title: Protein kinase-A affects sorting and conformation of the sodium-dependent glucose co-transporter SGLT1.
abstract: In Chinese hamster ovary cells expressing rabbit sodium-dependent glucose transporter (rbSGLT1) protein kinase A (PKA) activators (forskolin and 8-Br-cAMP) stimulated alpha-methyl D-glucopyranoside uptake. Kinetic analysis revealed an increase in both V(max) and affinity of the transport. Immunohistochemistry and biotinylation experiments showed that this stimulation was accompanied by an increased amount of SGLT1 localized into the plasma membrane, which explains the higher V(max) of the transport. Cytochalasin D only partly attenuated the effect of forskolin as did deletion of the PKA phosphorylation site of SGLT1 in transient transfection studies. Experiments using an anti-phosphopeptide antibody revealed that forskolin also increased the extent of phosphorylation of SGLT1 in the membrane fraction. These results suggested that regulation of SGLT1 mediated glucose transport involves an additional direct effect on SGLT1 by phosphorylation. To evaluate this assumption further, phosphorylation studies of recombinant human SGLT1 (hSGLT1) in vitro were performed. In the presence of the catalytic subunit PKA and [(32)P] ATP 1.05 mol of phosphate were incorporated/mol of hSGLT1. Additionally, phosphorylated hSGLT1 demonstrated a reduction in tryptophan fluorescence intensity and a higher quenching by the hydrophilic Trp quencher acrylamide, particularly in the presence of D-glucose. These results indicate that PKA-mediated phosphorylation of SGLT1 changes the conformation of the empty carrier and the glucose carrier complex, probably causing the increase in transport affinity. Thus, PKA-mediated phosphorylation of the transporter represents a further mechanism in the regulation of SGLT1-mediated glucose transport in epithelial cells, in addition to a change in surface membrane expression.
authors.affiliation: Max Planck Institute of Molecular Physiology, Otto-Hahn Strasse 11, 44227 Dortmund, Germany.
authors.forename: Supriya
authors.initials: S
authors.lastname: Subramanian
authors.forename: Petra
authors.initials: P
authors.lastname: Glitz
authors.forename: Helmut
authors.initials: H
authors.lastname: Kipp
authors.forename: Rolf K H
authors.initials: RK
authors.lastname: Kinne
authors.forename: Francisco
authors.initials: F
authors.lastname: Castaneda
mesh_terms: D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D008757:Methylglucosides / Q000378:metabolism; D010766:Phosphorylation; D011817:Rabbits; D051269:Sodium-Glucose Transporter 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D008757:Methylglucosides; D051269:Sodium-Glucose Transporter 1; D017868:Cyclic AMP-Dependent Protein Kinases