Isoform-specific subcellular targeting of glucose transporters in mouse fibroblasts.

Abstract

Abstract. GLUTI, the erythrocyte glucose transporter, and GLUT4, the adipose/muscle transporter, were each expressed in NIH-3T3 cells by retrovirus-mediated gene transfer. In fibroblasts overexpressing GLUT1, basal as well as insulin-stimulated deoxyglucose uptake was increased. Expression of GLUT4 was without affect on either basal or hormone stimulated hexose uptake. Localization of each of the transporters by indirect immunofluorescence revealed that, whereas GLUTI was found primarily on the cell surface, GLUT4 was directed to vesicles in a perinuclear distribution and throughout the cytoplasm. The GLUT4-containing compartment represented neither Golgi complex nor lysosomes, as evidenced by the failure of 1gp110 or Golgi mannosidase to co-localize. However, ONE of the best-characterized actions of the anabolic hormone insulin is its ability to rapidly and reversibly increase the rate of glucose metabolism in pe ripheral target tissues (8). The flux of sugars across the plasma membrane ofmammaliancells is catalyzed by a class of integral membrane glycoproteins, the facilitated hexose transporters . It is this process which is the primary site of action by which insulin modulates glucose uptake . A number of years ago, it became clear that in the basal state a substantial number of glucose transporters in adipose tissue and probably muscle reside within the cell, where they are inactive presumably solely because of their inaccessibility to extracellular sugars (36) . Shortly after exposure to insulin, the adipose cell redistributes its glucose transporters such that a greater number are located on the cell surface and contribute to the influx of hexoses. Whereas it remains unclear as to whether the "translocation" model explains all of the increase in hexose uptake in response to hormone, redistribution certainly occurs and is important to the overall augmented glucose flux. Therefore, a major part of understanding the mechanism by which insulin regulates hexose metabolism involves characterizing the pathways of subcellular trafficking of glucose transport proteins .Recently, the existence of a gene family encoding related glucose transport proteins has been firmly established (4) . The two isoforms relevant to the experiments discussed be- there was substantial overlap between the distribution of GLUT4 and the transferrin receptor, and some colocalization of the transporter isoform with the mannose-6-phosphate receptor. In addition, when FITCwheat germ agglutinin bound to the cell surface was allowed to internalize at 37°C, it concentrated in vesicular structures coincident with GLUT4 immunoreactivity.These data establish that GLUTI and GLUT4 contain within their amino acid sequences information which dictates targeting to distinct cellular compartments. Moreover, GLUT4 can be recognized by those cellular factors which direct membrane proteins to the endosomal pathway. low are GLUTI and GLUT4. cDNAs encoding the former were originally cloned from human hepatoma and rat brain, and probably also code for the wel...

Identification of the carboxy terminus as important for the isoform-specific subcellular targeting of glucose transporter proteins.

“…NIH 3T3 cells plated on glass coverslips 2 d before an experiment were washed with PBS and fixed in 3 % paraformaidehyde for immunnflnorescence using rabbit polyclonal antisera directed against the carboxy terminus of GLUT1 or GLUT4 (17). For some experiments, anisomycin (100 #M) or cycloheximide (20 #g/ml) was present for 0-4 h before fixation.…”

“…The retrovirai expression vector pDO-R (renamed pDOJ, a gift of Dr. Connie Cepko, Harvard Medical School, Boston, MA) has been described previously, as have the plasmids pDOJ-GT (the rat GLUTI cDNA inserted into DOJ) and pDOJ-SM (the rat GLUT4 cDNA inserted into DOJ) (17). Chimeric transporters were constructed by fusing portions of the GLUT1 and GLUT4 cDNAs at common restriction sites either present in the wild-type sequence or engineered by polymerase chain reaction-based mutagenesis (13,14,30).…”

“…Total cellular membranes were prepared and subjected to SDS-PAGE and Western blot analysis as described (17) except that 1% low-fat dried milk powder was used as a blocking agent. Blots were quantitated using a Molecular Dynamics (Sunnyvale, CA) phosphorimager equipped with ImageQuant software.…”

“…Cells transfected or infected with retrovirus containing the neo resistance gene were maintained in medium supplemented with 200 #g/ml active concentration Geneticin ((3418; GIBCO-BRL, Gaithersburg, MD). Retrovirus was harvested from pools of stably transfected Psi2 cells as described previously (17) and used to infect NIH 3T3 and PC12 ceils. Colonies of G418-resistant cells were identified 10-14 d after retrovirus infection and were expanded singularly as clones or together as pools in 200/~g/ml G418.…”

“…When cDNAs were introduced into minimally insulinresponsive cell lines, the transporters were found in distinct subcellular locales: predominantly perinuclear for GLUT4 and cell surface for GLUT1. The two isoforms have been expressed in mouse 3T3-L1 and NIH 3T3 fibroblasts (12,17), hamster CHO cells (23,26), human HepG2 cells (12) rat PCI2 cells (18). Even in such diverse cell types, each isoform shows a characteristic pattern of localization.…”