Differentiation of two classes of "A" system amino acid transporters.
Injection of mRNA from GF-14 cells (a mouse lymphocyte cell line) into Xenopus oocytes led to the expression of a transport activity characteristic of amino acid transport system A, i.e., Na+ dependent and recognizing aminoisobutyric acid and its methyl derivative (AIB and meAIB). Sucrose density gradient fractionation of GF-14 mRNA showed that the maximum level of expression of activity was associated with a 2.2-kb RNA fraction. Pretreatment of GF-14 cells with insulin caused a twofold increase in system A transport activity in the cells themselves and the mRNA from the insulin-treated cells induced a comparable increase in A system transport over control mRNA when expressed in oocytes. mRNA from cells treated with insulin in presence of actinomycin D did not show a response to insulin, suggesting that GF-14 cells synthesized new transporter in response to insulin. Similar experiments with mRNA from the Ehrlich ascites cells, showed that expression of system A type transport was associated chiefly with a 4.2-kb mRNA fraction. Although insulin treatment of Ehrlich cells also caused an increase in A system transport activity in the cells themselves (nearly twofold), the response to insulin was not blocked by actinomycin D or cycloheximide. Moreover, mRNA from insulin-treated and untreated Ehrlich cells gave the same level of expression in oocytes of A type amino acid transport. In contrast to GF-14 cells, Western blots of plasma membranes showed that insulin treatment of Ehrlich cells increased the amount therein of a 120- to 130-kDa peptide, previously associated with amino acid transport (Proc. Natl. Acad. Sci. USA 85, 7877, 1988), suggesting that in the Ehrlich cells, but not in GF-14, cells this polypeptide was translocated from intracellular sites in response to insulin. The data associate two distinctly different responses to insulin with A-type amino acid transporters and are consistent with the existence of more than a single A type amino acid transporter in mouse cells.
Lin
G
G
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
McCormick
J I
JI
Johnstone
R M
RM
D000596:Amino Acids / Q000378:metabolism*; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism; D002352:Carrier Proteins / Q000145:classification* / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D008214:Lymphocytes / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D012333:RNA, Messenger / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D014981:Xenopus
D000596:Amino Acids; D000621:Aminoisobutyric Acids; D002352:Carrier Proteins; D007328:Insulin; D008565:Membrane Proteins; D012333:RNA, Messenger; D003609:Dactinomycin; C100049:2-aminoisobutyric acid; D003513:Cycloheximide; D012964:Sodium