title: Cloning of a membrane-associated protein which modifies activity and properties of the Na(+)-D-glucose cotransporter.
abstract: An expression library from porcine kidney cortex was screened with a monoclonal antibody (R4A6) which stimulates high-affinity phlorizin binding in kidney and intestine but does not react with the membrane protein (SGLT1) which mediates Na(+)-coupled transport of D-glucose (Hediger, M.A., Coady, M.J., Ikeda, T.S., and Wright, E.M. (1987) Nature 330, 379-381). A cDNA (RS1) was obtained which codes for a hydrophilic M(r) 66,832 polypeptide and contains a predicted hydrophobic alpha-helix at the COOH terminus. After expression in Xenopus oocytes RS1 protein was found associated with the plasma membrane. RS1-homologous mRNAs were detected in renal outer cortex and outer medulla, small intestine, liver, and LLCPK1 cells, but not in skeletal muscle, heart muscle, Madin-Darby canine kidney (MDCK) cells, renal inner medulla, and Xenopus oocytes. After nondenaturing gel electrophoresis of renal brush-border membranes comigration of RS1- and SGLT1-homologous proteins as a high molecular weight complex was demonstrated. RS1 altered the expression of Na(+)-glucose cotransport by SGLT1 in Xenopus oocytes. There was no effect on the expression of the nonhomologous transporters for Na(+)-gamma-aminobutyric acid cotransport and for Na(+)-independent glucose transport. However, RS1 also changed the expression of the SGLT1-homologous Na(+)-myo-inositol cotransporter from MDCK cells. The Vmax of methyl-alpha-D-glucopyranoside (AMG) transport expressed after injection of a small amount of SGLT1-cRNA was increased 40-fold when a stoichiometric amount of RS1-cRNA was coinjected. In addition the voltage and glucose dependence of expressed AMG uptake and the concentration dependence of transport inhibition by phlorizin were changed when stoichiometric amounts of RS1-cRNA were coinjected with SGLT1-cRNA. Thus with SGLT1 one apparent transport site (K0.5 about 100 microM) and one apparent phlorizin inhibition site (Ki about 5 microM) was observed whereas with SGLT1 plus RS1 two apparent transport sites (K0.5(1) about 20 microM, K0.5(2) about 1 mM) and two apparent phlorizin inhibition sites (Ki(1) about 0.3 microM, Ki(2) about 30 microM) were found as has been described in brush-border membrane vesicles of kidney and intestine (see e.g. Koepsell, H., Fritzsch, G., Korn, K., and Madrala, A. (1990) J. Membr. Biol. 114, 113-132). The data suggest that the Na(+)-D-glucose cotransporter and possibly also other SGLT1-type Na(+)-cotransporters contain RS1-type regulatory subunits.
authors.affiliation: Max-Planck-Institut für Biophysik, Frankfurt am Main, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Veyhl
authors.forename: J
authors.initials: J
authors.lastname: Spangenberg
authors.forename: B
authors.initials: B
authors.lastname: Püschel
authors.forename: R
authors.initials: R
authors.lastname: Poppe
authors.forename: C
authors.initials: C
authors.lastname: Dekel
authors.forename: G
authors.initials: G
authors.lastname: Fritzsch
authors.forename: W
authors.initials: W
authors.lastname: Haase
authors.forename: H
authors.initials: H
authors.lastname: Koepsell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004285:Dogs; D005947:Glucose / Q000378:metabolism*; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009865:Oocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D051269:Sodium-Glucose Transporter 1; D013552:Swine; D014981:Xenopus
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D051269:Sodium-Glucose Transporter 1; D012964:Sodium; D005947:Glucose