Kidney international · 2005
Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Nashiki Kunitaka, Taketani Yutaka, Takeichi Tomoko, Sawada Naoki, Yamamoto Hironori, Ichikawa Masako, Arai Hidekazu, Miyamoto Ken-Ichi, Takeda Eiji
Аннотация
BACKGROUND: Parathyroid hormone (PTH) rapidly down-regulates type IIa sodium-dependent phosphate transporter (NaPi-IIa) via an endocytic pathway. Since the relationship between PTH signaling and NaPi-IIa endocytosis has not been explored, we investigated the role of membrane microdomains in this process.
METHODS: We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases.
RESULTS: We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH.
CONCLUSION: These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.
METHODS: We examined the submembrane localization of NaPi-IIa in opossum kidney (OK-N2) cells that stably expressed human NaPi-IIa, and searched for a PTH-induced specific phosphorylating substrate on their membrane microdomains by immunoblotting with specific antibody against phospho substrates of protein kinases.
RESULTS: We found that NaPi-IIa was primarily localized in low-density membrane (LDM) domains of the plasma membrane; PTH reduced the levels of immunoreactive NaPi-IIa in these domains. Furthermore, PTH activated both protein kinase A (PKA) and protein kinase Calpha (PKCa) and increased the phosphorylation of 250 kD and 80 kD substrates; this latter substrate was identified as ezrin, which a member of the ezrin-radixin-moesin (ERM) protein family. In response to PTH, ezrin was phosphorylated by both PKA and PKC. Dominant negative ezrin blocked the reduction in NaPi-IIa expression in the LDM domains that was induced by PTH.
CONCLUSION: These data suggest that NaPi-IIa and PTH-induced phosphorylated proteins that include ezrin are compartmentalized in LDM microdomains. This compartmentalization may play an important role in the down-regulation of NaPi-IIa via endocytosis.
Предметные рубрики MeSH
AnimalsAntineoplastic AgentsCell CompartmentationCell LineChlorpromazineCyclic AMP-Dependent Protein KinasesCyclodextrinsCytochalasin DCytoskeletal ProteinsDopamine AntagonistsDown-RegulationEndocytosisHumansImmunohistochemistryKidneyNocodazoleNucleic Acid Synthesis InhibitorsOpossumsParathyroid HormonePhosphatesPhosphoproteinsPhosphorylationProtein Kinase C-alphaProtein Structure, TertiarySodium-Phosphate Cotransporter Proteins, Type IIaEzrin