The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3 - salvage transporter human Na+-HCO3 - cotransport isoform 3

Meeyoung Park, Shigeru B H Ko, Joo Young Choi, Gaia Muallem, Philip J. Thomas, Alexander Pushkin, Myeong Sok Lee, Joo Young Kim, Min Goo Lee, Shmuel Muallem, Ira Kurtz

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Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO3 - secretion and HCO3 - salvage in secretory epithelia. At least two luminal transporters mediate HCO3 - salvage, the Na+/H+ exchanger (NHE3) and the Na+-HCO3 - cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na+-dependent, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO3 - transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO3 --transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO3 - transport by regulating the activity of all luminal HCO3 - secretion and salvage mechanisms of secretory epithelial cells.

Original languageEnglish (US)
Pages (from-to)50503-50509
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number52
DOIs
StatePublished - Dec 27 2002

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Salvaging
Cystic Fibrosis Transmembrane Conductance Regulator
Protein Isoforms
Submandibular Gland
Pancreas
PDZ Domains
Sodium-Hydrogen Antiporter
HEK293 Cells
Parotid Gland
Colforsin
Cyclic AMP-Dependent Protein Kinases
Glutathione Transferase
Human Activities
Scaffolds
Epithelium
Epithelial Cells

ASJC Scopus subject areas

  • Biochemistry

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The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3 - salvage transporter human Na+-HCO3 - cotransport isoform 3. / Park, Meeyoung; Ko, Shigeru B H; Choi, Joo Young; Muallem, Gaia; Thomas, Philip J.; Pushkin, Alexander; Lee, Myeong Sok; Kim, Joo Young; Lee, Min Goo; Muallem, Shmuel; Kurtz, Ira.

In: Journal of Biological Chemistry, Vol. 277, No. 52, 27.12.2002, p. 50503-50509.

Research output: Contribution to journalArticle

Park, Meeyoung ; Ko, Shigeru B H ; Choi, Joo Young ; Muallem, Gaia ; Thomas, Philip J. ; Pushkin, Alexander ; Lee, Myeong Sok ; Kim, Joo Young ; Lee, Min Goo ; Muallem, Shmuel ; Kurtz, Ira. / The cystic fibrosis transmembrane conductance regulator interacts with and regulates the activity of the HCO3 - salvage transporter human Na+-HCO3 - cotransport isoform 3. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 52. pp. 50503-50509.
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abstract = "Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO3 - secretion and HCO3 - salvage in secretory epithelia. At least two luminal transporters mediate HCO3 - salvage, the Na+/H+ exchanger (NHE3) and the Na+-HCO3 - cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na+-dependent, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO3 - transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO3 --transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO3 - transport by regulating the activity of all luminal HCO3 - secretion and salvage mechanisms of secretory epithelial cells.",
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AU - Choi, Joo Young

AU - Muallem, Gaia

AU - Thomas, Philip J.

AU - Pushkin, Alexander

AU - Lee, Myeong Sok

AU - Kim, Joo Young

AU - Lee, Min Goo

AU - Muallem, Shmuel

AU - Kurtz, Ira

PY - 2002/12/27

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N2 - Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO3 - secretion and HCO3 - salvage in secretory epithelia. At least two luminal transporters mediate HCO3 - salvage, the Na+/H+ exchanger (NHE3) and the Na+-HCO3 - cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na+-dependent, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO3 - transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO3 --transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO3 - transport by regulating the activity of all luminal HCO3 - secretion and salvage mechanisms of secretory epithelial cells.

AB - Cystic fibrosis transmembrane conductance regulator (CFTR) regulates both HCO3 - secretion and HCO3 - salvage in secretory epithelia. At least two luminal transporters mediate HCO3 - salvage, the Na+/H+ exchanger (NHE3) and the Na+-HCO3 - cotransport (NBC3). In a previous work, we show that CFTR interacts with NHE3 to regulate its activity (Ahn, W., Kim, K. W., Lee, J. A., Kim, J. Y., Choi, J. Y., Moe, O. M., Milgram, S. L., Muallem, S., and Lee, M. G. (2001) J. Biol. Chem. 276, 17236-17243). In this work, we report that transient or stable expression of human NBC3 (hNBC3) in HEK cells resulted in a Na+-dependent, DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid)- and 5-ethylisopropylamiloride-insensitive HCO3 - transport. Stimulation of CFTR with forskolin markedly inhibited NBC3 activity. This inhibition was prevented by the inhibition of protein kinase A. NBC3 and CFTR could be reciprocally coimmunoprecipitated from transfected HEK cells and from the native pancreas and submandibular and parotid glands. Precipitation of NBC3 or CFTR from transfected HEK293 cells and from the pancreas and submandibular gland also coimmunoprecipitated EBP50. Glutathione S-transferase-EBP50 pulled down CFTR and hNBC3 from cell lysates when expressed individually and as a complex when expressed together. Notably, the deletion of the C-terminal PDZ binding motifs of CFTR or hNBC3 prevented coimmunoprecipitation of the proteins and inhibition of hNBC3 activity by CFTR. We conclude that CFTR and NBC3 reside in the same HCO3 --transporting complex with the aid of PDZ domain-containing scaffolds, and this interaction is essential for regulation of NBC3 activity by CFTR. Furthermore, these findings add additional evidence for the suggestion that CFTR regulates the overall trans-cellular HCO3 - transport by regulating the activity of all luminal HCO3 - secretion and salvage mechanisms of secretory epithelial cells.

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