Characterization of potential TRPP2 regulating proteins in early Xenopus embryos

• Published in: Journal of cellular biochemistry 2018-12, Vol.119 (12), p.10338-10350
• Main Authors: Futel, Mélinée, Le Bouffant, Ronan, Buisson, Isabelle, Umbhauer, Muriel, Riou, Jean‐François
• Format: Article
• Language: English
• Subjects: Binding; Biochemistry, Molecular Biology; Calcium; Calcium (extracellular); Calcium (intracellular); Calcium (reticular); Calcium ions; Calcium signalling; Cations; Cilia; Dishevelled protein; dvl2; Embryo cells; Embryos; Endoplasmic reticulum; Fluorescence; Gene expression; golgin A2; Immunoblotting; Ion channels; Kidneys; Kinases; Life Sciences; Mass spectrometry; Mass spectroscopy; Mechanotransduction; primary cilium; PrKD1; pronephric field; Protein kinase; Proteins; Transient receptor potential proteins; TRPP2; Xenopus; Xenopus embryo
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.27376

Transient receptor potential cation channel‐2 (TRPP2) is a nonspecific Ca2+‐dependent cation channel with versatile functions including control of extracellular calcium entry at the plasma membrane, release of intracellular calcium ([Ca2+]i) from internal stores of endoplasmic reticulum, and calcium‐dependent mechanosensation in the primary cilium. In early Xenopus embryos, TRPP2 is expressed in cilia of the gastrocoel roof plate (GRP) involved in the establishment of left‐right asymmetry, and in nonciliated kidney field (KF) cells, where it plays a central role in early specification of nephron tubule cells dependent on [Ca2+]i signaling. Identification of proteins binding to TRPP2 in embryo cells can provide interesting clues about the mechanisms involved in its regulation during these various processes. Using mass spectrometry, we have therefore characterized proteins from late gastrula/early neurula stage embryos coimmunoprecipitating with TRPP2. Binding of three of these proteins, golgin A2, protein kinase‐D1, and disheveled‐2 has been confirmed by immunoblotting analysis of TRPP2‐coprecipitated proteins. Expression analysis of the genes, respectively, encoding these proteins, golga2, prkd1, and dvl2 indicates that they are likely to play a role in these two regions. Golga2 and prkd1 are expressed at later stage in the developing pronephric tubule where golgin A2 and protein kinase‐D1 might also interact with TRPP2. Colocalization experiments using exogenously expressed fluorescent versions of TRPP2 and dvl2 in GRP and KF reveal that these two proteins are generally not coexpressed, and only colocalized in discrete region of cells. This was observed in KF cells, but does not appear to occur in the apical ciliated region of GRP cells. Transient receptor potential cation channel‐2 (TRPP2) has been implicated in the control of Ca2+‐dependent processes in the early Xenopus embryo, both in the cilated cells of the gastrocoel roof plate involved in the establishment of left‐right asymmetry, and in the kidney field for renal cell specification. Proteins coimmunoprecipitating with TRPP2, potentially involved in its regulation in the embryo, have been identified using mass spectrometry. Characterization of three of them, golgin A2, protein kinase‐D1, and dvl2 suggest versatile functions of TRPP2 in the embryo.