Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain
Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca2+ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release mechanism, which only occurred in the presence of the TRPP2-IP3R interaction, and not via altered IP3R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca2+ microdomains necessary for initiating Ca2+-induced Ca2+ release. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease. Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca2+ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular Ca2+ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP3R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca2+ release in intact cells and IP3-induced Ca2+ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca2+ signal by a local Ca2+-induced Ca2+-release mechanism, which only occurred in the presence of the TRPP2-IP3R interaction, and not via altered IP3R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca2+ microdomains necessary for initiating Ca2+-induced Ca2+ release. The data strongly suggest that defects in this mechanism may account for the altered Ca2+ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease. IntroductionAutosomal dominant polycystic kidney disease (ADPKD) 4The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseaseACacidic clusterCICRCa2+-induced Ca2+ release[Ca2+]ERCa2+ concentration in the ER[Ca2+]cytcytosolic Ca2+ concentrationERendoplasmic reticulumIP3Rinositol 1,4,5-trisphosphate receptorIICRinositol 1,4,5-trisphosphate-induced Ca2+ releaseLBDligand-binding domainPKD1polycystin-1RyRryanodine receptorTGthapsigarginTRPP2polycystin-2aaamino acid(s)MOPS4-morpholinepropanesulfonic acidPipes1,4-piperazinediethanesulfonic acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolGSTglutathione S-transferaseTBSTris-buffered salineCTC-terminalNTN-terminalBAPTA1,2-bis(2-aminophenoxyl)ethane-N,N-N′,N′-tetraacetic acid. is an inherited human disorder that affects more than six million people worldwide and is the most common monogenic cause of kidney failure in humans (1Gabow P.A. Grantham J.J. Schrier R.W. Gottschalk C.W. Diseases of the Kidney. 6th Ed. Little Brown and Company, Boston, MA1997: 521-560Google Scholar). ADPKD results in end-stage renal disease in ∼50% of the affected individuals by the age of 60. ADPKD arises as a consequence of mutations of two genes PKD1 and PKD2, encoding integral membrane proteins polycystin-1 (PKD1, ∼460 kDa) and polycystin-2 (TRPP2, ∼110 kDa), respectively. Most mutations identified in affected families appear to truncate and (or) inactivate either of both proteins (2Mochizuki 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, 3Harris P.C. J. Am. Soc. Nephrol. 2009; 20: 1188-1198Crossref PubMed Scopus (49) Google Scholar, 4Wu G. Somlo S. Mol. Genet. Metab. 2000; 69: 1-15Crossref PubMed Scopus (101) Google Scholar, 5Torres V.E. Harris P.C. Kidney Int. 2009; 76: 149-168Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Mutations in PKD1 account for the vast majority (∼85%) of patients with ADPKD and are associated with a more severe clinical presentation and earlier onset of end-stage renal disease than the PKD2 phenotype (4Wu G. Somlo S. Mol. Genet. Metab. 2000; 69: 1-15Crossref PubMed Scopus (101) Google Scholar). However, in all other aspects, PKD1 and PKD2 mutations produce virtually indistinguishable disease manifestations, indicating that the two proteins might function in a common signaling pathway involved in maintaining the terminally differentiated state of renal epithelial cells.TRPP2 is a 968-amino acid (aa) protein with six predicted transmembrane domains and is highly conserved among multicellular organisms and widely expressed in various tissues (2Mochizuki 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). Structural analyses indicate that TRPP2 contains several functional domains in its C-terminal tail. There are two Ca2+-binding sites (aa 680–796) arranged in a typical and an atypical EF-hand motif, which could be involved in a Ca2+-mediated regulation of TRPP2 (6Schumann F. Hoffmeister H. Bader R. Schmidt M. Witzgall R. Kalbitzer H.R. J. Biol. Chem. 2009; 284: 24372-24383Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). An endoplasmic reticulum (ER) retention signal (aa 787–820) (7Cai 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) and a coiled-coil domain (aa 839–919), responsible for homo- and heterodimerization (8Celić A. Petri E.T. Demeler B. Ehrlich B.E. Boggon T.J. J. Biol. Chem. 2008; 283: 28305-28312Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 9Yu Y. Ulbrich M.H. Li M.H. Buraei Z. Chen X.Z. Ong A.C. Tong L. Isacoff E.Y. Yang J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11558-11563Crossref PubMed Scopus (142) Google Scholar), are also present. Recently, it was reported that this coiled-coil domain was responsible for formation of a TRPP2 trimer that interacts with PKD1 in the plasma membrane (9Yu Y. Ulbrich M.H. Li M.H. Buraei Z. Chen X.Z. Ong A.C. Tong L. Isacoff E.Y. Yang J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11558-11563Crossref PubMed Scopus (142) Google Scholar).There is a long-standing debate on the subcellular localization of TRPP2. TRPP2 has been detected (i) in the plasma membrane, where it is supposed to form a receptor-operated, non-selective cation channel (10Hanaoka 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), (ii) in the primary cilium, where it could act as a mechanosensitive channel, possibly in association with PKD1 (11Nauli 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 Google Scholar), L. T. E. G. Sukhatme V.P. Proc. Natl. Acad. Sci. U.S.A. 1999; PubMed Scopus Google Scholar, A. L. F. T. Tsiokas L. P. 2008; PubMed Scopus Google Scholar), (11Nauli 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 Google Scholar, M. B. F. X. M. R. T. R. M. A. Germino G.G. T. J. B. G. J. Biol. 2008; PubMed Scopus Google Scholar), in the where it is to function as an intracellular Ca2+-release channel P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google Scholar), also in and of cells in L. S. Ong PubMed Scopus Google Scholar, A. F. B. M. M. E. P. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). The of TRPP2 to subcellular is by (i) the protein (7Cai 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, L. Z. X. Cai Y. S. Somlo S. J. Sci. PubMed Scopus Google Scholar), (ii) with acidic cluster proteins and M. T. T. R. B. S. B. A. K. C.C. R. E. G. G. J. PubMed Scopus Google Scholar), and a of in the of TRPP2 M. T. T. R. B. S. B. A. K. C.C. R. E. G. G. J. PubMed Scopus Google Scholar, M. G. PubMed Scopus Google Scholar). However, the subcellular localization of TRPP2 is in the as by to and with proteins (7Cai 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, P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google between TRPP2 and the two intracellular Ca2+-release the receptor and the inositol 1,4,5-trisphosphate receptor (IP3R), has been and functional demonstrated that the N-terminal of TRPP2 is to the the C-terminal of TRPP2 only to it is in the function M. X. Somlo S. Ehrlich B.E. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). that TRPP2 interacts with the most its C-terminal Y. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Li Y. S. Qian F. Guggino W.B. J. Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated also that PKD1 with the IP3-induced Ca2+ release an of intracellular Ca2+ release that was to an of TRPP2 P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google Scholar, Y. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. G. Z. T. Qian L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. K. T. E. K. T. Y. T. T. Y. M. M. H. M. Y. J. Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar), the mechanism of was not the other and T. R. B. R. G. M. J. 2009; PubMed Scopus Google Scholar) that TRPP2 could also act as a channel in the the Ca2+ concentration in the which resulted in an and the of results that the mechanism which in and TRPP2 in Ca2+ signaling is not therefore a analysis of the molecular and functional between the IP3R and we identified a conserved positively charged cluster in the N-terminal domain of the IP3R and an acidic cluster the of the signal in the C-terminal tail of TRPP2 as for the interaction between both Moreover, in a of renal epithelial TRPP2−/− we a of both intracellular Ca2+ release in intact cells and in permeabilized cells TRPP2 analysis using pathological mutants of TRPP2 and that with the interaction between TRPP2 and the IP3R that the in both a functional TRPP2 channel and a interaction with the suggest that TRPP2 as a channel in the that in an intracellular signaling complex with the intracellular Ca2+ release a in the of of this was that TRPP2-IP3R signaling are responsible for the of The in this are: (i) the identification of residues responsible for the interaction, on TRPP2 as as on the which be to the (ii) the mechanism responsible for the Ca2+ which was to TRPP2 than to altered IP3R and that a association between TRPP2 and the IP3R to for a Ca2+ responsible for the of the that TRPP2 was not responsible for a Ca2+ the and the of via TRPP2 in a that an acidic cluster (aa in the C-terminal tail of TRPP2 and a positively charged cluster (aa in the of the in the of the IP3R for interaction. a functional consequence of this interaction, we that TRPP2 a Y. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) it was that the C-terminal of TRPP2 could the of the was on of the the in not for a of the IP3R as the of TRPP2 on in our where we to the Ca2+ release was most to of via TRPP2. was our that the of by TRPP2 was in using which the of the PubMed Scopus Google Scholar). The between Ca2+ is an to the of the local in a to Ca2+-release this by a local and the of the IP3R via a interaction with TRPP2 to be The was also our that a TRPP2, which the not this of Moreover, the was also by a competing for the An for the of a interaction between TRPP2 and the IP3R the that a as by not in an increased Ca2+ a where is and the which to a local that the TRPP2 channel as a experiments demonstrated regulation of the of the TRPP2 channel by Ca2+ Y. G. Z. T. Qian L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of TRPP2 the plasma membrane and the primary we suggest function for TRPP2 in the the of a signaling that a functional complex to by TRPP2. TRPP2 in an intracellular channel complex and intracellular Ca2+ signaling. has been in experiments that TRPP2 is a cation channel, a on P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google Scholar, Y. G. Z. T. Qian L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the via TRPP2 was reported to Y. G. Z. T. Qian L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the predicted the of the IP3R the PubMed Scopus Google Scholar, E. 20: Full Text Full Text PDF PubMed Scopus Google Scholar), a for Ca2+ may TRPP2 to local in on the of the EF-hand the of TRPP2 the that Ca2+ (6Schumann F. Hoffmeister H. Bader R. Schmidt M. Witzgall R. Kalbitzer H.R. J. Biol. Chem. 2009; 284: 24372-24383Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, A. Petri E.T. Demeler B. Ehrlich B.E. Boggon T.J. J. Biol. Chem. 2008; 283: 28305-28312Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, the between Ca2+ to the EF-hand of TRPP2 and its channel regulation has not been demonstrated and of of TRPP2 the of TRPP2 channel to the Y. G. Z. T. Qian L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). our results the with the acidic cluster of TRPP2 to the IP3R may be of the responsible for the in to Ca2+ Ca2+ signaling by TRPP2 is for various of TRPP2 results in increased in ADPKD and this is of the to formation V.E. Harris P.C. Kidney Int. 2009; 76: 149-168Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar, Ong A.C. 2008; PubMed Scopus Google Scholar). has been reported that ADPKD cells a to a of TRPP2 channel function T. S.J. J. Am. Soc. Nephrol. PubMed Scopus Google Scholar). of and genes as are also a common in the of ADPKD ADPKD Ca2+ in an and T. S. Grantham J.J. Kidney Int. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of the of Ca2+ in the regulation of via via it has been that in intracellular Ca2+ account for the in V.E. Harris P.C. Kidney Int. 2009; 76: 149-168Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). is to that the increased Ca2+ signaling in microdomains may be in by Ca2+ the strongly suggest that the form for only also is an that is in this might be a consequence than a cause of Recently, T. R. B. R. G. M. J. 2009; PubMed Scopus Google Scholar) a for TRPP2 in that TRPP2 the Ca2+ to our data and P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google Scholar, Y. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that TRPP2 and Ca2+ by IP3R a Ca2+ in TRPP2 it that cells a Ca2+ release IP3R we could a in in Ca2+ and and we an in in our is that we not TRPP2, expression with of TRPP2 the TRPP2 channel is to be and by the protein L. W. Maeda Y. X. Z. Ehrlich B.E. Somlo S. Proc. Natl. Acad. Sci. U.S.A. 2008; PubMed Scopus Google Scholar). TRPP2 is it is that the of is not to this of we a interaction between TRPP2 and the IP3R and identified a conserved positively charged cluster in the N-terminal domain of the IP3R and an acidic cluster the of the signal in the of TRPP2 as for interaction. TRPP2 was re-introduced in TRPP2−/− mouse renal epithelial was a of agonist-induced intracellular Ca2+ release in intact cells and in permeabilized cells. analysis using pathological mutants of TRPP2 and competing that this on was on the TRPP2 channel function and on the interaction with the a in which the TRPP2 channel is as a channel by a local by that a signaling complex involving TRPP2 and the IP3R is for intracellular Ca2+ signaling. of this interaction, which in mutants of TRPP2, to altered intracellular Ca2+ and might contribute to the development of ADPKD by loss-of-function mutations in TRPP2. IntroductionAutosomal dominant polycystic kidney disease (ADPKD) 4The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseaseACacidic clusterCICRCa2+-induced Ca2+ release[Ca2+]ERCa2+ concentration in the ER[Ca2+]cytcytosolic Ca2+ concentrationERendoplasmic reticulumIP3Rinositol 1,4,5-trisphosphate receptorIICRinositol 1,4,5-trisphosphate-induced Ca2+ releaseLBDligand-binding domainPKD1polycystin-1RyRryanodine receptorTGthapsigarginTRPP2polycystin-2aaamino acid(s)MOPS4-morpholinepropanesulfonic acidPipes1,4-piperazinediethanesulfonic acidBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolGSTglutathione S-transferaseTBSTris-buffered salineCTC-terminalNTN-terminalBAPTA1,2-bis(2-aminophenoxyl)ethane-N,N-N′,N′-tetraacetic acid. is an inherited human disorder that affects more than six million people worldwide and is the most common monogenic cause of kidney failure in humans (1Gabow P.A. Grantham J.J. Schrier R.W. Gottschalk C.W. Diseases of the Kidney. 6th Ed. Little Brown and Company, Boston, MA1997: 521-560Google Scholar). ADPKD results in end-stage renal disease in ∼50% of the affected individuals by the age of 60. ADPKD arises as a consequence of mutations of two genes PKD1 and PKD2, encoding integral membrane proteins polycystin-1 (PKD1, ∼460 kDa) and polycystin-2 (TRPP2, ∼110 kDa), respectively. Most mutations identified in affected families appear to truncate and (or) inactivate either of both proteins (2Mochizuki 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, 3Harris P.C. J. Am. Soc. Nephrol. 2009; 20: 1188-1198Crossref PubMed Scopus (49) Google Scholar, 4Wu G. Somlo S. Mol. Genet. Metab. 2000; 69: 1-15Crossref PubMed Scopus (101) Google Scholar, 5Torres V.E. Harris P.C. Kidney Int. 2009; 76: 149-168Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar). Mutations in PKD1 account for the vast majority (∼85%) of patients with ADPKD and are associated with a more severe clinical presentation and earlier onset of end-stage renal disease than the PKD2 phenotype (4Wu G. Somlo S. Mol. Genet. Metab. 2000; 69: 1-15Crossref PubMed Scopus (101) Google Scholar). However, in all other aspects, PKD1 and PKD2 mutations produce virtually indistinguishable disease manifestations, indicating that the two proteins might function in a common signaling pathway involved in maintaining the terminally differentiated state of renal epithelial cells.TRPP2 is a 968-amino acid (aa) protein with six predicted transmembrane domains and is highly conserved among multicellular organisms and widely expressed in various tissues (2Mochizuki 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). Structural analyses indicate that TRPP2 contains several functional domains in its C-terminal tail. There are two Ca2+-binding sites (aa 680–796) arranged in a typical and an atypical EF-hand motif, which could be involved in a Ca2+-mediated regulation of TRPP2 (6Schumann F. Hoffmeister H. Bader R. Schmidt M. Witzgall R. Kalbitzer H.R. J. Biol. Chem. 2009; 284: 24372-24383Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). An endoplasmic reticulum (ER) retention signal (aa 787–820) (7Cai 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) and a coiled-coil domain (aa 839–919), responsible for homo- and heterodimerization (8Celić A. Petri E.T. Demeler B. Ehrlich B.E. Boggon T.J. J. Biol. Chem. 2008; 283: 28305-28312Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 9Yu Y. Ulbrich M.H. Li M.H. Buraei Z. Chen X.Z. Ong A.C. Tong L. Isacoff E.Y. Yang J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11558-11563Crossref PubMed Scopus (142) Google Scholar), are also present. Recently, it was reported that this coiled-coil domain was responsible for formation of a TRPP2 trimer that interacts with PKD1 in the plasma membrane (9Yu Y. Ulbrich M.H. Li M.H. Buraei Z. Chen X.Z. Ong A.C. Tong L. Isacoff E.Y. Yang J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11558-11563Crossref PubMed Scopus (142) Google Scholar).There is a long-standing debate on the subcellular localization of TRPP2. TRPP2 has been detected (i) in the plasma membrane, where it is supposed to form a receptor-operated, non-selective cation channel (10Hanaoka 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), (ii) in the primary cilium, where it could act as a mechanosensitive channel, possibly in association with PKD1 (11Nauli 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 Google Scholar), L. T. E. G. Sukhatme V.P. Proc. Natl. Acad. Sci. U.S.A. 1999; PubMed Scopus Google Scholar, A. L. F. T. Tsiokas L. P. 2008; PubMed Scopus Google Scholar), (11Nauli 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 Google Scholar, M. B. F. X. M. R. T. R. M. A. Germino G.G. T. J. B. G. J. Biol. 2008; PubMed Scopus Google Scholar), in the where it is to function as an intracellular Ca2+-release channel P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google Scholar), also in and of cells in L. S. Ong PubMed Scopus Google Scholar, A. F. B. M. M. E. P. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). The of TRPP2 to subcellular is by (i) the protein (7Cai 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, L. Z. X. Cai Y. S. Somlo S. J. Sci. PubMed Scopus Google Scholar), (ii) with acidic cluster proteins and M. T. T. R. B. S. B. A. K. C.C. R. E. G. G. J. PubMed Scopus Google Scholar), and a of in the of TRPP2 M. T. T. R. B. S. B. A. K. C.C. R. E. G. G. J. PubMed Scopus Google Scholar, M. G. PubMed Scopus Google Scholar). However, the subcellular localization of TRPP2 is in the as by to and with proteins (7Cai 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, P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google between TRPP2 and the two intracellular Ca2+-release the receptor and the inositol 1,4,5-trisphosphate receptor (IP3R), has been and functional demonstrated that the N-terminal of TRPP2 is to the the C-terminal of TRPP2 only to it is in the function M. X. Somlo S. Ehrlich B.E. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). that TRPP2 interacts with the most its C-terminal Y. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Li Y. S. Qian F. Guggino W.B. J. Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar) demonstrated also that PKD1 with the IP3-induced Ca2+ release an of intracellular Ca2+ release that was to an of TRPP2 P. Cai Y. L. Maeda Y. S. Witzgall R. Ehrlich B.E. Somlo S. Nat. Biol. PubMed Scopus Google Scholar, Y. Qian F. Germino G.G. Guggino W.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. G. Z. T. Qian L. R. Ehrlich B.E. Somlo S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. K. T. E. K. T. Y. T. T. Y. M. M. H. M. Y. J. Biol. Chem. 2009; 284: Full Text Full Text PDF PubMed Scopus Google Scholar), the mechanism of was not the other and T. R. B. R. G. M. J. 2009; PubMed Scopus Google Scholar) that TRPP2 could also act as a channel in the the Ca2+ concentration in the which resulted in an and the of results that the mechanism which in and TRPP2 in Ca2+ signaling is not therefore a analysis of the molecular and functional between the IP3R and we identified a conserved positively charged cluster in the N-terminal domain of the IP3R and an acidic cluster the of the signal in the C-terminal tail of TRPP2 as for the interaction between both Moreover, in a of renal epithelial TRPP2−/− we a of both intracellular Ca2+ release in intact cells and in permeabilized cells TRPP2 analysis using pathological mutants of TRPP2 and that with the interaction between TRPP2 and the IP3R that the in both a functional TRPP2 channel and a interaction with the suggest that TRPP2 as a channel in the that in an intracellular signaling complex with the intracellular Ca2+ release a in the of