The high-affinity sulfonylurea receptor: distribution, glycosylation, purification, and immunoprecipitation of two forms from endocrine and neuroendocrine cell lines.
The high-affinity sulfonylurea receptor, a novel member of the ATP-binding cassette superfamily, is one component of the ATP-sensitive K+ channel. The protein is critical for regulation of insulin secretion from pancreatic beta-cells, and mutations in the receptor have been linked to familial hyperinsulinemia, a disorder characterized by unregulated insulin release despite severe hypoglycemia. The sulfonylurea receptor is present in membranes from a number of endocrine and neuroendocrine cell lines, including HIT-T15, RINm5f, alpha TC-6, AtT-20, and GH3 cells. Two forms of the receptor are present in RINm5f and alpha TC-6 cells, with apparent SDS gel molecular masses of 140 and 150 kDa. The two forms have equally high affinity, KD approximately 3 nM, for an iodinated derivative of glyburide, an anti-diabetic sulfonylurea. The receptor is a glycoprotein; treatment of RINm5f or alpha TC-6 cells with tunicamycin reduces the 140 and 150 kDa species to a single approximately 137 kDa protein. The 140 and 150 kDa receptors bind differentially to concanavalin A and wheat germ agglutinin, and lectin-affinity chromatography is ideal for the initial stages of receptor purification. After lectin-affinity chromatography, the same methods can be applied for purifying the 150 kDa form as for the 140 kDa receptor. A transiently expressed receptor with a histidine-tagged carboxy-terminus was purified by Ni-agarose chromatography, and this variant was used to demonstrate that the 140 kDa polypeptide is full length. Anti-peptide antibodies directed against the amino-terminus of the receptor and antibodies against the nucleotide binding folds immunoprecipitate both receptor forms. The results indicate the 140 and 150 kDa receptors are differentially glycosylated forms of the same polypeptide chain.
Nelson
D A
DA
Department of Biology, University of North Carolina at Charlotte 28223, USA.
Bryan
J
J
Wechsler
S
S
Clement
J P
JP
Aguilar-Bryan
L
L
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004702:Endocrine Glands / Q000166:cytology / Q000378:metabolism*; D006031:Glycosylation; D037102:Lectins; D009490:Neurosecretory Systems / Q000166:cytology / Q000378:metabolism*; D015221:Potassium Channels / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D011233:Precipitin Tests; D011955:Receptors, Drug / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D064233:Sulfonylurea Receptors
D018528:ATP-Binding Cassette Transporters; D018076:DNA, Complementary; D037102:Lectins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D064233:Sulfonylurea Receptors