TY - JOUR
T1 - Rab3D, a small GTP-binding protein implicated in regulated secretion, is associated with the transcytotic pathway in rat hepatocytes
AU - Larkin, Janet M.
AU - Woo, Bonnie
AU - Balan, Vijayan
AU - Marks, David L.
AU - Oswald, Barbara J.
AU - LaRusso, Nicholas F.
AU - McNiven, Mark A.
N1 - Funding Information:
Acknowledgment: The authors thank Drs. G. Baldini (Columbia University) for anti-rab3D antibody and adipocytes; B. Goud (Pasteur Institute) for purified rab1, -2, -4, -6, and antibodies to these proteins; J. Anderson (Yale University School of Medicine) for polyclonal anti–ZO-1 antibody; M. Farquhar (UCSD) for anti–α mannosidase II antibody; and K. Kirk (University of Alabama at Birmingham) for rab3B fusion protein and anti-rab3B antibody. Special thanks go to Dr. Ann Hubbard (Johns Hopkins University School of Medicine) for providing paraformaldehyde-fixed liver tissue and monoclonal anti-ZO-1 antibody, as well as critically reading the manuscript. S. Bronk and S. Kuntz provided excellent technical assistance in culturing isolated hepatocytes and cutting some of the 2-µm-thick frozen liver sections, respectively. Dr. Lar-kin thanks the Center for Basic Research in Digestive Diseases at the Mayo Clinic for funding and supporting the initial stages of this project, the Department of Biological Sciences at Barnard College for continued support, and the Department of Chemistry at Barnard College for their support of BW and her biochemistry honors thesis.
Funding Information:
Abbreviations: GTP, guanosine triphosphate; ZO-1, zonula occludens 1; HRP, horse-radish peroxidase; RT-PCR, reverse transcriptase polymerase chain reaction; bp, base pair; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; CVCF, crude vesicle carrier fraction; PBS, phosphate-buffered saline; pIgA-R, polymeric immunoglobulin A receptor; BDL, bile duct ligation. From 1Barnard College, Department of Biological Sciences, New York, NY; and 2Center for Basic Research in Digestive Diseases, Mayo Foundation, Rochester, MN. Received December 8, 1999; accepted May 11, 2000. Supported by the National Institutes of Health grants AA09227 and DK44650 awarded to M. McNiven; and an AGA/Industry Scholar Award and National Institutes of Health grant DK52553 awarded to J. Larkin. Address reprint requests to: Janet M. Larkin, Ph.D., Department of Biological Sciences, Barnard College/Columbia University, 3009 Broadway, New York, NY 10027. E-mail: jlarkin@barnard.columbia.edu; fax: 212-854-1950. Copyright © 2000 by the American Association for the Study of Liver Diseases. 0270-9139/00/3202-0024$3.00/0 doi:10.1053/jhep.2000.9110
PY - 2000
Y1 - 2000
N2 - Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of ~25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.
AB - Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of ~25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.
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U2 - 10.1053/jhep.2000.9110
DO - 10.1053/jhep.2000.9110
M3 - Article
C2 - 10915742
AN - SCOPUS:0033859259
SN - 0270-9139
VL - 32
SP - 348
EP - 356
JO - Hepatology
JF - Hepatology
IS - 2
ER -