Role of membrane microdomains in PTH-mediated down-regulation of NaPi-IIa in opossum kidney cells.
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.


CONCLUSIONS
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.
Nashiki
Kunitaka
K
Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan.
Taketani
Yutaka
Y
Takeichi
Tomoko
T
Sawada
Naoki
N
Yamamoto
Hironori
H
Ichikawa
Masako
M
Arai
Hidekazu
H
Miyamoto
Ken-Ichi
K
Takeda
Eiji
E
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002451:Cell Compartmentation / Q000502:physiology*; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins; D018492:Dopamine Antagonists / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology*; D015739:Nocodazole / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D009893:Opossums; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000378:metabolism; D017434:Protein Structure, Tertiary; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099149:Ezrin
D000970:Antineoplastic Agents; D002746:Chlorpromazine; D017868:Cyclic AMP-Dependent Protein Kinases; D003505:Cyclodextrins; D015638:Cytochalasin D; D003598:Cytoskeletal Proteins; D018492:Dopamine Antagonists; D015739:Nocodazole; D019384:Nucleic Acid Synthesis Inhibitors; D010281:Parathyroid Hormone; D010710:Phosphates; D010750:Phosphoproteins; D051571:Protein Kinase C-alpha; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D000099149:Ezrin; C493554:SLC34A1 protein, human