Swainsonine对胰岛素受体功能影响的研究

以Ｓｗａｉｓｏｎｉｎｅ（Ｓｗ）作为高尔基体Ｎ－糖链加工酶系中α－甘露糖苷酶ＩＩ的特异抑制剂。研究Ｎ－糖链结构和胰岛素受体（Ｉｎｓ－Ｒ）功能的关系,发现Ｓｗ不影响细胞生长和３Ｈ－亮氨酸参入ＳＭＭＣ７７２１细胞,但明显促进３Ｈ－甘露糖参入细胞总糖蛋白和表面糖蛋白,并使后者的ＣｏｎＡ强结合组分显著增加,提示Ｓｗ使Ｉｎｓ－Ｒ的Ｎ－糖链变成杂合型及高甘露糖型,胰岛素结合试验后作Ｓｃａｔｃｈａｒｄ分析,发现Ｓ     

佛波酯能改变细胞表面糖蛋白N－糖链的结构

利用标记Ｎ－糖链的凝集素亲和层析法研究了佛波醇肉桂酸乙酸酯（ＰＭＡ）对人肝癌细胞ＳＭＭＣ－７７２１表面糖蛋白上Ｎ－糖链结构的影响,发现１００ｎｍｏｌ／Ｌ的ＰＭＡ处理５天后,可使细胞表面Ｎ－糖链中高甘露糖型和杂合型以及四天线、Ｃ２Ｃ２,６三天线复杂型的比例增高,而二天线复杂型降低。此结果与我们曾报道的视黄酸（ＲＡ）和双丁配环磷酸腺苷（ｄｂ－ｃＡＭＰ）对该细胞表面Ｎ－糖链的影响相反。因ＲＡ和ｄｂ－ｃＡＭＰ是ＳＭＭＣ－７７２１细胞的分化诱导剂,可抑制细胞生长;而ＰＭＡ是该细胞的增殖促进剂,故细胞表面Ｎ－糖链的变化与细胞的分化和增殖密切相关。     

视黄酸对人肝癌细胞表面N糖链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切糖苷酶处理研究了在视黄酸作用１－５天过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ糖结构的变化。结果表明,ＲＡ促进＾３Ｈ－甘露糖参入细胞表面Ｎ糖链,使高甘露糖型Ｎ糖的百分比下降,复杂型百分比长升,并促进二天线Ｎ糖链的生物合成,使多天线特别是四天线和Ｃ２,Ｃ２１ｂ三天线Ｎ糖链的合成减少。结果提示,Ｎ糖链结构的这些变化可能是ＲＡ诱导ＳＭＭＣ－７７２１细胞向正常方向     

佛波酯能改变细胞表面糖蛋白N—糖链的结构

利用标记Ｎ－糖链的凝集素亲和层析法研究了佛波醇肉桂酸乙酸酯对人肝癌细胞ＳＭＭＣ－７７２１表面糖蛋白上Ｎ－糖链结构的影响,发现１００ｎｍｏｌ／Ｌ的ＰＭＡ处理５天后,可使细胞表面Ｎ－糖链中高甘露糖型和杂合型以及四天线,Ｃ２Ｃ２,６三天线复杂型的比例增高,而二天线复杂型降低,此结果与我们曾报道的视黄酸和双丁酰环磷酸腺苷对该细胞表面Ｎ－糖链的影响相反。因ＲＡ和ｄｂ－ｃＡＭＰ是ＳＭＭＣ－７７２１细胞的分化诱     

地衣霉素对细胞膜表面运铁蛋白受体功能的影响

应用抑制糖蛋白Ｎ－糖链合成的地衣霉素处理ＳＭＭＣ－７７２１人肝癌细胞,３Ｈ甘露糖掺入实验显示细胞表面糖蛋白Ｎ－糖链的合成受到显著抑制,但细胞膜表面运铁蛋白受体内吞再循环的过程无显明变化,进一步的研究表明受体与运铁蛋白的亲和力亦无改变,但细胞膜表面运铁蛋白受体数减少。结果提示用地衣霉素处理细胞后,在内质网合成的无Ｎ－糖链的运铁蛋白受体影响其运输到细胞膜表面表达。     

双丁酰环烯酸腺苷对人肝癌细胞株SMMC—7721表面N—糖…

本文采用系列凝集素柱层析法,并配合外切糖苷酶研究了在双丁酰环磷酸腺苷（ｄＢ－ｃＡＭＰ）作用１－５过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ－糖链类型及复杂型糖链天线数的变化。结果表明,ｄＢ－ｃＡＭＰ促进＾３Ｈ－Ｍａｎ参人细胞表面Ｎ－糖链,使高甘露糖型Ｎ－糖链的百分比下降,并促进二天线Ｎ－糖链的生物合成,使多天线特别是四天线和Ｃ２Ｃ２Ｃ６三天线Ｎ－糖链的百分比减少。结果提示,Ｎ－糖链结构的这些变     

视黄酸对胰岛素受体表达的影响

ＳＭＭＣ－７７２１细胞经１０μｍｏｌ／Ｌ视黄酸（ＲＡ）处理１－５天后,完整细胞或纯化胰岛素受体（Ｉｎｓ－Ｒ）对胰岛素（Ｉｎｓ）的结合容量逐日降低。＾％３５Ｓ－甲硫氨酸参人Ｉｎｓ－Ｒ明显减少,特别是β亚基。部分纯化Ｉｎｓ－Ｒ的酷氨酸蛋白激酶（ＴＰＫ）活力也随ＲＡ处理时间延长而逐步降低。结果表明ＲＡ降低Ｉｎｓ－Ｒ的表达。但ＲＡ并不改变Ｉｎｓ－Ｒ与Ｉｎｓ结合的亲和力和它的负协同效应。     

地衣霉素对细胞膜表面运铁蛋白受体功能的影响

应用抑制糖蛋白Ｎ－糖链合成的地衣霉素处理ＳＭＭＣ－７７２１人肝癌细胞,３Ｈ甘露糖掺入实验显示细胞膜表面糖蛋白Ｎ－糖链的合成受到显著抑制,但细胞膜表面运铁蛋白受体内吞再循环的过程无显明变化,进一步的研究表明受体与运铁蛋白的亲和力亦无改变,但细胞膜表面运铁蛋白受体数减少。结果提示用地衣霉素处理细胞后,在内质网合成的无Ｎ－糖链的运铁蛋白受体影响其运输到细胞膜表面表达。     

双丁酰环磷酸腺苷耐人肝癌细胞株SMMC－7721表面N－糟链类型及天线数的影响

本文采用系列凝集素柱层析法,并配合外切精苷酶研究了在双丁酰环磷酸腺苷（ｄＢ－ｃＡＭＰ）作用１～５天过程中人肝癌细胞株ＳＭＭＣ－７７２１细胞表面Ｎ－糖链类型及复杂型糖链天线数的变化。结果表明,ｄＢ－ｃＡＭＰ促进３Ｈ—Ｍａｎ参入细胞表面Ｎ－糖链,使高甘露糖型Ｎ－糖链的百分比下降,并促进二天线Ｎ－糖链的生物合成。使多天线特别是四天线和Ｃ２Ｃ２Ｃ６三天线Ｎ－糖链的百分比减少．结果提示,Ｎ－糖链结构的这些变化可能是ｄＢ－ｃＡＭＰ诱导ＳＭＭＣ－７７２１细胞向正常方向分化的结果。     

衣霉素对人肝癌细胞表面胰岛素受体的影响

衣霉素是一种糖蛋白Ｎ－连接型糖链合成的抑制剂。它可抑制人肝癌细胞株ＳＭＭＣ－７７２１的生长,其抑制率和剂量及处理时间有相关性。衣霉素处理细胞１８ｈ尚可显著抑制＾３Ｈ－亮氨酸的参入。这些标记化合物的参入抑制都有量效关系。０．１μｇ／ｍＬ衣霉素处理１８ｈ后,细胞表面胰岛素受体和胰岛素的结合容量下降,而对照细胞和处理细胞的胰岛素竞争结合曲线基本平行。这主要由于衣霉素抑制新合成的胰岛素受体的糖基化所致。我     

N-glycosylation is critical for the stability and intracellular trafficking of glucose transporter GLUT4

The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wild-type GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K(m) or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.     

Characterization of affinity-purified insulin receptor/kinase. Effects of dithiothreitol on receptor/kinase function

The insulin receptor/kinase was purified to near homogeneity from human placenta. The purified kinase exhibited a specific activity of 300 nmol/min/mg of protein at 30 degrees C using the synthetic peptide, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, as substrate in the presence of insulin. Treatment of the receptor/kinase with dithiothreitol (DTT) reduced insulin binding by 40-50% and also inhibited tyrosine kinase activity. Phosphorylation and activation of the receptor/kinase did not prevent the DTT-induced loss of binding but completely protected it from the deleterious effects of reducing agent on enzymic activity. Analyses of the structure of the receptor/kinase following phosphorylation and treatment with DTT indicated that the class I disulfide bonds were reduced under the conditions employed, but the tetrameric structure of the receptor/kinase was essentially unaltered. These findings indicate that intact class I disulfides are required for insulin binding but are not necessary for maintenance of the preactivated kinase. DTT was also found to enhance the autoactivation of the insulin receptor/kinase and to promote the reversal of the autophosphorylation reaction. Thus disulfide bonds appear to have multiple roles in the function of the insulin receptor/kinase.     

糖链结构不同的纤连蛋白与HT1080细胞结合研究

人血浆纤连蛋白（Ｆｉｂｒｏｎｅｃｔｉｎ,Ｆｎ）与人胎盘纤连蛋白两者在肽链结构上基本相同,但人血浆Ｆｎ的Ｎ－糖链结构为二天线结构,而人胎盘Ｆｎ不仅Ｎ－糖链的数量增加,同时还含有多天线结构,分别用~（１２５）Ｉ标记这两种具有不同糖链结构的Ｆｎ,观察两者与ＨＴ１０８０细胞的饱和结合的亲和性,结果发现,在４℃,人血浆Ｆｎ与ＨＴ１０８０细胞的饱和结合为１２９ｎｇ／１０~５细胞,解离常数为２．８３×１０~（－８）ｍｏｌ／Ｌ,人胎盘Ｆｎ与ＨＴ１０８０细胞的饱和结合为１３３ｎｇ／１０~６细胞,解离常数为２．６４×１０~（－８）ｍｏｌ／Ｌ．因而,人血浆Ｆｎ与人胎盘Ｆｎ上Ｎ－糖链的不同并未影响其与受体的结合．     

衣霉素对人肝癌细胞表面胰岛素受体的影响

衣霉素(Tunicamycin)是一种糖蛋白N-连接型糖链合成的抑制剂。它可抑制人肝癌细胞抹SMMC-7721的生长,其抑制率和剂量及处理时间有相关性。衣霉素处理细胞18h尚可显著抑制~3H-甘露糖和~3H-氨基葡萄糖参入细胞,但仅轻度抑制~3H-亮氨酸的参入。这些标记化合物的参入抑制都有量效关系。0.1gg/mL衣霉素处理18h后,细胞表面胰岛素受体和胰岛素的结合容量下降,而对照细胞和处理细胞的胰岛素竞争结合曲线基本平行。这主要由于衣霉素抑制新合成的胰岛素受体的糖基化所致。我们对糖基化障碍引起细胞膜胰岛素受体结合容量降低的机理作了讨论。     

Insulin receptor binding kinetics: modeling and simulation studies

Biological actions of insulin regulate glucose metabolism and other essential physiological functions. Binding of insulin to its cell surface receptor initiates signal transduction pathways that mediate cellular responses. Thus, it is of great interest to understand the mechanisms underlying insulin receptor binding kinetics. Interestingly, negative cooperative interactions are observed at high insulin concentrations while positive cooperativity may be present at low insulin concentrations. Clearly, insulin receptor binding kinetics cannot be simply explained by a classical bimolecular reaction. Mature insulin receptors have a dimeric structure capable of binding two molecules of insulin. The binding affinity of the receptor for the second insulin molecule is significantly lower than for the first bound insulin molecule. In addition, insulin receptor aggregation occurs in response to ligand binding and aggregation may also influence binding kinetics. In this study, we develop a mathematical model for insulin receptor binding kinetics that explicitly represents the divalent nature of the insulin receptor and incorporates receptor aggregation into the kinetic model. Model parameters are based upon published data where available. Computer simulations with our model are capable of reproducing both negative and positive cooperativity at the appropriate insulin concentrations. This model may be a useful tool for helping to understand the mechanisms underlying insulin receptor binding and the coupling of receptor binding to downstream signaling events.     

Importance of the solvent-exposed residues of the insulin B chain alpha-helix for receptor binding

Conjointly, the solvent-exposed residues of the central alpha-helix of the B chain form a well-defined ridge, which is flanked and partly overlapped by the two described insulin receptor binding surfaces on either side of the insulin molecule. To evaluate the importance of this interface in insulin receptor binding, we developed a new powerful method that allows us to introduce all the naturally occurring amino acids into a given position and subsequently determine the receptor binding affinities of the resulting insulin analogues. The total amino acid scanning mutagenesis was performed at positions B9, B10, B12, B13, B16, and B17, and the vast majority of the insulin analogue precursors were expressed and secreted in amounts close to that of the wild-type (human insulin) precursor. The analogue binding data revealed that positions B12 and B16 were the two positions most affected by the amino acid substitutions. Interestingly, the receptor binding affinities of the B13 analogues were also markedly affected by the amino acid substitutions, suggesting that GluB13 indeed is a part of insulin's binding surface. The B10 library screen generated analogues covering a wide range of (20-340%) of relative binding affinities, and the results indicated that a structural stabilization of the central alpha-helix and thereby a more rigid presentation of the binding epitope at the insulin receptor is important for receptor recognition. In conclusion, systematic amino acid scanning mutagenesis allowed us to confirm the importance of the B chain alpha-helix as a central recognition element serving as a linker of a continual binding surface.     

Properties of the insulin receptor of rat pancreatic islet

Rat pancreatic islets have been shown to possess specific binding sites for ¹²⁵I-labeled insulin. Enzymatic and chemical modification of islets are used to reveal important structures and chemical groups for insulin binding. Pretreatment with trypsin, neuraminidase, 1-ethyl-3(3-dimethylamino)carbodiimide (a carboxyl reagent), tetranitromethane (a tyrosyl and thiol reagent), and 1,3-difluoro-4,6-dinitrobenze (modification of protein functional groups) decreased binding of insulin. This was due to the diminuation of the receptor number; in the case of trypsin-pretreatment also the receptor affinity was decreased. Inhibition of insulin binding was in each case associated with a decrease of the inhibitory effect of exogenous insulin on glucose-induced insulin secretion (not measured in the case of difluorodinitrobenzene and tetranitromethane). Phospholipase A₂ (cleavage of phospholipids) did not affect these parameters. 5,5′-dithiobis(2-nitrobenzoic acid) (Ellman's reagent) and possibly p-chloromercuribenzoate (both thiol reagents) increased the number of receptors and decreased receptor affinity, but did not influence the inhibitory effect of insulin on insulin release. It is concluded that protein functional groups, sialic acid, carboxyl and tyrosyl groups, but not phospholipids and probably not sylfhyryl groups are important for the interaction of insulin with insulin receptors of rat pancreatic islets.     

Aldosterone impairs insulin responsiveness in U-937 human promonocytic cells via the downregulation of its own receptor

In an earlier study, we have reported an inhibition of insulin receptor (IR) mRNA levels and insulin binding by aldosterone in U-937 human promonocytic cells. In the present extension of our studies, we demonstrate that this inhibition by aldosterone had no effects on basal glucose transport or on basal thymidine incorporation into DNA, while the cell responsiveness reflected by the maximal response to insulin was decreased by 23% for glucose transport and by 31% for DNA synthesis after the aldosterone treatment. We also prove that this inhibition of the insulin response by aldosterone is mediated by a downregulation of the levels of mineralocorticoid receptors (MRs) (50% decrease) and their mRNA (50% decrease). In addition, the mineralocorticoid antagonist spironolactone reversed the decrease in MR mRNA levels elicited by aldosterone, which suggests the involvement of this receptor in the process.     

Cross-linking of insulin receptors to MHC antigens in human B lymphocytes: evidence for selective molecular interactions

Molecular interactions between insulin receptors and MHC antigens were investigated in human B cells. Two B lymphoblastoid cell lines, IM-9 and 526, chosen for their high insulin binding capacity, were found to express 15,000 and 25,000 insulin receptors per cell, respectively. Insulin receptors were labeled with a 125I-photoreactive insulin analogue, and all other surface proteins by lactoperoxidase-catalyzed radioiodination. Neighbor proteins were cross-linked with a cleavable homobifunctional reagent dithio-bis-(succinimidyl propionate) (DSP) and solubilized before immunoprecipitation by anti-HLA monoclonal antibodies. Gel analysis of the precipitated proteins showed that 90% of insulin receptors precipitable by anti-insulin receptor antibodies were precipitated by anti-class I antibodies (anti-heavy chain and anti-beta 2-microglobulin) after cross-linking with 2 mM DSP. In neither IM-9- nor 526 cells could HLA antigens be precipitated by anti-insulin receptor antibodies, suggesting that the concentration of class I antigens largely exceeds the concentration of insulin receptors at the cell surface. In 526 lymphocytes, class I MHC antigens were also found to adjoin class II antigens, since both molecules could be coprecipitated with anti-HLA A, B, C and with anti-HLA-DR antibodies after chemical cross-linking. Down-regulation of insulin receptors by chronic exposure of IM-9 cells to insulin did not affect the amount of MHC molecules present on the cell surface, and conversely, class I MHC molecules were internalized in 526 cells irrespective of the presence of insulin. These results thus show that insulin receptors and MHC antigens form multimolecular complexes in the plasma membrane of cultured human B cells. These interactions, which do not appear to influence the regulation of these proteins on the cell surface, may be involved in the mechanism of hormone signaling.