Biomedical subjects

O M Rosen

Publications and source records attributed to O M Rosen.

At least 19 recordsLinked to original sources

PubMed1991

The role of insulin receptor autophosphorylation in signal transduction.

We have examined the role of autophosphorylation in insulin signal transmission by oligonucleotide directed mutagenesis of seven potential tyrosine autophosphorylation sites in the human insulin receptor. Chinese hamster ovary cells transfected with these receptors were analyzed for insulin stimulated 2-deoxyglucose uptake, thymidine incorporation, endogenous substrate phosphorylation, and in vitro kinase activity. We found that phosphorylation on tyrosine residues 953, 1316, and 1322 were not necessary for receptor-mediated signal transduction. Mutation of tyrosine 960 reduced but did not abolish the signaling capabilities of the receptor. Finally, the simultaneous mutation of tyrosine residues 1146, 1150, and 1151 (the numbering system is that of Ullrich et al. (Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761) resulted in a biologically inactive receptor, suggesting that the insulin receptor can be inactivated by removal of key autophosphorylation sites.

Animals↗

PubMed1991

Phosphorylation of the insulin receptor by a casein kinase I-like enzyme.

A serine protein kinase that phosphorylates the beta-subunit of the insulin receptor has been partially purified 5,000-fold from HeLa cell membranes. The enzyme has been purified by ion-exchange and hydroxylapatite chromatography and sucrose gradient centrifugation; it has an apparent molecular weight of 36,000-43,000 daltons. It exhibits the following properties: (a) it catalyzes the phosphorylation of the autophosphorylated insulin receptor more efficiently than the nonautophosphorylated insulin receptor, (b) it decreases insulin receptor phosphorylation of tubulin but has no effect on insulin receptor phosphorylation of microtubule-associated proteins or reduced and carboxyamidomethylated lysozyme. The enzyme also phosphorylates casein and ribosomal protein S6 and shares many properties with casein kinase I: (a) similar molecular weight, (b) utilization of ATP but not GTP as phosphoryl donor, and (c) sensitivity to inhibition by heparin. Based on several criteria the receptor serine kinase is neither protein kinase C nor the cAMP-dependent protein kinase.

Animals↗

PubMed1991

Protein kinase C isotypes and signaling in neutrophils. Differential substrate specificities of a translocatable calcium- and phospholipid-dependent beta-protein kinase C and a phospholipid-dependent protein kinase which is inhibited by long chain fatty acyl coenzyme A.

Neutrophils possess a classical Ca2+, phosphatidyl serine (PS) and diglyceride (DG)-dependent protein kinase C (beta-PKC) which was translocatable from cytosol to membrane in response to elevated Ca2+ in the physiologic range or to pretreatment with phorbol myristate acetate (PMA). The translocatable beta-PKC was purified from neutrophil membranes prepared in the presence of Ca2+, eluted with EGTA and subjected to hydroxyapatite chromatography. An 80-kDa protein possessing Ca/DG/PS-dependent histone phosphorylating activity was recognized by a monoclonal antibody to beta-PKC but not to alpha-PKC or gamma-PKC. A cytosolic kinase activity remaining after Ca(2+)-induced translocation of beta-PKC was dependent on PS and DG but did not require Ca2+. This novel Ca(2+)-independent, PS/DG-dependent kinase, termed nPKC, eluted from hydroxyapatite between alpha-PKC and beta-PKC, ran as a 76-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and was reactive to a polyclonal consensus antibody but not to monoclonal antibodies to alpha-PKC, beta-PKC, or gamma-PKC. Long chain fatty acyl-CoA, but not the corresponding free fatty acids, inhibited nPKC in the 1-10 microM range. The chemotactic peptide fMet-Leu-Phe triggered prompt but transient increases in neutrophil long chain fatty acid acyl-CoA, suggesting that nPKC is regulated by fatty acyl-CoA as well as DG during neutrophil activation. Purified beta-PKC phosphorylated a number of cytosolic proteins in a Ca(2+)-dependent manner, including a major 47-kDa cytosolic protein, which may be implicated in superoxide anion generation. In contrast, nPKC did not phosphorylate the 47-kDa protein, but phosphorylated numerous cytosolic proteins in a Ca(2+)-independent manner, including a 66-kDa protein which was not phosphorylated by beta-PKC. Differences in location, substrate specificity, and cofactor dependence between nPKC and beta-PKC suggest these kinases may play selective roles in the activation sequence of the neutrophil.

Acyl Coenzyme A↗

PubMed1991

Expression of inducible membrane-anchored insulin receptor kinase enhances deoxyglucose uptake.

We have transfected mouse L cells with a recombinant membrane-anchored insulin receptor kinase (called MARK), under the transcriptional control of a glucocorticoid-responsive element. The transfected construct includes only 15 extracellular residues, with the transmembrane and intracellular kinase domains of the human insulin receptor cDNA (amino acid residues -27 to 12 and 915 to 1343 (see Ullrich, A., Bell, J. R., Chen, E. Y., Herrera, R., Petruzzelli, L. M., Dull, T. J., Gray, A., Coussens, L., Liao, Y. C., Tsubokawa, M., Mason, A., Seeburg, P. H., Grunfeld, C., Rosen, O. M., and Ramachandran, J. (1985) Nature 313, 756-761), predicted Mr = 56,000 with signal sequence, 53,000 without). Transfected cells which are exposed to dexamethasone express two proteins of approximate Mr = 54,000 which (a) react with anti-peptide antisera raised to human insulin receptor sequences, (b) localize to the membrane fraction, and (c) possess ligand (insulin)-independent tyrosine kinase activity. In extracts of steroid-treated MARK cells, a phosphotyrosine-containing protein of Mr = 185,000 is detected, which corresponds in size to a known endogenous substrate for the insulin receptor. Control studies were performed with the nontransfected parent line, and with L cells transfected with an inactive human insulin receptor protein tyrosine kinase. Dexamethasone induced no change in the proteins detected by an anti-phosphotyrosine antibody in the two control lines. In studies of deoxyglucose uptake, dexamethasone stimulated increase in deoxyglucose uptake 2-fold in the MARK cells compared to the same cells studied in the absence of dexamethasone. Dexamethasone had no effect in the control cell lines. These studies demonstrate that a membrane-anchored insulin receptor kinase, devoid of virtually the entire extracellular domain of the insulin receptor, is sufficient to induce enhanced deoxyglucose uptake.

Animals↗

PubMed1991

A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs.

We show that D- but not L-hexoses modulate the accumulation of radioactive vinblastine in injected Xenopus laevis oocytes expressing the murine Mdr1b P-glycoprotein. We also show that X. laevis oocytes injected with RNA encoding the rat erythroid/brain glucose transport protein (GLUT1) and expressing the corresponding functional transporter exhibit a lower accumulation of [3H]vinblastine and show a greater capacity to extrude the drug than do control oocytes not expressing the rat GLUT1 protein. Cytochalasin B and phloretin, two inhibitors of the mammalian facilitative glucose transporters, can overcome the reduced drug accumulation conferred by expression of the rat GLUT1 protein in Xenopus oocytes but have no significant effect on the accumulation of drug by Xenopus oocytes expressing the mouse Mdr1b P-glycoprotein. These drugs also increase the accumulation of [3H]vinblastine in multidrug-resistant Chinese hamster ovary cells. Cytochalasin E, an analog of cytochalasin B that does not affect the activity of the facilitative glucose transporter, has no effect on the accumulation of vinblastine by multidrug-resistant Chinese hamster cells or by oocytes expressing either the mouse Mdr1b P-glycoprotein or the GLUT1 protein. In all three cases, the drug verapamil produces a profound effect on the cellular accumulation of vinblastine. Interestingly, although immunological analysis indicated the presence of massive amounts of P-glycoprotein in the multidrug-resistant cells, immunological and functional studies revealed only a minor increase in the expression of a hexose transporter-like protein in resistant versus drug-sensitive cells. Taken together, these results suggest the participation of the mammalian facilitative glucose transporter in the development of drug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Functional expression of murine multidrug resistance in Xenopus laevis oocytes.

The development of multidrug resistance (MDR) is associated with the overproduction of a plasma membrane glycoprotein, P glycoprotein. Here we report the functional expression of a member of the murine mdr family of proteins and show that Xenopus oocytes injected with RNA encoding the mouse mdr1b P glycoprotein develop a MDR-like phenotype. Immunological analysis indicated that oocytes injected with the mdr1b RNA synthesized a protein with the size and immunological characteristics of the mouse mdr1b P glycoprotein. These oocytes exhibited a decreased accumulation of [3H]vinblastine and showed an increased capacity to extrude the drug compared to control oocytes not expressing the P glycoprotein. In addition, competition experiments indicated that verapamil, vincristine, daunomycin, and quinidine, but not colchicine, can overcome the rapid drug efflux conferred by the expression of the mouse P glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1990

Mn2(+)-binding properties of a recombinant protein-tyrosine kinase derived from the human insulin receptor.

The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd greater than 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in alpha-helical content. The addition of ATP to Mn2(+)-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.

Circular Dichroism↗

PubMed1990

Glucose transporters serve as water channels.

Water traverses the plasma membranes of some eukaryotic cells faster than can be explained by the water permeability of their lipid bilayers. This has led to a search for a water channel. Our previous work identified glucose transporters as candidates for such a channel. We report here that Xenopus laevis oocytes injected with mRNA encoding the brain/Hep G2, adult skeletal muscle/adipocyte, or liver forms of the glucose transporter exhibit an osmotic water permeability of their plasma membranes larger than that of untreated oocytes. The osmotic water permeability component attributable to glucose transporters increased an average of 4.8-fold in the injected oocytes. These studies provide direct evidence that the facilitative, sodium-independent mammalian glucose transporters serve as membrane water channels.

Animals↗

PubMed1990

Reconstitution of an insulin signaling pathway in Xenopus laevis oocytes: coexpression of a mammalian insulin receptor and three different mammalian hexose transporters.

We report the functional expression of the mammalian muscle-adipocyte insulin-sensitive hexose transporter in Xenopus laevis oocytes. Oocytes microinjected with RNA synthesized in vitro showed enhanced hexose transport activity compared with uninjected controls. However, like the endogenous oocyte hexose transporter, activity was stimulated only twofold by 1 microM insulin. X. laevis oocytes injected with in vitro-synthesized RNA encoding the human insulin proreceptor expressed a functionally active insulin receptor that enhanced the insulin sensitivity of injected oocytes. This increase was not observed in oocytes expressing a mutant insulin receptor that lacked protein tyrosine kinase activity. In the presence of the coexpressed human insulin receptor, insulin induced a two- to threefold increase in hexose transport. The muscle-, brain-, and liver-type hexose carriers normally expressed in tissues with different responses to insulin exhibited the same insulin sensitivity when expressed in oocytes. This was observed whether or not the insulin signal was transduced through a coexpressed human insulin receptor or the endogenous oocyte insulin-like growth factor I receptor. We conclude that the expressed human insulin receptor is able to couple efficiently with preexisting postreceptor regulatory pathways in oocytes and that the regulation of hexose transport in these cells can be mediated through the combined actions of the expressed human insulin receptor and the endogenous oocyte insulin-like growth factor I receptor.

Animals↗

PubMed1990

Insulin-receptor approaches to studying protein kinase domain.

Protein tyrosine kinase activity found in the beta-subunit of the insulin receptor provides a mechanism by which insulin binding on the outside of the cell transmits its signal across the plasma membrane into the cytosol. The autophosphorylation of the insulin receptor on tyrosyl residues activates the intrinsic tyrosine kinase of the receptor, rendering its ligand independent. Evidence suggests that phosphorylation of tyrosyl residues 1146, 1150, and 1151 in the kinase domain of the beta-subunit play a role in activation. Point mutations in the cytoplasmic portion of the beta-subunit confirm the above suggestions and indicate that additional sites are important for receptor function. We present methodology for overproducing the cytoplasmic domain of the receptor in the Baculovirus expression system. The protein, produced in insect cells and larvae, is soluble and fully active on autophosphorylation. Like the intact receptor, its autophosphorylation is intramolecular. Because greater than or equal to 10 mg of pure protein can be isolated from 10(10) insect cells infected with the recombinant Baculovirus encoding the human insulin-receptor kinase domain, sufficient enzyme is available for various studies, including physicochemical analysis. Isolation of beta-subunit defects found in the receptors of patients with various forms of diabetes mellitus also implicates the insulin-receptor kinase in insulin action. Finally, a potential model system for the genetic analysis of the insulin-insulin-receptor system with Drosophila melanogaster is noted. Conservation of the deduced amino acid sequence for both alpha- and beta-subunit sequences between humans and insects highlights the significance of this manner of signal transduction throughout nearly 1 billion years of evolution.

Amino Acid Sequence↗

PubMed1990

Hydrolysis of glycosyl-phosphatidylinositol in response to insulin is reduced in cells bearing kinase-deficient insulin receptors.

A glycosyl-phosphatidylinositol (GPI) has been previously identified that serves as a precursor of the polar head group that mimics and may mediate some of the intracellular actions of insulin. Since many of the biological activities of insulin may depend upon the activity of the insulin receptor kinase, we evaluated the requirement for this activity in insulin-dependent GPI hydrolysis. For the analysis we used stably transfected CHO cell lines, expressing either the wild-type human insulin receptor or a mutant receptor that lacks tyrosine kinase activity (Chou et al., 1987) and a stably transfected CHO cell line, expressing the wild-type human insulin-like growth factor I (IGF-1) receptor (Steele-Perkins et al., 1988). A GPI was identified in both types of transfected cells and in both sets of parental cells by metabolic labeling with [3H]glucosamine or [3H]galactose. The isolated glycolipid was sensitive to hydrolysis by phospholipase C and to deamination by nitrous acid. Insulin induced a time- and dose-dependent hydrolysis of the GPI in the parental line and in the transfected cell types. Cells bearing normal human receptors hydrolyzed up to 70% of their radiolabeled GPI within 2 min of the addition of 0.1 nM insulin, whereas parental cells and cells expressing the mutant receptor hydrolyzed only 20-30% in response to 100 nM insulin. IGF-1 (5-50 nM) had little effect on GPI hydrolysis in these cells as well as in CHO cells expressing the human IGF-1 receptor. It is concluded that insulin-dependent GPI hydrolysis is mediated by the normal but not by a kinase-deficient insulin receptor.

Animals↗

PubMed1989

Complete activation of protein kinase C by an antipeptide antibody directed against the pseudosubstrate prototope.

It has been proposed that the regulatory domain of protein kinase C contains a pseudosubstrate site between amino acid residues 19 and 36 (House, C., and Kemp, B. E. (1987) Science 238, 1726-1728). Antiserum raised against this peptide sequence has now been shown to completely activate protein kinase C in the absence of calcium and phospholipids. Pre-clearing the antiserum with resin-immobilized pseudosubstrate peptide eliminates the ability of the serum to activate protein kinase C. Activation is not the result of degradation of the enzyme to a calcium- and phospholipid-independent fragment; the activated protein kinase remains intact. Although there are minor sequence differences in the pseudosubstrate region, the three principal protein kinase C isoforms (alpha, beta, and gamma) are recognized and apparently activated by the same pseudosubstrate antiserum. These results provide strong evidence that the pseudosubstrate region, presumably by interacting with the substrate binding site, is responsible for maintaining the catalytic domain in an inactive state. We propose that incubation of protein kinase C with the pseudosubstrate antiserum renders the catalytic domain accessible to protein substrates in a manner analogous to the conformational changes induced by physiological activators such as phospholipids.

Amino Acid Sequence↗

PubMed1989

Another version of the human insulin receptor kinase domain: expression, purification, and characterization.

We have overexpressed another insulin receptor kinase molecule, which consists of residues 941-1343 inclusive of the human insulin receptor, by using the baculo-virus expression vector pVL941. Unlike the two previous preparations of insulin receptor kinase in this expression system, this molecule contains the complete unmodified sequence of the cytoplasmic domain of the human insulin receptor beta subunit. Our construct allows high-level expression of the recombinant protein in cultured Sf9 cells and in cabbage looper (Trichoplusia ni) larvae. An improved purification procedure yields greater than or equal to 95% pure protein in 55% yield from the cells. The specific activity of this purified protein is 3.5-fold greater than from the previously described baculovirus insulin receptor kinase (that included residues 946-1343 from the proreceptor). Like the latter molecule, the insulin receptor kinase molecule reported here sediments as a monomer, and its autophosphorylation occurs by an intramolecular process. Preliminary data about the spectroscopic features of the cytosolic domain of the human insulin receptor are presented.

Animals↗

PubMed1989

Functional expression of mammalian glucose transporters in Xenopus laevis oocytes: evidence for cell-dependent insulin sensitivity.

We report the functional expression of two different mammalian facilitative glucose transporters in Xenopus oocytes. The RNAs encoding the rat brain and liver glucose transporters were transcribed in vitro and microinjected into Xenopus oocytes. Microinjected cells showed a marked increase in 2-deoxy-D-glucose uptake as compared with controls injected with water. 2-Deoxy-D-glucose uptake increased during the 5 days after microinjection of the RNAs, and the microinjected RNAs were stable for at least 3 days. The expression of functional glucose transporters was dependent on the amount of RNA injected. The oocyte-expressed transporters could be immunoprecipitated with anti-brain and anti-liver glucose transporter-specific antibodies. Uninjected oocytes expressed an endogenous transporter that appeared to be stereospecific and inhibitable by cytochalasin B. This transporter was kinetically and immunologically distinguishable from both rat brain and liver glucose transporters. The uniqueness of this transporter was confirmed by Northern (RNA) blot analysis. The endogenous oocyte transporter was responsive to insulin and to insulinlike growth factor I. Most interestingly, both the rat brain and liver glucose transporters, which were not insulin sensitive in the tissues from which they were cloned, responded to insulin in the oocyte similarly to the endogenous oocyte transporter. These data suggest that the insulin responsiveness of a given glucose transporter depends on the type of cell in which the protein is expressed. The expression of hexose transporters in the microinjected oocytes may help to identify tissue-specific molecules involved in hormonal alterations in hexose transport activity.

Animals↗

PubMed1989

Insulin and insulinlike growth factor 1 (IGF-1) receptors during central nervous system development: expression of two immunologically distinct IGF-1 receptor beta subunits.

Insulin and insulinlike growth factor 1 (IGF-1) receptors are present in brain, yet their function remains obscure. Expression of these tyrosine kinase-bearing growth factor receptors during rat brain development was examined by using three antipeptide antibodies directed against epitopes in the beta subunits (AbP2, AbP4, and AbP5). All three antibodies recognized both insulin and IGF-1 receptors. Membranes were prepared from fetal brains (14 to 21 days of gestation), neonatal brain (postnatal day 1), and adult brain. Immunoblot analyses using AbP4 and AbP5 revealed a 92-kilodalton (kDa) protein that corresponded to the beta subunit of the insulin and IGF-1 receptors. Densitometric scanning of immunoblots indicated that receptor proteins were 4- to 10-fold more abundant in fetal brain membranes than in membranes from adult brain. Expression was highest during 16 to 18 days of gestation and declined thereafter to the relatively low level found in adult brain. Immunoblot analyses with AbP2 as well as ligand-activated receptor autophosphorylation revealed an additional protein of 97 kDa. This protein was phosphorylated in response to IGF-1 and was not directly recognized by AbP4 or AbP5. The covalent association of the 97-kDa protein with the 92-kDa beta subunit was indicated by the ability of AbP4 and AbP5 to immunoprecipitate both proteins under nonreducing conditions but only the 92-kDa protein after reduction. In contrast, AbP2 immunoprecipitated both proteins regardless of their association. This immunospecificity remained unchanged after deglycosylation of the isolated proteins. Two-dimensional tryptic phosphopeptide analysis showed that the 92- and 97-kDa subunits of the IGF-1 receptor are related but distinct proteins. Taken together, the data suggest that the 92- and 97-kDa subunits differ in primary amino acid sequence. Thus, two distinct beta subunits may be present in a single IGF-1 receptor in brain. These subunits have in common an epitope recognized by an antibody to the tyrosine kinase domain (AbP2) but differ in regions thought to be important in receptor kinase regulation and signal transduction.

Animals↗

PubMed1989

Banting lecture 1989. Structure and function of insulin receptors.

The actions of insulin are mediated by an integral plasma membrane protein, the insulin receptor. The processed receptor is a tetramer composed of two alpha-subunits that bind insulin and two beta-subunits that traverse the plasma membrane and are, in their cytosolic domains, protein tyrosine kinases. The insulin proreceptor cDNA has been cloned and its complete amino acid sequence deduced. The availability of cDNA permitted an analysis of both the role of protein tyrosine kinase activity in insulin action and the autophosphorylation sites that regulate kinase activity. The human cDNA probe has also been used to identify a putative Drosophila insulin receptor. This work is reviewed, and approaches that may be used to identify physiological substrates for the receptor kinase are suggested.

Animals↗

PubMed1988

Synthesis, purification, and characterization of the cytoplasmic domain of the human insulin receptor using a baculovirus expression system.

The cytoplasmic domain of the beta subunit of the human insulin receptor has been overexpressed in insect cells using the baculovirus expression system. A recombinant baculovirus (BIR-2) was constructed by inserting the human insulin proreceptor cDNA fragment that encodes the cytoplasmic domain of the receptor into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Synthesis of the protein (baculovirus insulin receptor kinase (BIRK), Mr 48,000) in BIR-2-infected Spodoptera frugiperda (Sf9) cells was detected 24 h after infection and maximal accumulation (2-3% of the cytosolic protein) was achieved 48-72 h post-infection. The expressed protein is active as a soluble protein tyrosine kinase, both in Sf9 cells and in vitro. Rapid purification to near homogeneity was accomplished by sequential chromatography on Fast-Q-Sepharose and phenyl-Superose with an overall yield of 35% and a specific activity with histone as substrate of 20 nmol/min/mg protein. Autophosphorylation activated the intrinsic kinase activity of BIRK and decreased its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using a combination of tryptic digestion and immunoprecipitation with specific antipeptide antisera, it was ascertained that 30-40% of the 32P incorporated into BIRK by autophosphorylation is in the carboxyl-terminal domain (that includes tyrosyl residues 1316 and 1322 of the human proreceptor). Of the remaining radioactivity, 75% is in the amino-terminal domain (that includes tyrosyl residues 953, 960, 972, 999, and 1075) and 25% is in the conserved autophosphorylation domain (including tyrosyl residues 1146, 1150, and 1151). Limited digestion of BIRK with trypsin yielded a fragment, Mr 38,000, that lacks the carboxyl-terminal domain. This fragment exhibits protein tyrosine kinase activity that is stimulated by autophosphorylation. The properties of the soluble, monomeric BIRK are similar to those of the intact, activated, oligomeric insulin receptor kinase with respect to specificity, immunoreactivity, divalent cation requirements, and specific activity. These observations coupled with the ease of producing 0.4 mg of purified enzyme from 100 ml of suspension culture suggest that BIRK will be useful for biochemical and biophysical analysis of the insulin receptor protein tyrosine kinase.

Chromatography↗

PubMed1988

Structure, biosynthesis, and function of the hexose transporter in Chinese hamster ovary cells deficient in N-acetylglucosaminyltransferase 1 activity.

We have used a Chinese hamster ovary cell line deficient in N-acetylglucosaminyltransferase 1 activity (Lec1) to study the effects of altered asparagine-linked oligosaccharides on the structure, biosynthesis, and function of glucose transporter protein. Immunoblots of membranes of Lec1 cells show a glucose transporter protein of Mr 40,000, whereas membranes of wild-type (WT) cells contain a broadly migrating Mr 55,000 form similar to that observed in several other mammalian tissues. The total content of immunoreactive glucose transporters in Lec1 cells is 3.5-fold greater than that of WT cells. Digestion with endoglycosidases, treatment with inhibitors of glycosylation, and interactions with agarose-bound lectins demonstrate that glucose transporters of both cell lines derive from a similar Mr 38,000 core polypeptide and that both contain asparagine-linked oligosaccharide. Transporters in Lec1 cells contain primarily "undecorated" but "trimmed" mannose-type asparagine-linked oligosaccharides, while the protein in WT cells contains a mixture of "decorated" and "trimmed" asparagine-linked oligosaccharides. Biosynthetic and turnover studies demonstrate that Lec1 cells, in contrast to WT cells, are unable fully to process the core asparagine-linked oligosaccharides of maturing glucose transporters. When radiolabeled in methionine-deficient medium both Lec1 and WT cells show similar rates of synthesis and turnover of glucose transporter proteins. It should be noted, however, that starvation for a critical amino acid may alter the ability of the cell to synthesize or degrade proteins. The abilities of Lec1 and WT cells to transport hexoses and to interact with the inhibitor cytochalasin B are very similar. The results indicate that, although altered asparagine-linked glycosylation can affect the content and biogenesis of glucose transporters, these changes do not greatly modify cellular hexose uptake. The possibility that alterations in asparagine-linked glycosylation may change the cell surface localization or acquisition of a "functional conformation" of the glucose transporter is also suggested.

Animals↗