Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes.
Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.
Wadzinski
B E
BE
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Shanahan
M F
MF
Seamon
K B
KB
Ruoho
A E
AE
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001665:Binding Sites; D005576:Colforsin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D003571:Cytochalasin B / Q000378:metabolism; D004224:Diterpenes; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation
D000345:Affinity Labels; D001386:Azides; D004224:Diterpenes; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; C054433:3-iodo-4-azidophenethylamido-7-O-succinyldeacetylforskolin; D005576:Colforsin; D003571:Cytochalasin B