Renal proximal tubule and intestinal brush border membranes contain transporters which catalyze active cotransport of Na+ and glucose. Using immunological and functional criteria, we have identified a 75 kD glycoprotein in renal brush border membranes as a subunit of the Na+/glucose symporter, purified the 75 kD protein to homogeneity and obtained functional reconstitution in proteoliposomes. Our monoclonal and polyclonal antibodies also recognize the 75 kD subunit in LLC-PK1 cells, a long term line from renal proximal tubule. The overall goals of the continuation of this project are to further characterize the structure and function of the renal Na+/D- glucose symporter and to investigate the regulation of its differentiated expression in cell culture. The cDNA encoding the 75 kD subunit of the renal Na+/glucose symporter will be cloned and sequenced. An LLC-PK1 lambda gt11 expression library enriched in symporter cDNA will be screened using our polyclonal antibody to the symporter; alternately, a LLC- PK1 lambda gtlO library will be screened using synthetic oligonucleotides corresponding to the sequence of a peptide derived from our pure symporter preparation. A predicted amino acid sequence and secondary structure will be calculated based on the cDNA sequence. The oligomeric structure of the symporter will be investigated by gel filtration and comparative sedimentation in sucrose gradients made from D2O and H2O and by cross-linking studies. The possibility that Na+, glucose or phlorizin affect oligomeric structure will also be tested. Epitopes of the symporter on the inner and outer surface of the membrane and transmembrane segment will be mapped using monoclonal antibodies to the purified protein and site-directed polyclonal antibodies. Site-directed mutagenesis studies will be initiated to investigate functional domains of the symporter. The regulation of Na+/glucose symporter expression in LLC-PK1 cells will be investigated using specific antibodies to monitor biosynthesis and turnover of the protein and cDNA probes to measure specific mRNA levels, rates of transcription and half-life. These studies will focus on the dramatic (20-fold) induction of symporter activity achieved after treatment with the differentiation inducer hexamethylene bisacetamide (HMBA).

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK027400-12
Application #
3483495
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1980-12-01
Project End
1993-08-31
Budget Start
1992-09-01
Budget End
1993-08-31
Support Year
12
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Type
Schools of Medicine
DUNS #
City
Houston
State
TX
Country
United States
Zip Code
77225
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
Yet, S F; Kong, C T; Peng, H et al. (1994) Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells. J Cell Physiol 158:506-12
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

Showing the most recent 10 out of 16 publications