TY - JOUR
T1 - Ezrin-radixin-moesin-binding phosphoprotein 50 is expressed at the apical membrane of rat liver epithelia
AU - Fouassier, L.
AU - Cheng Ying Duan, Ying Duan
AU - Feranchak, A. P.
AU - Yun, C. H C
AU - Sutherland, E.
AU - Simon, F.
AU - Fitz, J. G.
AU - Doctor, R. B.
N1 - Funding Information:
Abbreviations: ERM, ezrin-radixin-moesin; EBP50, ERM-binding phosphoprotein 50; NHE-RF, Na/H exchanger-regulatory factor; NRC, normal rat cholangiocyte; BEC, biliary epithelial cell; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; LAP, leucine amino peptidase; PDZ, PSD-95/DIg/ ZO-1. From the 1University of Colorado Health Sciences Center, Division of Gastroenterology and Hepatology, Denver, CO and the 2Johns Hopkins University School of Medicine, Division of Gastroenterology, Baltimore, MD. Received July 26, 2000; accepted October 19, 2000. Supported by post-doctoral fellowship to L. Fouassier from INSERM, France; NIH grant (RO1 DK-44484) to CHC Yun; NIH (R01 DK-15851); and VA Merit grants to FR Simon, NIH grants (R01 DK 46082 and DK 43278) to JG Fitz, and American Liver Foundation (ALF PN 9801-014) Liver Scholar Award to RB Doctor. Address reprint requests to: R.B. Doctor, Ph.D., University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B158, Denver, CO 80262. E-mail: brian.doctor@uchsc.edu; fax: 303-315-3507. Copyright © 2001 by the American Association for the Study of Liver Diseases. 0270-9139/01/3301-0022$3.00/0 doi:10.1053/jhep.2001.21143
PY - 2001
Y1 - 2001
N2 - Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) and NHE3 Kinase A regulatory protein (E3KARP) are membrane-cytoskeleton linking proteins that utilize 2 PSD-95/DIg/ZO-1 (PDZ) domains and an ERM binding site to coordinate cyclic adenosine monophosphate (cAMP)-regulated ion transport in a number of distinct epithelia. ERM family members serve to anchor EBP50 and E3KARP to the actin cytoskeleton and sequester protein kinase A (PKA) to these protein complexes. In hepatocytes and cholangiocytes, the epithelial cells of the bile secretory unit, cAMP-activated PKA stimulates secretion and bile formation, but the molecular mechanisms, including the potential contribution of EBP50 and E3KARP, remain undetermined. The present studies evaluated the comparative expression and localization of EBP50 and E3KARP in rat hepatocytes and cholangiocytes. Complementary DNAs encoding rat EBP50 and E3KARP were identified by reverse transcription-polymerase chain reaction in both epithelial cell types and subsequently sequenced. Northern and Western analysis showed the presence of EBP50 messenger RNA and protein in both hepatocytes and cholangiocytes. Confocal immunofluorescence revealed EBP50 was concentrated at the apical domain of both cell types. E3KARP was also expressed in cholangiocytes but had a distinct cytoplasmic/nuclear distribution. In dominant-negative transfection studies, patch clamp analysis of Mz-ChA1 cholangiocarcinoma cells showed that expression of the PDZl domain of EBP50 selectively decreased the endogenous cAMP-mediated Cl- secretory response. The apical expression of EBP50, presence of specific ERM proteins, and functional effects of PDZl expression on cholangiocyte secretion suggest EBP50 is positioned to contribute to the organization and regulation of bile secretory proteins in both hepatocytes and cholangiocytes.
AB - Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) and NHE3 Kinase A regulatory protein (E3KARP) are membrane-cytoskeleton linking proteins that utilize 2 PSD-95/DIg/ZO-1 (PDZ) domains and an ERM binding site to coordinate cyclic adenosine monophosphate (cAMP)-regulated ion transport in a number of distinct epithelia. ERM family members serve to anchor EBP50 and E3KARP to the actin cytoskeleton and sequester protein kinase A (PKA) to these protein complexes. In hepatocytes and cholangiocytes, the epithelial cells of the bile secretory unit, cAMP-activated PKA stimulates secretion and bile formation, but the molecular mechanisms, including the potential contribution of EBP50 and E3KARP, remain undetermined. The present studies evaluated the comparative expression and localization of EBP50 and E3KARP in rat hepatocytes and cholangiocytes. Complementary DNAs encoding rat EBP50 and E3KARP were identified by reverse transcription-polymerase chain reaction in both epithelial cell types and subsequently sequenced. Northern and Western analysis showed the presence of EBP50 messenger RNA and protein in both hepatocytes and cholangiocytes. Confocal immunofluorescence revealed EBP50 was concentrated at the apical domain of both cell types. E3KARP was also expressed in cholangiocytes but had a distinct cytoplasmic/nuclear distribution. In dominant-negative transfection studies, patch clamp analysis of Mz-ChA1 cholangiocarcinoma cells showed that expression of the PDZl domain of EBP50 selectively decreased the endogenous cAMP-mediated Cl- secretory response. The apical expression of EBP50, presence of specific ERM proteins, and functional effects of PDZl expression on cholangiocyte secretion suggest EBP50 is positioned to contribute to the organization and regulation of bile secretory proteins in both hepatocytes and cholangiocytes.
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U2 - 10.1053/jhep.2001.21143
DO - 10.1053/jhep.2001.21143
M3 - Article
C2 - 11124833
AN - SCOPUS:0035192915
SN - 0270-9139
VL - 33
SP - 166
EP - 176
JO - Hepatology
JF - Hepatology
IS - 1
ER -