Abstract

The overall goals of this project are to identify and isolate the renal Na+/D-glucose transporter(s) and to investigate the regulation of its expression in the established renal epithelial cell line LLC-PK1 of proximal tubule origin using specific antisera. Antibodies to the renal Na+/D-glucose transporter will be prepared by two different strategies. One approach is based on the formation of anti-idiotype antibodies prepared against antibodies to a phlorizin-bovine serum albumin conjugate. The other approach is based on production of monoclonal antibodies after immunization with partially - purified preparations of the Na+/D-glucose transporter. Immuno-affinity methods will be utilized to identify and isolate the renal Na+/D-glucose transporter(s) from pig kidney and from LLC-PK1 cell cultures. The isolated protein will be characterized by functional and molecular criteria. Cross-linking studies will be carried out to investigate its possible oligomeric structure in cell membranes. The biosynthesis and regulation of the expression of the Na+/D-glucose transporter in LLC-PK1 cultures will be investigated using these antibody probes. Biosynthetic labelling and immunoprecipitation studies, cell-free translation, immunoelectroblot analysis, crossed immunoelectrophoresis and immunofluorescense histochemistry will be carried out to explore the basis for the dramatically-increased Na+/D-glucose transport activity which develops after cell confluence and after addition of differentiation inducers.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK027400-07
Application #
3228282
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1980-12-01
Project End
1988-08-31
Budget Start
1987-05-01
Budget End
1988-08-31
Support Year
7
Fiscal Year
1987
Total Cost
Indirect Cost

  Institution

Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225

  Publications

Loflin, Paul; Lever, Julia E (2002) Cyclic nucleotide-dependent screening of a lamda expression library for nucleic acid binding activities. Biotechniques 32:1020, 1022, 1024-5 passim
Loflin, P; Lever, J E (2001) A cis-dominant cyclic nucleotide-dependent regulatory domain in the 3'-untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA. FEBS Lett 492:233-7
Loflin, P; Lever, J E (2001) HuR binds a cyclic nucleotide-dependent, stabilizing domain in the 3' untranslated region of Na(+)/glucose cotransporter (SGLT1) mRNA. FEBS Lett 509:267-71
Lee, W Y; Loflin, P; Clancey, C J et al. (2000) Cyclic nucleotide regulation of Na+/glucose cotransporter (SGLT1) mRNA stability. Interaction of a nucleocytoplasmic protein with a regulatory domain in the 3'-untranslated region critical for stabilization. J Biol Chem 275:33998-4008
Clancey, C J; Lever, J E (2000) Differential regulation of three glucose transporter genes in a renal epithelial cell line. J Cell Physiol 185:244-52
Panayotova-Heiermann, M; Loo, D D; Kong, C T et al. (1996) Sugar binding to Na+/glucose cotransporters is determined by the carboxyl-terminal half of the protein. J Biol Chem 271:10029-34
Peng, H; Lever, J E (1995) Regulation of Na(+)-coupled glucose transport in LLC-PK1 cells. Message stabilization induced by cyclic AMP elevation is accompanied by binding of a M(r) = 48,000 protein to a uridine-rich domain in the 3'-untranslated region. J Biol Chem 270:23996-4003
Peng, H; Lever, J E (1995) Post-transcriptional regulation of Na+/glucose cotransporter (SGTL1) gene expression in LLC-PK1 cells. Increased message stability after cyclic AMP elevation or differentiation inducer treatment. J Biol Chem 270:20536-42
Wu, J S; Lever, J E (1994) N-linked glycosylation is not required for Na+/glucose symport activity in LLC-PK1 cells. Biochim Biophys Acta 1192:289-92
Mackenzie, B; Panayotova-Heiermann, M; Loo, D D et al. (1994) SAAT1 is a low affinity Na+/glucose cotransporter and not an amino acid transporter. A reinterpretation. J Biol Chem 269:22488-91

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