PMC:4925210 / 10786-14082 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4925210","sourcedb":"PMC","sourceid":"4925210","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4925210","text":"TRPP2 mediates WNT-induced whole cell currents in MEFs\nWild type MEFs express PKD111 and TRPP2 (Fig. 6a and b and Supplementary Fig. 2d) and deletion of Pkd2 is expected to cause an upregulation of the WNT/β-catenin pathway and constitutive activation of p38-MAPK37. Consistently, phospho-β-catenin levels were slightly decreased, whereas phospho-p38MAPK were slightly increased in Pkd2-null MEFs (Supplementary Fig. 5b,c). WNT3A induced activation of the canonical WNT/β-catenin pathway in Pkd2 mutant cells and this increase was 2-3-fold higher compared to wild type cells (Supplementary Fig. 5g). Overexpression of ZNRF3 suppressed this effect (Supplementary Fig. 5h). Expression levels of Fzd1/2/6/7/8, and Ryk mRNAs or LRP6 and ROR2 proteins (LRP5 and ROR1 are not expressed in MEFs) were not different between wild type and Pkd2-null MEFs (Supplementary Fig. 5a,e,f). Expression level of DVL1 or DVL2 was similar between Pkd2+/+ and Pkd2−/− cells, but slightly reduced in Pkd1−/− cells (Supplementary Fig. 5d). In sum, Pkd2-null cells showed higher activation of the canonical WNT/β-catenin pathway compared to wild type cells and no obvious differences in the expression levels of FZDs, ROR2, LRP6, and RYK receptors.\nWhen intracellular Ca2+ was clamped at 50 nM, WNT9B induced large currents in wild type (Fig. 4a, black filled squares, c, red filled squares), but not Pkd2-null cells (Fig. 4a and c, blue open circles). WNT9B-induced currents showed a reversal potential at 0 mV and were reversibly blocked by La3+, as was seen in CHO-K1 cells transfected with PKD1 and TRPP2 (Fig. 2e,h). In zero intracellular Ca2+, WNT9B-induced whole cell currents showed outward rectification and a reversal potential close to −20 mV (Fig. 4d-f), as was seen in CHO-K1 cells transfected with PKD1 and TRPP2 under the same conditions (Fig. 3b). WNT9B did not induce a significant current in Pkd2−/− cells (Fig. 4d and f, blue open circles). Therefore, these experiments demonstrated that WNT9B was able to induce a similar current in native MEFs or CHO-K1 cells transfected only with PKD1/TRPP2, but not in Pkd2-null MEFs. The differential effect of WNT9B in wild type and Pkd2-null cells was specific, since both cell types responded similarly to extracellular ATP (Supplementary Fig. 6a-f). Adding-back wild type TRPP2 in mutant cells restored WNT9B-induced currents (Fig. 4g-i), whereas adding-back TRPP2Kv1.3 restored only outward currents, but not inward currents (Fig. 4j-l), indicating that TRPP2 was directly involved in WNT9B-induced currents. Overexpression of ZNRF3 did not have an effect on WNT9B-induced currents (Fig. 4m-o) indicating that WNT9B-induced TRPP2-mediated currents were independent of FZDs and/or LRP6, as shown in CHO-K1 and Drosophila S2 cells.\nBinding experiments showed that WNT3A was able to interact with the LRR-WSC domain of PKD1 (Supplementary Fig. 1). However, canonical WNT3A was not expected to induce Ca2+ signaling. At zero intracellular Ca2+, WNT3A induced whole cell currents in wild type cells (Fig. 5a-c), but with a noticeable delay (by about a min) compared to WNT9B-induced currents (Fig. 4a,g). Expression of ZNRF3 did not have significant effects on current amplitude, whereas WNT3A failed to induce whole cell currents in Pkd2-null cells (Fig. 5g-i).","divisions":[{"label":"Title","span":{"begin":0,"end":54}}],"tracks":[{"project":"2_test","denotations":[{"id":"27214281-19939939-73835250","span":{"begin":263,"end":265},"obj":"19939939"},{"id":"27214281-19939939-73835250","span":{"begin":263,"end":265},"obj":"19939939"}],"attributes":[{"subj":"27214281-19939939-73835250","pred":"source","obj":"2_test"},{"subj":"27214281-19939939-73835250","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93ebec","default":true}]}]}}