cellular functions, particularly cell prolifera-tion. In normal renal tubular epithelial cells, c-Raf (Raf-1) kinase activatesMEK/ERK1/2 signaling to prevent or reduce the activation of cell proliferativepathways. In cystic PKD epithelial cells, PKA has been shown to activateB-Raf kinase to promote increased cell proliferation, increased fluid secretion,and reduced tubulogenesis. V2R, vasopressin V2 receptor; EP2/EP4, E-prostanoid2 or 4 receptor; A3AR, adenosine A3 receptor; SSTR, somatostatin receptor; B2R,bradykinin type 2 receptor; ETB, endothelin type B receptor; �, activate; �,inhibit.

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adenylyl cyclase through G�i-activation, but its role has notbeen evaluated in PKD.

At this time, the role of the adrenergic receptor system as itrelates to the pathogenesis of cyst progression in PKD remainsto be fully determined. In fact, there is recent evidence thatdeficits in renal function in ADPKD patients may be precededby extrarenal pathologies to the heart, since the �-adrenergicsystem has been shown to be perturbed in terms of its ability toexpress calcium handling proteins (84). Further studies areneeded to better understand the importance of the adrenergicreceptors in PKD.

Somatostatin receptors. The somatostatin receptors (SSTR)represent another set of GPCR that could activate the inhibitoryG�i-subunits to reduce adenylyl cyclase activity and decreasecAMP production. In mice, rat and human kidneys, there arefive known types of SSTR, and their distribution could belocalized to each segment of the nephron, although the collect-ing duct is the most conserved segment to express SSTR2 (7,12). Under normal conditions, somatostatin could inhibit renaltubular phosphate transports in both humans and rats (136, 177,182) and partially antagonize the vasopressin V2 receptor-mediated changes in water transport by reducing cAMP pro-duction (191). In the cystic epithelia of PKD patients, thispathway is believed to be involved in the dysregulation oftubular epithelial cell function and promotes the transformationto a cystic phenotype. There is evidence that SSTR3 is asso-ciated with the cilia (71), but for therapeutic purposes, shortpeptide somatostatin analogs with longer circulating half-liveshave been developed, such as octreotide, lanreotide, and pasir-eotide, which could have their own distinct SSTR-bindingaffinity (58, 103). Currently, these SSTR agonists are widelyevaluated in clinical trials as potential anticystic agents toattenuate cystic disease progression in PKD (21, 67). Althoughthere have been some promising results to slow cystic diseasein the liver, the kidney has exhibited somewhat varied re-sponses to SSTR agonists (20, 21, 66, 67, 181). To furtherclarify the role of the SSTR system as an anticystic therapy forthe kidney, there is an on-going clinical trial to focus on theeffects of lanreotide on improving renal function in ADPKDpatients (104).

However, the differences in the biological effect of soma-tostatin agonists in the liver vs. kidney need further investiga-tion but may be associated with the differential abundance andpattern of expression of SSTR and its associated adenylylcyclase partners within the nephron. In cystic cholangiocytes,SSTR1 and SSTR2 expression is markedly reduced in cysticcholangiocytes compared with normal cells and that chronicstimulation with either octreotide or pasireotide results in acompensatory increase in the receptor levels for SSTR1 andSSTR2 similar to levels observed in normal cells (103). Similartypes of studies will need to be performed in PKD kidneys toconfirm whether the use of SSTR agonists could augment themagnitude of adenylyl cyclase inhibition by upregulating re-ceptor expression and/or localization to slow renal cystic dis-ease progression.

Adenosine receptors. Adenosine is a nucleoside that hasbeen shown to play an important role in the control of renalfunction by balancing blood flow with the constant energydemands in the kidney (156, 179). Catabolism of cAMP, ATP,and AMP could lead to conversion into adenosine, which couldactivate one of four distinct GPCR, known as adenosine

receptor A1 (A1AR), A2AAR, A2BAR, and A3AR (179). InPC-1-deficient cells, a selective induction of the A3AR relativeto the other AR isoforms has been detected (1). A3AR reducescAMP production by activating G�i-subunits and coincideswith reduced cystic epithelial cell numbers following stimula-tion of the A3AR using a selective agonist (1). Similar to otherpathways that control adenylyl cyclase, phosphorylation ofERK1/2 is decreased in the presence of an A3AR agonist,which suggests that PKA is involved in stimulating Raf/MEK/ERK signaling. Alternatively, A3AR may stimulate phospho-lipase C� (PLC) to reduce ERK1/2 activation via PI3K/Aktsignaling (106), which could also involve either G�q or pos-sibly unbound G��-dimers. Further studies will be required todetermine the relative importance of adenosine on cAMPproduction compared with the other adenylyl cyclase-regulat-ing hormonal systems.

In all, these studies demonstrate that multiple hormonalsystems integrate into the regulation of the cAMP system andthat the use of a single inhibitor to control cAMP productionwould be compromised by the multifactorial input of numeroushormones.

G�q and Calcium Regulation

Although the list of interacting signaling pathways expandsfor each G protein subunit, G�q-subunits are classically knownto be directly involved in regulating intracellular calcium levelsby activating PLC (147). The pathways involved in G�q-signaling are illustrated in Fig. 2. PLC catabolized phosphati-dylinositol-4,5-bisphosphate (PIP2) into two products, inositoltriphosphate (IP3) and diacylglycerol (DAG). IP3 traverses tothe sarcoplasmic reticulum to stimulate calcium channel activ-ity and promote the movement of the calcium into the cyto-plasm. DAG activates protein kinase C to negatively affect theactivation of G�i-subunits. Unconventionally, G�q could alsopromote signaling cross talk between GPCR and the phosphor-ylation of growth factor receptors to activate downstreamsignaling pathways (29). In this section, we focus upon the roleof G�q in the renin-angiotensin, kallikrein-kinin, endothelin,and EGFR systems.

Renin-angiotensin system. The renin-angiotensin system(RAS) remains one of the best-characterized hormonal systemsin the field of cardio-renal biology, and its role in bloodpressure regulation and renal cystic disease progression inPKD continues to be an area of intense investigation (88).

The effector hormone angiotensin II (ANG II) is generatedby a proteolytic cascade initiated by the cleavage of theprecursor protein, angiotensinogen, which is an �2-globulinformed primarily in the liver and, to a lesser extent, in thekidney. Angiotensinogen is subsequently catabolized by theendopeptidase renin to form angiotensin I (ANG I). Renin issynthesized and released into the circulation from the juxta-glomerular apparatus in the kidney and can be regulated in partby the adrenergic �1/G�s/cAMP pathway (35). ANG I ispredominantly converted to ANG II by angiotensin-convertingenzyme (ACE), but chymase can also perform this function toa lesser degree. At this point, ANG II exhibits multifunctionaleffects due to its ability to interact with two distinct GPCRsubtypes, AT1 (AT1R) and AT2 (AT2R), which activate adiverse set of G�-subunits throughout the kidney, including thevasculature, tubular epithelial cells of the nephron, and inter-

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stitial cells (96, 97, 108). In fact, all of the other components ofRAS, including angiotensinogen, renin, ACE, and ANG II, areproduced in the kidney (96, 97). Moreover, there is evidence inpolycystic kidney disease (PCK) rat kidneys and biopsiedhuman ADPKD kidneys that various components of the RAS,particularly renin, are elevated compared with their noncysticcounterparts (50, 51, 172).

In PKD, investigators studying the biological functions ofANG II have focused almost entirely upon elucidating themechanisms involved in blood pressure regulation and itsimpact on renal cyst progression. ANG II has potent vasocon-strictor effects in the systemic circulation via the AT1R/G�q/11 pathways (69, 108). In the kidney, ANG II could alsopromote Na�/H� exchange to enhance reabsorption of sodiumand water by activating apical AT1R in the proximal tubules(69). Since the etiology associated with hypertension in hu-mans with PKD remains largely undetermined (75, 88, 96), theprimary treatment modality has been to administer inhibitors toeither angiotensin converting enzymes (ACEI) or antagoniststo angiotensin receptors (ARB), particularly the AT1R (23).Unfortunately, the recently completed HALT-PKD trial wasunable to prove definitively that cystic disease progression wasslowed in either early (150) and late-stage (170) ADPKDpatients following treatment of either ACEI (Lisinopril) aloneor in combination with an ARB (telmistartan). Similarly, ro-dent models of PKD have shown only modest to little efficacyon cystic disease progression following blockade of the RASwith Lisinopril, even with reduced blood pressures (70, 143).Interestingly, a selective reduction in angiotensinogen levelsusing antisense oligonucleotide technology did markedly at-tenuate the increase in cyst area within the Pkd1 null kidneyswith only a mild reduction in systolic blood pressure comparedwith the effects of Lisinopril (143). From these studies, itappears the RAS may play a direct mitogenic role in tubularepithelial cells, but additional studies are needed to dissect thedirect ANG II-mediated tubular effects from those changesattributed to physical factors, such as blood pressure and renalblood and tubular luminal flow. These future studies will likelyrequire the evaluation of subpressor doses of ANG II orservo-controlled renal perfusion pressure in the presence ofANG II in rodent models of PKD to determine whether renalcystic disease progression can be modulated by RAS.

Regardless, there are other recent studies that have describedan effect by the RAS on tubular epithelial cells with defectivepolycystin or ciliary proteins. In human ADPKD cystic epithe-lial cells, defective PC-1 function negatively affected the shearstress-increased transient response to cytoplasmic Ca2� con-centration. However, the effect of ANG II was essentiallyretained at near normal Ca2� responses as control normalkidney cells (194), which would suggest that the receptorlocalization and signaling pathways associated with the ANGII/AT1R axis is essentially intact during cystic disease progres-sion. In cilia-deficient collecting duct epithelial cells, ANG IIstimulation of the apical AT1R was also able to increase cAMPproduction significantly, similar in magnitude as the costimu-lation of both apical and basolateral AT1R activation (142).Previous studies focusing on urine-concentrating mechanismshave observed that there is a cooperative effect on cAMPproduction between ANG II and AVP on their respectiveAT1R and V2R, respectively (89, 157). A genetic loss of theAT1R in collecting duct reduces the maximal urine-concen-

trating capacity through decreased production of AQP2 waterchannels (157) and insertion of AQP2 into the apical mem-brane (89). To date, it is unclear how ANG II/AT1R activatesa calcium-dependent pathway to increase cAMP production,but it may involve the activation of alternate subtypes ofadenylyl cyclase, such as AC3 and AC5. However, neither ofthose AC subtypes in the collecting ducts affects urine con-centration under basal conditions (78, 138). Alternatively,AT1R may also activate other downstream signaling pathwaysby interacting with G�12/�13 (48, 141, 162, 178) or evendirectly stimulating the Raf/MEK/ERK1/2 pathway (169, 185).It may be possible that the AT2R, which is not well describedin PKD, may need to be evaluated before we fully comprehendthe importance of AT1R and its associated heterotrimeric Gprotein signaling in PKD. The AT2R has been shown tooppose the AT1R-mediated vasoconstrictor effects exerted bypromoting the production of bradykinin and exaggerate thevasodilator response of smooth muscle cells (153). Addition-ally, AT2R could modulate other AT1R functions by regulat-ing sodium excretion (153), cell growth, and anti-inflammatorypathways (33, 34).

These studies clearly demonstrate that the RAS could acti-vate a diverse signaling network, which interacts with a num-ber of other hormonal systems to dysregulate blood pressureand potentially regulate a network of signaling pathways thatcould lead to changes in tubular epithelial cell architecture andfunction. Further studies are needed to isolate systematicallyeach potential signaling partner and identify how they alter theexpression profile, localization, and functional effects of bothAT1R and AT2R in renal cells during the cystogenic process.

Kallikrein-kinin system. Under normal conditions, the pri-mary functions of bradykinin are involved in regulating vas-cular tone and sodium and water balance in the kidney (72).The biological role of the kallikrein-kinin system (KKS) needsfurther exploration, especially since the catabolism of activebradykinin is regulated by ACE (also known as kininase II),which is the same enzyme involved in the production of ANGII (72). The KKS in PKD has not aroused much interest, sinceone study found was no detectable change in the KKS (16),although this finding was observed only in a small group ofhuman ADPKD patients. However, in the heterozygous Han:SPRD rats, a nonorthologous model of ADPKD, urinary kal-likrein and bradykinin levels were actually detected at higherlevels compared with age-matched wild-type controls (16, 17).

Bradykinin elicits its biological function through the activa-tion of one or both receptor subtypes, B1 (B1R) and B2 (B2R)(110). Both receptor subtypes have been identified in collectingduct epithelial cells in embryonic (39) and postnatal Oak Ridgepolycystic kidney (Orpk) mouse kidneys (18). Functionally,blockade of the B2R reduces proteinuria and albuminuria in theHan:SPRD rat (17), similar to the findings observed with ACEinhibition (17, 37, 38, 75). In mice, the loss of the B2R did notlead to abnormal renal architecture or function, but high saltintake during the period of kidney development led to tubulardilation. In some cases, the formation of cystic cells wasdescribed in the kidneys (39). The signaling pathways per-turbed in KKS during cyst formation remain largely unknown.Generally, the B2R interacts with G�q-subunits to promotePLC signaling and increase intracellular Ca2�, which couldreduce adenylyl cyclase activity or increase production of nitricoxide/cGMP second messengers to reduce vascular tone (110).

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Alternatively, the B2R could be involved in the control ofproliferation in collecting duct cells through the transactivationof the EGFR/ERK1/2 signaling axis by G�q-subunits (112).

Although there is a paucity of data regarding the dysregu-lation of the KKS in PKD, further exploration using mice thatare genetically deficient of bradykinin receptor subtypes wouldbe essential to obtain an unequivocal answer to the question ofwhether the KKS is a fundamentally important hormone thatmodulates cystic signaling pathways.

Endothelin receptors. Endothelin (ET)-1 exerts potent vaso-constriction in the systemic and renal circulation and promotesnatriuresis and diuresis by the kidneys (30). Over the years,evidence has accumulated that endothelin, specifically ET-1,plays a role in blood pressure regulation and cystic diseaseprogression in PKD (63–65, 107, 113, 117, 123, 183). Inhypertensive ADPKD patients, plasma ET-1 levels are ele-vated compared with healthy patients (107, 183), and endothe-lin has been detected in cyst fluid (113). Moreover, renal ET-1mRNA (117) and protein content (64, 65) has been detected ata higher level in rodent models of ADPKD, and ET-1 produc-tion is localized to renal cystic epithelial cells in mice, rats, andhumans (63, 65, 123) and in human vascular smooth musclecells (123). Intriguingly, the transgenic overexpression of ET-1in mice results in the formation of cysts in the kidney (63).

The biological actions by ET-1 are elicited by its ability tobind and activate distinct receptor subtypes, ETA and ETB (4,145). ETA mRNA and protein are localized to vascular smoothmuscle cells in renal blood vessels and glomeruli (167, 188)and protein expression in the distal tubules and collecting ductcells (188). ETB is expressed in the endothelial cell part of theblood vessels (188) and epithelial cells in the proximal tubule(188) and inner medullary collecting ducts (167, 188). Therenal ETA and ETB mRNA (117), protein levels (123), orligand binding (64) has been shown to be either higher or lowerdepending upon the PKD species being investigated. Stimula-tion of ETA and ETB could activate a number of G protein�-subunit partners, including G�q, G�12/13, and G�i, depend-ing on the cell type and the context of its activation (30). Aspart of the global signaling network in Fig. 2, the mostcommon ETA/ETB signaling is illustrated in epithelial andvascular cells. More descriptive signaling pathways for renalETA/ETB can be found elsewhere (80). Functionally, receptorblockade of the endothelin receptors to attenuate cystic diseaseprogression has resulted in conflicting results. Pharmacologicalblockade of ETB increased renal cAMP levels, which wasassociated with accelerated cystic disease progression (22). Inaddition, ETB blockade enhanced urine concentration, whichsuggested that there may be an integration of signaling path-ways with the V2R/G�s-axis. This observation may be con-sistent with an earlier study that showed ETB inhibited cAMPproduction by a G�i-dependent mechanism, possibly throughincreased production of PGE2 (81). Interestingly, concomitantblockade of ETA receptors could prevent the pathogenic ef-fects mediated by the ETB receptor (22). Conversely, receptorantagonism of the ETA, and not ETB, was instrumental inexaggerating tubular epithelial cell proliferation and cysticdisease progression in Han:SPRD (cy/�) rats. Similar to themouse study, coantagonism of ETA and ETB receptors atten-uated the effects by ET-1 on cystic disease progression (62).These somewhat contradictory results have suggested that acritical balance between ETA and ETB function may be

required to maintain normal kidney structure and function.However, further development in endothelin receptor blockersand also their use in orthologous models of PKD is needed toclarify whether ETA or ETB is the crucial receptor that isinvolved in the cystic disease process.

Tyrosine kinase receptors. In PKD, growth factors, includ-ing epidermal growth factor (EGF), are well established tointeract with single transmembrane-spanning tyrosine kinasereceptors, which initiate a process in which homo- or het-erodimerization occurs leading to the recruitment of a numberof adaptor proteins (13). This results in the subsequent activa-tion of downstream proliferative signaling pathways, includingthe canonical Raf/MEK/ERK1/2 pathway (164). In this signal-ing paradigm, EGFR functions as a downstream signalingeffector system, which could be transactivated by heterotri-meric G protein subunits, namely G�q-subunits (29). As de-scribed earlier, bradykinin and ANG II are bound to theirrespective GPCR to promote EGFR phosphorylation (112),and this may be the mode of action mediated by G�q. Itremains to be determined whether the cross talk mediated byG�q between various GPCR with the EGFR system occurswith basal membrane receptors or the receptors that are mis-targeted to the apical membrane, which are known to have aprocystic role in PKD (163, 165). In the end, ligand-activatedGPCR with the EGFR system likely provides additional pleio-tropic signal diversity within the cell to further complicate thetherapeutic development to treat PKD.

EMERGING AREAS OF HETEROTRIMERIC G PROTEINSIGNALING

In this section, we will describe GPCR-dependent and -in-dependent signaling pathways that have been implicated morerecently in PKD (Fig. 2). At present, the role of the heterotri-meric G proteins in these pathways has not been fully de-scribed other than in nonrenal systems, which provide potentialmechanisms of action that may be relevant in the pathogenesisof PKD.

Hedgehog Signaling

Recent evidence has suggested that the Hedgehog (Hh)signaling pathway may function through the regulation of Gprotein signaling and control cystic disease progression (174,175). Multiple components of the Hh system, includingPatched (Ptch), the putative receptor for Hh ligands, are local-ized to the cilia (114). In the absence of Hh, ciliary Ptch isinactivated. Upon interaction with Hh ligands, Ptch exits thecilia and removes its repressive effects on Smoothened (Smo),a seven-transmembrane GPCR associated with G�i-subunits(152). Smo facilitates the transmission of signaling effectsinitiated by Hh by accumulating in the cilia. This activates aseries of signaling events converting full-length glioblastoma(Gli) transcription factors, Gli2 and Gli3, from transcriptionalrepressors to transcriptional activators for cellular proliferation(192).

In multiple rodent models of PKD, including a Pkd1 mutantmouse, aberrantly high expression of all three Gli subtypes hasbeen detected (175). Blockade of Hh signaling reduces cysticdisease progression, but the noncanonical role of Smo-G�i-signaling has not been studied. In some invertebrate andmammalian cells, the activation of Gli2/3 may (56, 122, 139)

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or may not (100) be dependent upon the availability of G�i-dependent signaling. There is evidence that Smo can activateeffector targets normally associated with G�i activation, in-cluding PI3-K dependent cell migration (133) and inhibition ofadenylyl cyclase to reduce cAMP levels (152). However,blockade of Hh signaling did not affect the activity of CREB,which is a marker for cAMP production, in explanted cystickidneys, suggesting that the inhibition of G�i signaling maynot be involved (175). Further studies will be required toconfirm whether trimeric G protein signaling is a crucial playerin Hh signaling in orthologous model of PKD. These findingswould suggest that Hh signaling may provide a relationshipbetween ciliary dysfunction with cyst formation.

Wingless/Int1 and Frizzled Receptor

Frizzled (Fz) receptors, which have the structure-functionrelationship similar to a GPCR, are an integral component forthe wingless/int1 (Wnt) signaling pathway (36). At present, 10different Fz receptors have been identified to bind and processthe signaling of various Wnt ligands into the cell, and theirgeneral biological activity has been reviewed in greater detailelsewhere (47, 74). In brief, Wnt signaling is generally cate-gorized as either �-catenin dependent (canonical) or �-cateninindependent (noncanonical). In canonical Wnt signaling,�-catenin is a major signaling effector, which accumulates andinduces transcriptional activation of Wnt target genes. In thenoncanonical pathway, Wnt/Fz signaling could stimulate oneof two pathways, which are involved in calcium signaling orplanar cell polarity pathways (36). The role of heterotrimeric Gproteins in the Wnt signaling pathways has not been welldescribed, particularly in the context of PKD, but there is anaccumulating body of evidence in nonrenal cells that bothcanonical and noncanonical Wnt signaling regulates theactivation of multiple classes of G�-subunits, includingG�i, G�q, G�s, and G�12 (93, 120, 154), by interactingwith Fz receptors and other Wnt signaling components,disshevelled and axin (2, 94, 158). These G protein-depen-dent pathways are subsequently involved in various path-ways associated with proliferation, cytoskeletal changes,and cell motility (36).

At present, there are an increasing number of studies dem-onstrating perturbed regulation in Wnt signaling pathways inrenal cystic disease (47, 73, 74, 184). Loss of Wnt9b has beenshown to disrupt planar cell polarity to promote renal cystformation through noncanonical signaling (73), whereas a lossof aquaporin-1 expression accelerated cystic disease progres-sion in a mouse model of ADPKD by promoting canonicalWnt/�-catenin signaling (184). However, the role of Wntsignaling to regulate cystogenesis is not universal (160) andmay be attributed to the use of different genetic strains in thesestudies. Alternatively, there is a plethora of Wnt ligands thatcould interact with numerous types of Fz receptors, so thepotential network of diverse intertwining signaling branchescould be quite expansive. Therefore, it will be extremelychallenging to unravel, particularly if one or more subtypes ofheterotrimeric G protein complexes, which have yet to beidentified, which are involved in the process of the variousextrinsic cues.

G�i-Subunits in Mitotic Spindle Regulation

Proper orientation of mitotic spindles is a central mechanismthat enables tubular epithelial cells to maintain their comple-ment of polarity proteins and complete a round of symmetriccell division (10). Oriented cell division involves tight regula-tion of mitotic spindle alignment using protein complexescontrolling either apico-basal polarity or planar cell polarity.Defective control of the mitotic spindles was believed to be acausative factor in the initiation and progression of cystic kidneydisease, but the findings are not universal (42, 73, 101, 121, 131).Moreover, the signaling pathways involved in the misalignmentof the mitotic spindles are not well established, even though it wasbelieved to be attributed to dysfunctional planar cell polaritypathway. There are, however, compelling data in MDCK cellsthat alterations in the localization and composition of apico-basolateral polarity protein complexes could be a critical factor inthe orientation of the mitotic spindles (207). In tubular epithelialcells, the alignment of the mitotic spindles is perpendicular to theapico-basal direction (207). To achieve this proper alignmentduring mitosis, it is essential for the formation of a core tripartitecomplex composed of: 1) G�i-subunits of heterotrimeric inhibi-tory G proteins; 2) Leu-Gly-Asn (LGN), a cytosolic scaffoldprotein containing GoLoco/G protein regulatory (GPR) motifs;and 3) nuclear mitotic apparatus (NuMA), a nuclear coiled coilmicrotubule-binding protein. During mitosis, G�i-subunits arelocalized to the basolateral cortex, and LGN captures the inactiveforms of G�i-GDP by direct protein interaction at the COOH-terminal GoLoco/GPR motifs in LGN. At this point, nucleardissolution allows for NuMA translocation to the lateral cortex bybinding to the NH2-terminal TPR domains in LGN. Subsequently,dynein is recruited and accumulates at the cell cortex by bindingto NuMA, which promotes spindle elongation. LGN plays acentral role in maintaining proper epithelial cell architecture fol-lowing cell division. Loss of LGN expression or a lack of lateralcortex targeting of LGN leads to increased epithelial cells with anabnormal number of lumens that exhibit aberrant tilting of themitotic spindles in the apico-basal direction (207).

More recent studies demonstrate that LGN could be a pivotalswitch between the apico-basal and planar cell polarity com-plexes to control the orientation of the mitotic spindles (9,140). LGN has the capability to shift its binding affinity fromNuMA in the apico-basal complex to Disks large homolog 1(Dlg1), which leads to the regulation of the mitotic spindle inthe PCP direction. From these studies, it would be difficult toconclude which pathways would be disrupted during thechanges in morphogenetic programming in the normal tubularepithelial cell as it transitions to a cystic phenotype.

At present, the localization of LGN in normal and PKDkidneys is thought to be exclusively in distal tubular epithelialcells (91), but its biological role remains largely undefined invivo. However, a close homolog of LGN, known as activatorof G protein signaling 3 (AGS3), has a beneficial role inattenuating cystic disease progression (86). Similar to LGN,AGS3 is normally distributed throughout the cytoplasm but canlocalize to the spindle poles in the presence of G�i (115). Itremains to be determined whether the same complement ofpolarity proteins can interchangeably bind to AGS3 as they dofor LGN. However, it is clear that heterotrimeric G�i-subunitscould play a pivotal role in the absence of GPCR activation to

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control mitotic spindle orientation through their interactionwith accessory proteins, such as LGN/AGS3.

G��-Signaling

To date, G��-dimers, the native binding partner of G�-subunits, have not been as well characterized compared withG�-subunits as regulators of signal transduction pathways inPKD. G�� has been shown to have a diverse array of functionswithin the cell, including the ability to regulate ion channelactivity and PI3K activation (26, 155). As described earlier andillustrated in Fig. 1B, full-length PC1 bound to G�i/o-subunitsleads to the regulation of Ca2� and K� channels through aG��-dependent mechanism (31, 32). Similarly, AGS3 func-tions to bind G�i-subunits and release G��-dimers that in-crease the heteromeric PC1/PC2 ion channel activity (86). Inother studies, the availability of G��-dimers mediated byAGS3 could control mitotic spindle orientation (146). The poolof G��-dimers by AGS3 could be regulated near the cilia(200), but further studies are needed to confirm whether G��has a key role in the transformation of normal tubular intocystic epithelial cells. In vertebrate and mammalian cells, Wntligand activation of selective Fz, in particular Fz2 and Fz7,elicits induction of calcium transients through a G�i-dependentmechanism. Downstream signaling is controlled by the releaseof G��-dimers, resulting in the activation of PI3-K activity(154), protein kinase C translocation (151), calmodulin-depen-dent protein-kinase II (83), and also cdc42 activity (132).Further studies are needed to confirm the role of Wnt/Fz andthe activation of G��-dependent pathways in PKD, but there isemerging evidence that cdc42 knockout in mice can producefocal cysts in the kidney (25). As mentioned earlier, PC-1could also induce Akt function to prevent apoptotic signalingby interacting with active forms of G�i-subunits, which ulti-mately leads to an increased pool of unbound G��-subunits(14). The G�� would subsequently activate the p110� subunitof PI-3K (85, 166), which would result in the activation of Akt.

Although these downstream signaling pathways may seemdistinct, a common theme that has arisen appears to be that thesequestration of G�i-subunits promotes the hyperactivity ofG��. In this scenario, the G�i-subunit functions more like anentity that is designed to inactivate G��, rather than pursue itsown functional role to regulate other effector systems, such asinhibiting adenylyl cyclase.

PERSPECTIVES AND CONCLUSIONS

Heterotrimeric G protein signaling remains an integral partof the cell signaling network during the establishment ofnormal tubular epithelial cell homeostasis but also in theadaptive process by the cell in response to continual extrinsicstressors. To date, the main method to control G proteinactivation has been predominantly through the design anddevelopment of pharmacological drugs targeted to specificGPCR, including those abnormally active in PKD. This type ofcontrol mechanism is primarily used to regulate the activity ofthe G�-subunits. As described in this review, there is increaseddiversity by which heterotrimeric G protein signaling can occurwithin the cell, especially with the accumulating evidence ofnonconventional mechanisms to control G�-subunit function.Moreover, changes in G�� function within the cell could havea fundamentally important role in the control of tubular epi-

thelial cell biology by either complementing G�-directed sig-naling or, in some cases, being the major driving force depend-ing upon the specific G�-subunit to regulate downstreamsignaling cascades.

It has been nearly two decades since the initial observationthat PC-1 could bind to heterotrimeric G proteins (129), andnearly two-thirds of all humans diagnosed with ADPKD havemutations in the PKD1 gene. At present, there are �60 clini-cally identified variants in the germline and somatic cellswithin the G protein-binding domain (amino acids 4110–4183) (http://pkdb.mayo.edu/). There is yet a paucity of infor-mation regarding the importance of how mutant forms of PC-1can dysregulate tubular epithelial cell biology. Moreover, therole of heterotrimeric G protein binding and function in PKDhas not yet been fully resolved and likely requires additionalareas of investigation. First, remove the G protein-bindingdomain in PC-1 to assess whether this region is an essentialpart of the pathogenic process to either initiate or exacerbatetubular epithelial cell transformation into cystic cells. Second,determine the G�-subtype and its preferred state of activity(i.e., GTP- or GDP-bound) for efficient binding to PC-1. Third,define whether the G� or G�� is the primary signaling effectorfollowing interaction with PC-1 and whether there is a positiveor negative influence on signaling output depending upon theactivation of each individual G protein subunit. Fourth, deter-mine whether subcellular targeting of the COOH-terminalfragment of PC-1 to the nucleus can be influenced by G proteinbinding, which could modify transcriptional profiling. Last,identify the composition of the G��-complex within thecystic epithelial cell to determine whether selective interactionsoccur to drive signal processing toward cyst progression.

In all, heterotrimeric G proteins are clearly an important partof the cellular repertoire to control signal transduction withinthe tubular epithelial cell. However, the regulation by whichheterotrimeric G proteins are controlled continues to expandfar beyond the membrane-associated GPCRs. In fact, hetero-trimeric G proteins may function, albeit through unconven-tional modes of action, by altering its normal subcellularlocation (i.e., the plasma membrane) to other sites, such as themitotic spindle organizing center, through novel interactionswith cytoplasmic proteins. As the data continue to accumulateregarding the role of G proteins in PKD, the design anddevelopment of selective G� and/or G�� modulators willenable researchers to determine whether specific G proteinsubunits can be therapeutically targeted to attenuate the pro-cystic signaling pathways in vascular and tubular epithelialcells in the kidney.

ACKNOWLEDGMENTS

Current address for T. Hama: Dept. of Pediatrics, Wakayama MedicalUniv., Wakayama, Japan.

GRANTS

This work was supported by National Institute of Diabetes and Digestiveand Kidney Diseases Grant DK-90123 (to F. Park).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

T.H. and F.P. prepared figures; T.H. and F.P. drafted manuscript; T.H. andF.P. edited and revised manuscript; T.H. and F.P. approved final version ofmanuscript.

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