A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7
2009; Elsevier BV; Volume: 284; Issue: 49 Linguagem: Inglês
10.1074/jbc.m109.015149
ISSN1083-351X
AutoresKyoko Miyagi, Shigeki Kiyonaka, Kazunori Yamada, Takafumi Miki, Emiko Mori, Kenta Kato, Tomohiro Numata, Yuichi Sawaguchi, Takuro Numaga, Tōru Kimura, Yoshikatsu Kanai, Mitsuhiro Kawano, Minoru Wakamori, Hideki Nomura, Ichiro Koni, Masakazu Yamagishi, Yasuo Mori,
Tópico(s)Renal Diseases and Glomerulopathies
ResumoMutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD. Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD. IntroductionAutosomal dominant polycystic kidney disease (ADPKD) 3The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseasePC2polycystin-2PC1polycystin-1TRPtransient receptor potentialTRPCTRP canonicalERendoplasmic reticulummAChRmuscarinic acetylcholine receptorCChcarbacholPMplasma membraneWTwild typeEGFPenhanced green fluorescent proteinRIPA bufferradioimmune precipitation assay bufferGSTglutathione S-transferaseERKextracellular signal-regulated kinase. is a genetically heterogeneous Mendelian inheritance disorder affecting ∼1 in 1000 live births (1.Gabow P.A. N. Engl. J. Med. 1993; 329: 332-342Crossref PubMed Scopus (842) Google Scholar). ADPKD is characterized clinically by progressive formation and enlargement of renal cysts that demonstrate abnormalities in cell growth, fluid secretion, and extracellular matrix with other common complications (2.Harris P.C. Torres V.E. Annu. Rev. Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar). Linkage analyses have shown that either PKD1 or PKD2 loci are responsible for almost all ADPKD pedigrees. Nearly 85% of ADPKD pedigrees have been linked to PKD1, and ∼15% have been linked to PKD2 (2.Harris P.C. Torres V.E. Annu. Rev. Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar, 3.Nilius B. Owsianik G. Voets T. Peters J.A. Physiol. Rev. 2007; 87: 165-217Crossref PubMed Scopus (1118) Google Scholar). In PKD2 cases end-stage renal disease develops at a mean age of 10–15 years later than in PKD1 cases, although heterogeneity in clinical phenotype is seen among PKD2 mutations (4.Hateboer N. v Dijk M.A. Bogdanova N. Coto E. Saggar-Malik A.K. San Millan J.L. Torra R. Breuning M. Ravine D. Lancet. 1999; 353: 103-107Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar).The PKD2 gene, consisting of 15 exons, encodes a 968-amino acid integral transmembrane protein polycystin-2 (PC2, TRPP2) (5.Mochizuki T. Wu G. Hayashi T. Xenophontos S.L. Veldhuisen B. Saris J.J. Reynolds D.M. Cai Y. Gabow P.A. Pierides A. Kimberling W.J. Breuning M.H. Deltas C.C. Peters D.J. Somlo S. Science. 1996; 272: 1339-1342Crossref PubMed Scopus (1159) Google Scholar). Recent reports suggested that the C terminus of TRPP2 interacts with the coiled-coil domain of the PKD1 gene product, polycystin-1 (PC1) (6.Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (663) Google Scholar). TRPP2 has homology to polycystin-L (TRPP3) (7.Nomura H. Turco A.E. Pei Y. Kalaydjieva L. Schiavello T. Weremowicz S. Ji W. Morton C.C. Meisler M. Reeders S.T. Zhou J. J. Biol. Chem. 1998; 273: 25967-25973Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), another member of the polycystin superfamily that has been shown to conduct Ca2+-permeable cation currents (8.Chen X.Z. Vassilev P.M. Basora N. Peng J.B. Nomura H. Segal Y. Brown E.M. Reeders S.T. 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Invertebrate and vertebrate TRP homologues of the so-called "canonical" TRPC subfamily are characterized by activation induced upon stimulation of phospholipase C-coupled receptors (11.Abramowitz J. Birnbaumer L. FASEB J. 2009; 23: 297-328Crossref PubMed Scopus (263) Google Scholar). TRPC channels have been originally proposed as store-operated channels activated by Ca2+ depletion of stores, whereas closely related TRPC homologues, TRPC3, TRPC6, and TRPC7, showed activation sensitivity to the membrane-delimited action of diacylglycerol (12.Boulay G. Zhu X. Peyton M. Jiang M. Hurst R. Stefani E. Birnbaumer L. J. Biol. Chem. 1997; 272: 29672-29680Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 13.Hofmann T. Obukhov A.G. Schaefer M. Harteneck C. Gudermann T. Schultz G. Nature. 1999; 397: 259-263Crossref PubMed Scopus (1237) Google Scholar, 14.Okada T. Inoue R. Yamazaki K. Maeda A. Kurosaki T. Yamakuni T. Tanaka I. Shimizu S. Ikenaka K. Imoto K. Mori Y. J. Biol. 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Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (663) Google Scholar) suggested that PC1 and TRPP2 coassemble at the plasma membrane (PM) to produce a new channel and regulate renal tubular morphology and function. Nauli et al. (20.Nauli S.M. Alenghat F.J. Luo Y. Williams E. Vassilev P. Li X. Elia A.E. Lu W. Brown E.M. Quinn S.J. Ingber D.E. Zhou J. Nat. Genet. 2003; 33: 129-137Crossref PubMed Scopus (1602) Google Scholar) proposed that PC1 and TRPP2 form mechanosensitive channels in the primary cilium of kidney cells. Naturally occurring pathogenic mutations of TRPP2, which disrupt their associations through their C-terminal tails (30.Tsiokas L. Kim E. Arnould T. Sukhatme V.P. Walz G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6965-6970Crossref PubMed Scopus (417) Google Scholar), result in the defect in translocation of TRPP2 to PM. In contrast, a role of TRPP2 as a subunit of intracellular channels with the endoplasmic reticulum (ER)-targeting sequence, whose deletion induces trafficking to PM in pathogenic PKD2 mutants, has been suggested (26.Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (555) Google Scholar, 27.Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Another possibility is that TRPP2 functions appropriately both as Ca2+ release channels in ER and, under certain defined conditions, as PM Ca2+ entry channels (31.Vassilev P.M. Guo L. Chen X.Z. Segal Y. Peng J.B. Basora N. Babakhanlou H. Cruger G. Kanazirska M. Ye Cp. Brown E.M. Hediger M.A. Zhou J. Biochem. Biophys. Res. Commun. 2001; 282: 341-350Crossref PubMed Scopus (199) Google Scholar). However, it is still unclear how the C terminus-truncated TRPP2 mutant proteins behave after being mistranslocated from ER to the PM independently of the interaction with PC1. Thus, the physiological function of normal and pathogenic mutant TRPP2 as well as its operating subcellular site is yet to be established.In the present study we have identified a novel PKD2 gene mutation (2092delA) that generates a TRPP2 product (697fsX) with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 (Glu697) at the C terminus in a Japanese family. The recombinant TRPP2 mutant 697fsX was examined for subcellular localization as well as molecular and functional properties in HEK293 cells. 697fsX localized at the PM elicited a physical association with the TRPC3 or TRPC7 protein and muscarinic acetylcholine receptor (mAChR)-activated Ca2+ influx in HEK293 cells coexpressing TRPC3 or TRPC7, whereas wild-type (WT) TRPP2 localized at the ER significantly enhanced mAChR-activated Ca2+ release. In polarized kidney epithelial LLC-PK1 cells, confocal image analysis revealed codistribution of native TRPC3 with transfected 697fsX in the apical membrane but not with WT TRPP2 distributed in the basolateral membrane area. These observations suggest dual impacts of PKD2 mutations producing TRPP2 proteins deleted with the C-terminal tail in the pathogenesis of ADPKD.DISCUSSIONCoimmunoprecipitation, GST pulldown, and cross-linking experiments suggest that the C terminus-truncated TRPP2 mutant 697fsX undergoes physical association with TRPC3 and TRPC7. In electrophysiological recordings, coexpression of 697fsX but not that of WT TRPP2 caused a depolarizing shift of reversal potentials and an increased single-channel amplitude of mAChR-induced currents in TRPC3-expressing cells. These altered ion permeation properties of TRPC3-mediated cation currents after coexpression of 697fsX suggest that TRPC3 and 697fsX form heteromultimeric cation channels in which both TRPC3 and 697fsX function as pore-forming subunits, and pore-lining residues are different from those of homomultimeric TRPC3 channels. Consistent with this notion, HEK293 cells coexpressing 697fsX with WT TRPC3 and those with E632Q TRPC3 mutant showed single-channel currents with different amplitudes, excluding a possibility that TRPC3 proteins, which form channels independently of 697fsX proteins, support co-translocation of 697fsX via indirect mechanisms, for example, by sharing the same membrane microdomain.Previously, the TRPP2 C-terminal tail containing the coiled-coil domain 772–796 was shown to be responsible for the assembly of TRPP2 with PC1 using the C-terminal tail-truncated mutant R742X (6.Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (663) Google Scholar). Because the coiled-coil domain is also deleted from the TRPP2 sequence in the 697fsX mutant, 697fsX can be incapable of forming a complex with PC1. In LLC-PK1 cells, native TRPC3 proteins were colocalized with transfected 697fsX in the apical membrane area but not with WT TRPP2 distributed in the basolateral membrane and adjacent intracellular areas (Fig. 7). Therefore, in native tissues of ADPKD patients with 697fsX, a deficiency of PC1 interaction can lead to the formation of heteromultimeric TRPP2/TRPC3 channels with ion permeation properties different from those of TRPC3-containing channels in normal tissues. Interestingly, cation influx activity is increased when 697fsX is coexpressed with TRPC3. It is, therefore, possible that 697fsX enhances trafficking of TRPC3 channels through protein multimerization, as previously reported for TRPV4 associated with TRPP2 (47.Köttgen M. Buchholz B. Garcia-Gonzalez M.A. Kotsis F. Fu X. Doerken M. Boehlke C. Steffl D. Tauber R. Wegierski T. Nitschke R. Suzuki M. Kramer-Zucker A. Germino G.G. Watnick T. Prenen J. Nilius B. Kuehn E.W. Walz G. J. 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Genet. 2005; 14: 1587-1603Crossref PubMed Scopus (98) Google Scholar).TRPP2 abundantly expressed in ER membrane has been suggested to act as a Ca2+ release channel of intracellular stores (26.Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (555) Google Scholar) and to functionally interact with inositol 1,4,5-trisphosphate receptor-induced Ca2+ release (52.Li Y. Wright J.M. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. 2005; 280: 41298-41306Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). TRPP2 is also reported to form protein complexes with PC1 or TRPC1 to induce Ca2+ influx at the PM (6.Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (663) Google Scholar, 41.Tsiokas L. Arnould T. Zhu C. Kim E. Walz G. Sukhatme V.P. Proc. Natl. Acad. Sci. U.S.A. 1999; 96: 3934-3939Crossref PubMed Scopus (268) Google Scholar). However, because our data clearly indicate that overexpression of TRPP2 potentiates receptor-activated Ca2+ release but not Ca2+ influx in HEK293 cells, TRPP2 should mainly function as a Ca2+ release channel. In this study we detected endogenous expression of PC1, TRPP2, TRPC1, TRPC3, TRPC4, and TRPC6 in HEK293 cells (supplemental Fig. 2). Interestingly, our data suggest a higher expression level of TRPP2 compared with those of PC1 and TRPC1, further suggesting that endogenous PC1 and TRPC1 are fully associated with endogenous TRPP2 and that PC1 and TRPC1 are not freely accessible for recombinant TRPP2. With regard to the augmentation of Ca2+ influx in HEK293 cells recombinantly expressing 697fsX, endogenous TRPC3 but not TRPC7 was detected in HEK293 cells. Other TRPCs such as TRPC1, TRPC4, and TRPC6 are also endogenously expressed in HEK293 cells but fail to show enhancement of Ca2+ influx by 697fsX when recombinantly expressed (Fig. 5B). Therefore, endogenous TRPC3 may interact with 697fsX at the PM.The present study reveals that the TRPP2 C terminus-truncating mutations exert a dual pathogenic impact at different subcellular sites, the ER and PM, on receptor-induced [Ca2+]i mobilization. At the ER, the potentiation effect of WT TRPP2 on receptor-activated Ca2+ release from ER was abolished by the 697fsX mutation in HEK293 cells. This is consistent with the previous report of Cai et al. (27.Cai Y. Maeda Y. Cedzich A. Torres V.E. Wu G. Hayashi T. Mochizuki T. Park J.H. Witzgall R. Somlo S. J. Biol. Chem. 1999; 274: 28557-28565Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar), which suggested that the C terminus-truncated mutation (R742X) abolishes retention of TRPP2 at the ER membrane, where TRPP2 regulates Ca2+ release (26.Koulen P. Cai Y. Geng L. Maeda Y. Nishimura S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Cell Biol. 2002; 4: 191-197Crossref PubMed Scopus (555) Google Scholar). At the PM, physical and functional association of 697fsX TRPP2 proteins with TRPC3 or TRPC7 prominently enhanced receptor-activated Ca2+ entry into HEK293 cells. This finding clarifies the interaction target of C terminus-truncated TRPP2 proteins and their activation mechanism as ion channels, which were unresolved issues. Previous reports only described the disrupted assembly of TRPP2 with PC1 and consequent PM mistranslocation of TRPP2 by the R742X mutation (6.Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. Nature. 2000; 408: 990-994Crossref PubMed Scopus (663) Google Scholar) as well as the abnormal spontaneous [Ca2+]i-independent cation current via the R742X TRPP2 mutant (29.Chen X.Z. Segal Y. Basora N. Guo L. Peng J.B. Babakhanlou H. Vassilev P.M. Brown E.M. Hediger M.A. Zhou J. Biochem. Biophys. Res. Commun. 2001; 282: 1251-1256Crossref PubMed Scopus (66) Google Scholar). Furthermore, compared with the proposed loss of Ca2+ influx by ablation of the association between PC1 and TRPP2s, the enhanced Ca2+ influx via complexation with TRPC3 or TRPC7 proteins is a gain of function more likely consistent with the dominant phenotype of the PKD mutations. Thus, the obtained data may provide an important clue to address the discrepancy between seemingly conflicting hypotheses with regard to the subcellular sites of the pathogenic action of TRPP2 C terminus mutants (6.Hanaoka K. Qian F. Boletta A. Bhunia A.K. Piontek K. Tsiokas L. Sukhatme V.P. Guggino W.B. Germino G.G. 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Notably, during preparation of this manuscript, it has been reported that the heteromeric channels comprised of TRPP2 and TRPC1 are activated in response to receptor stimulation (66.Bai C.X. Giamarchi A. Rodat-Despoix L. Padilla F. Downs T. Tsiokas L. Delmas P. EMBO Rep. 2008; 9: 472-479Crossref PubMed Scopus (141) Google Scholar). It is, therefore, speculated that C-terminal truncations disrupt the PC1-TRPP2 assembly as well as the formation of heteromultimeric TRPC1/TRPP2 channels in the basolateral membrane (65.Roitbak T. Ward C.J. Harris P.C. Bacallao R. Ness S.A. Wandinger-Ness A. Mol. Biol. Cell. 2004; 15: 1334-1346Crossref PubMed Scopus (123) Google Scholar). The heteromultimerization may consequently induce mislocalization of TRPP2 proteins and the formation of heteromultimeric TRPC3/TRPP2 channels in the apical membrane, finally leading to transduction of pathogenic signals in ADPKD tissues. Taking into consideration that TRPC3 and TRPC7 are different from TRPC1 in molecular and functional properties (11.Abramowitz J. Birnbaumer L. FASEB J. 2009; 23: 297-328Crossref PubMed Scopus (263) Google Scholar), a conclusion based on TRPC1 should not be directly extrapolated to TRPC3 and TRPC7. This is consistent with an idea that TRPP2 mutants associated with TRPC3 and TRPC7 indeed play unique pathophysiological roles in signal transduction. We can raise a different possibility that unknown regulatory mechanisms, which suppress the action of the TRPP2 C terminus, may induce translocation to the apical membrane and subsequent heteromultimerization with WT TRPP2 and TRPC3 also in normal tissues under certain cellular conditions. In this scenario it is conceivable that the 697fsX mutant is constantly present at the apical membrane to transmit constitutively active aberrant signals in ADPKD tissues.At the cellular level ADPKD inherited in a dominant manner has been explained by a recessive mechanism, leading to the complete loss of function through somatic mutations in the normal PKD2 allele (the two-hit model). However, TRPP2 (PC2) is frequently observed in renal cy
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