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 Ca(2+) signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP(3)R), an intracellular Ca(2+) 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 IP(3)R 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 Ca(2+) release in intact cells and IP(3)-induced Ca(2+) release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca(2+) signal by a local Ca(2+)-induced Ca(2+)-release mechanism, which only occurred in the presence of the TRPP2-IP(3)R interaction, and not via altered IP(3)R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca(2+) microdomains necessary for initiating Ca(2+)-induced Ca(2+) release. The data strongly suggest that defects in this mechanism may account for the altered Ca(2+) signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.
Sammels
Eva
E
Department of Molecular Cell Biology, Laboratory of Molecular and Cellular Signaling, KU Leuven, Campus Gasthuisberg O&N1, Herestraat 49 bus 802, B-3000 Leuven, Belgium.
Devogelaere
Benoit
B
Mekahli
Djalila
D
Bultynck
Geert
G
Missiaen
Ludwig
L
Parys
Jan B
JB
Cai
Yiqiang
Y
Somlo
Stefan
S
De Smedt
Humbert
H
D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry*; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000737:chemistry* / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D051379:Mice; D009154:Mutation; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D050396:TRPP Cation Channels / Q000378:metabolism*
D002118:Calcium; D004247:DNA; D005982:Glutathione Transferase; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D050396:TRPP Cation Channels