转化淋巴细胞胰岛素受体酪氨酸蛋白激酶活性及细胞内源性底物的研究

本文对增殖期的淋巴细胞胰岛素依赖性酪氨酸蛋白激酶活性及内源性废物进行了分析研究。在纯化的健康人淋巴细胞中加入适量的植物血凝素(PHA),经过72h培养即成为转化淋巴细胞(增殖期淋巴细胞)。应用~(32)P参入实验,证实转化淋巴细胞胰岛素受体具有胰岛素依赖性的酪氨酸蛋白激酶活性,与未转化的对照组相比其活性增加约9倍。Scatchard分析表明转化后淋巴细胞膜表面胰岛素受体数增加3.5倍。应用抗酪氨酸磷酸酯抗体,对胰岛素作用前后的转化与未转化淋巴细胞内,酪氨酸残基磷酸化的蛋白进行了鉴定,结果表明:除了95kD受体β亚基自身磷酸化外,45kD蛋白质也明显磷酸化,我们命名它为PP45。我们认为PP45可能是淋巴细胞中胰岛素受体酪氨酸蛋白激酶的主要内源性废物,它的磷酸化是胰岛素信息传递过程级联反应的初始步骤。     

胰岛素受体酪氨酸激酶与胰岛素作用

关于胰岛素的作用机制,目前有一种假说,认为胰岛素与其受体的α亚基结合后将信息传递给β亚基,引起β亚基酪氨酸激酶自身磷酸化而激活。酪氨酸激酶可以使细胞内其他蛋白质磷酸化,从而启动一系列细胞内事件发挥胰岛素的多重调节作用。通过近六年来的深入研究,已经了解胰岛素受体酪氨酸激酶在介导胰岛素的绝大多数生物学效应中起着关键作用。本文对这方面的研究进展情况及前景作一简要综述。 (一)胰岛素受体的结构近年来采用亲和标记、免疫沉淀、亲和层析、分子克隆等技术,对胰岛素受体的结构进行了研究。现在已了解胰岛素受体分子由两个13.5万分子量的α亚基、两个9.5万分子量的β亚基,三对二硫键连接而成。胰岛素受体的α及β亚基是由一1382个氨基酸组成的前受体原     

胰岛素受体信号传递

胰岛素受体是具有酪氨酸蛋白激酶活性的膜受体。胰岛素与靶细胞相应受体结合后,引起受体酪氨酸残基自身磷酸化及β亚基酪氨酸蛋白激酶活化,后者使靶细胞内底物如IRS1或Hhc的酪氨酸残基磷酸化,酪氨酸蛋白激酶在胰岛素受体信号传递中发挥重要作用。胰岛素信号所激发的信号传递途径主要有二：一为Ras-MAP激酶途径,一为PI3－激酶途径,胰岛素的作用与此有关。     

胰岛素受体结构与功能研究概况

胰岛素受体(IR)是由两个α-亚基和两个β-亚基构成的跨膜糖蛋白。α-亚基位于细胞表面,是胰岛素结合区域。β-亚基的1/3也位于细胞表面,其余2/3则跨膜并插入胞浆中,后者是IR的活力区域,具有受胰岛素调节的酪氨酸蛋白激酶活性,此活性受多位点磷酸化的调节并表现出变构酶行为。不同组织的IR在分子结构,化学性质和生理功能上均有差异,其中脑IR代表了IR的一个结构和功能亚型。IR的生物合成类似于胰岛素的生物合成。     

β-TC3细胞膜与胰岛素受体结合G蛋白的鉴定

目的:用免疫共沉淀的方法检测β-TC3(小鼠胰岛β细胞瘤细胞)细胞膜中与胰岛素受体结合的G蛋白.方法:提取β-TC3细胞膜蛋白,通过免疫共沉淀及蛋白质印迹的方法,检测G蛋白α及β亚基的表达.结果:抗胰岛素受体抗体沉淀胰岛素受体结合的G蛋白复合物后,分别用抗胰岛素受体抗体、抗G蛋白α亚基抗体及抗G蛋白β亚基抗体,检测到胰岛素受体、G蛋白α亚基及G蛋白β亚基的表达.结论:在β-TC3细胞膜中,胰岛素受体与G蛋白共存,G蛋白α亚基及β亚基与胰岛素受体可能存在直接的相互作用.     

胰岛素受体胞外区三维结构的模建及结构功能关系

利用计算机辅助分子设计技术模建了人胰岛素受体α亚基N端三个结构域的空间结构。实验研究证明胰岛素受体结合胰岛素的主要决定要素在这三个结构域,在模建的结构中,三个结构域围成了一个很大的开放的空洞,这个空洞的体积大小足以容纳一个胰岛素分子,这个空洞可能就是胰岛素受体与底物的作用的部位,另外,突变实验研究表明在胰岛素受体中有一个结合底物的“footprint”,由4个在一级结构上不连续的片段组成,从三维结构角度看,这4个片段中,有3个出现在邻近的平行折叠股,在我们的模型中,胰岛素受体分子中有一个两性表面,这个表面位于L1结构域,并且面向三个结构域围成的空洞,这个表面可能就是受体识别胰岛素和与底物发生初始作用的部位。“foot-print”和两性表面的大部分残基是相同的。     

牛脑Ca~(2+)/CaM依赖性蛋白激酶Ⅱ的纯化和鉴定

通过一系列层析法,首次从牛脑纯化得到胶凝电泳匀一的Ca~(2+)/CaM PKⅡ。凝胶过滤法测定全酶分子量为550kD,SDS-PAGE法测定亚基分子量为55kD,推测牛脑Ca~(2+)/CaM PK Ⅱ由十个相同的亚基组成。该酶活性绝对依赖于Ca~(2+)和CaM,以63kD PDE同工酶为底物,其AC_(50)分别为0.85μmol/L和0.18μmol/L;以酪蛋白为底物,其AC_(50)分别为0.22μmol/L和0.06μmol/L。牛脑Ca~(2+)/CaM PK Ⅱ旣能催化63kD PDE同工酶等多种蛋白或酶磷酸化,又能进行自身磷酸化。该酶催化63kD PDE同工酶最大磷酸参入量为1mol/mol亚基。磷酸化型63kD PDE同工酶的Ca~(2+)的AC_(50)高于非磷酸化型。     

肥胖及2型糖尿病大鼠Alzheimer病样Tau蛋白过度磷酸化修饰及机制探讨

Tau蛋白异常过度磷酸化修饰在阿尔茨海默病(Alzheimerdisease,AD)发病机理中起非常重要的作用,而2型糖尿病是AD的风险因素之一.采用蛋白质印迹研究2型糖尿病及单纯肥胖大鼠脑中海马回tau蛋白磷酸化程度,发现在这两种大鼠模型中海马tau蛋白在多个位点上都呈现过度磷酸化状态.同时,胰岛素信号传导系统中的关键酶糖原合成激酶-3β(glycogensynthasekinase-3β,GSK-3β)活性在这两种大鼠模型的海马回中明显增高,经脑立体定位法向大鼠海马回注射GSK-3β抑制剂氯化锂(LiCl),可阻止2型糖尿病及肥胖大鼠模型中的GSK-3β激活,但仅阻止单纯肥胖大鼠海马回tau蛋白过度磷酸化.另外,海马神经细胞膜上胰岛素受体β亚基水平在两种实验模型中显著下降.研究结果表明,2型糖尿病及肥胖可能通过增高胰岛素抵抗,从而导致GSK-3β激活和tau蛋白的过度磷酸化来提高AD的发病风险.2型糖尿病脑中低下的葡萄糖代谢也可能在tau蛋白的过度磷酸化起一定作用.     

小鼠抗人胰岛素受体单克隆抗体的制备、纯化及鉴定

 M11D杂交瘤细胞株是由人胎盘细胞膜纯化所得胰岛素受体免疫BALB/C小鼠后,取其脾细胞与同系小鼠骨髓瘤细胞株NS-1细胞融合所得。该杂交瘤细胞分泌的抗体经ELISA及放射免疫沉淀法证实为胰岛素受体特异的单克隆抗体。该抗体经Protein A-Sepharose亲和层析分离、纯化,SDS-聚丙烯酰胺梯度凝胶电泳鉴定得分子量分别为53000及23000的两条区带,免疫双扩证明为IgGl。该抗体特异地沉淀125Ⅰ-人胎盘细胞膜胰岛素受体,沉淀经SDS-聚丙烯酰胺凝胶电泳后放射自显影得分子量为135000的特异显影带,与胰岛素受体α亚基分子量相同,说明M11D为抗胰岛素受体α亚基的单克隆抗体。     

花生种子发育和萌发过程中贮藏蛋白的合成和降解

以花生品种汕油5 2 3种子为材料,分离纯化花生球蛋白的41 kD和38.5 kD两种主要亚基及伴花生球蛋白的6 0.5 KD亚基并制备抗体.We stern blot分析表明,3种亚基在花生胚组织分化期的胚轴和子叶中就开始合成,其中60.5 kD亚基是最先在胚轴和子叶中大量合成和积累的贮藏蛋白,41 kD和38.5 kD亚基在随后的发育中积累量不断增加;种子萌发时这3种亚基的降解进程不一样,胚轴和子叶中41 kD和38.5kD亚基的降解均先于60.5 kD亚基.     

Purification and characterization of the human brain insulin receptor

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.     

Purification and characterization of the receptor for insulin-like growth factor I

The receptor for insulin-like growth factor I (IGF-I) was purified from the rat liver cell line BRL-3A by a combination monoclonal anti-receptor antibody column and a wheat germ agglutinin column. Analyses of these receptor preparations on reduced sodium dodecyl sulfate-polyacrylamide gels yielded protein bands of Mr 136K (alpha subunit) and Mr 85K and 94K (beta subunit). These receptor preparations bound 5 times more IGF-I than insulin, and the binding of both labeled ligands was more potently inhibited by unlabeled IGF-I than by insulin. These results indicate that these receptor preparations contained predominantly the IGF-I receptor. This highly purified receptor preparation was found to possess an intrinsic kinase activity; autophosphorylation of the receptor beta subunit was stimulated by low concentrations of IGF-I (half-maximal stimulation at 0.4 nM IGF-I). Twentyfold higher concentrations of insulin were required to give comparable levels of stimulation. A monoclonal antibody that inhibits the insulin receptor kinase was found to inhibit the IGF-I receptor kinase with the same potency with which it inhibits the insulin receptor. In contrast, monoclonal antibodies to other parts of the insulin receptor only poorly recognized the IGF-I receptor. A comparison of V8 protease digests of the insulin and IGF-I receptors again revealed some similarities and also some differences in the structures of these two receptors. Thus, the IGF-I receptor is structurally, antigenically, and functionally similar to but not identical with the insulin receptor.     

Purification of insulin receptor with full binding activity

Insulin receptor was purified 2400-fold with an overall yield of 40% from human placental membranes by affinity chromatography on wheat germ agglutinin-Sepharose and insulin-Sepharose. The receptor was eluted from insulin-Sepharose using mild conditions, eliminating urea, so that it was stable and retained full insulin-binding activity. Chromatofocusing and gel filtration analysis indicated that the receptor preparation was apparently pure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three high molecular weight protein bands with Mr = 320,000, 300,000, and 270,000 under nonreducing conditions and two major protein bands with Mr = 135,000 and 90,000 under reducing conditions. The purified receptor showed a curvilinear Scatchard plot with maximum insulin binding of 28.5 micrograms per mg of protein. In comparison, the receptor eluted from insulin-Sepharose with previously used conditions in the presence of urea resulted in maximum insulin binding of only 6 micrograms per mg of protein. This indicates that a 4-to 5-fold increase in specific activity can be obtained by using the new elution conditions.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.     

Abnormality of insulin binding and receptor phosphorylation in an insulin-resistant melanoma cell line

The insulin receptor possesses an insulin-stimulated tyrosine-kinase activity; however, the significance of receptor phosphorylation in terms of the binding and signaling function of the receptor is unclear. To help clarify this problem, we have studied insulin binding and receptor phosphorylation in a Cloudman S91 melanoma cell line and two of its variants: the wild type (1A) in which insulin inhibits cell growth, an insulin-resistant variant (111) in which insulin neither stimulates or inhibits growth, and a variant (46) in which insulin stimulates cell growth. 125I-insulin binding to intact cells was similar for the wild-type 1A and insulin-stimulated variant 46. The insulin-resistant variant 111, in contrast, showed approximately 30% decrease in insulin binding. This was due to a decrease of receptor affinity with no major difference in receptor number. When the melanoma cells were solubilized in 1% Triton X-100 and the insulin receptor was partially purified by chromatography on wheat germ agglutinin-agarose, a similar pattern of binding was observed. Phosphorylation was studied by incubation of the partially purified receptor with insulin and [gamma-32P]ATP, and the receptor was identified by immunoprecipitation and NaDodSO4 PAGE. Insulin stimulated phosphorylation of the 95,000-mol- wt beta-subunit of the receptor in all three cells types with similar kinetics. The amount of 32P incorporated into the beta-subunit in the insulin-resistant cell line 111 was approximately 50% of that observed with the two other cell lines. This difference was reflected throughout the entire dose-response curve (10(-9) M to 10(-6) M). Qualitatively similar results were obtained when phosphorylation was studied in the intact cell. Peptide mapping of the beta-subunit using tryptic digestion and reverse-phase high-performance liquid chromatography column separation indicated three sites of phosphorylation in receptor from the wild type and variant 46, but only two major sites of phosphorylation of variant 111. These data suggest that the insulin- resistant variant melanoma 111 possesses a specific defect in the insulin receptor which alters both its binding and autophosphorylation properties, and also suggests a possible role of receptor phosphorylation in both the binding and the signaling function of the insulin receptor.     

Purification of the type I insulin-like growth factor receptor from human placenta

The type I IGF receptor from human placental membranes was purified to near homogeneity by affinity chromatography on IGF I-Sepharose. SDS-polyacrylamide gel electrophoresis of the affinity purified type I IGF receptor demonstrated a high molecular weight protein with Mr greater than or equal to 300,000 under non-reducing conditions. After reduction with 2-mercaptoethanol two protein bands were found of Mr = 125,000 and 95,000, representing the alpha- and beta-subunits of the receptor molecule, respectively. A co-purification of the insulin receptor through the IGF I-affinity column could be avoided by a preincubation step with insulin.     

Purification and properties of insulin-activated nitric oxide synthase from human erythrocyte membranes

A membrane bound form of nitric oxide synthase of human erythrocytes that could be activated by insulin was purified to homogeneity by detergent solubilization of the purified membrane preparation of these cells. The purified enzyme (M(r) 230 KD) was found to be composed of one heavy chain (M(r) 135 KD) and one light chain (Mr 95 KD) held together by disulphide bond(s). Scatchard plot analysis of insulin binding to the purified enzyme showed the presence of 2 different populations of the binding sites and the activation were directly related to the hormone binding to the protein. Line weaver Burk plot of the purified enzyme showed that the stimulation of the enzymic activity by insulin was related to the decrease of K(m) with simultaneous increase of V(max). Treatment of the purified enzyme with anti insulin receptor antibody inhibited the activation of the enzyme and the binding of the hormone to the protein. Furthermore NO itself, at low concentration (<0.4 microM) activated the enzyme, but at higher concentration (>0.8 microM) had no effect on the activation. Incubation of the purified enzyme with insulin simultaneously stimulated the tyrosine kinase and nitric oxide synthase activities of the preparations, that could be inhibited by genistein (an inhibitor of tyrosine kinase). These results indicated that the insulin activated nitric oxide synthase could be the insulin receptor itself.     

Antibody against nuclear thyroid hormone receptors

Rat liver nuclear thyroid hormone receptor was purified to 700-1600 pmol T3 binding capacity/mg protein by sequentially using hydroxylapatite column, ammonium sulfate precipitation, Sephadex G-150 gel filtration, DNA-cellulose column, DEAE-Sephadex A-50 column, and heparin-Sepharose column. Serum from a mouse immunized using this purified receptor preparation caused a shift of [125I]T3-receptor peak on glycerol density gradient sedimentation from 3.4 S to approximately 7 S. [125I]T3-receptor complex was immunoprecipitated using this serum and goat anti-mouse IgG. The serum showed reduced ability to immunoprecipitate the globular T3 binding fragment with Stokes radius of 22 A produced by trypsin digestion, a receptor fragment which has core histone and hormone binding but not DNA binding activity. These data indicate the production of anti-nuclear thyroid hormone receptor antibody which mainly recognized epitopes unrelated to hormone and core histone binding domain.     

The rat liver insulin receptor

Using insulin affinity chromatography, we have isolated highly purified insulin receptor from rat liver. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the rat liver receptor contained the Mr 125,000 alpha-subunit, the Mr 90,000 beta-subunit, and varying proportions of the Mr 45,000 beta'-subunit. The specific insulin binding of the purified receptor was 25-30 micrograms of 125I-insulin/mg of protein, and the receptor underwent insulin-dependent autophosphorylation. Rat liver and human placental receptors differ from each other in several functional aspects: (1) the adsorption-desorption behavior from four insulin affinity columns indicated that the rat liver receptor binds less firmly to immobilized ligands; (2) the 125I-insulin binding affinity of the rat liver receptor is lower than that of the placental receptor; (3) partial reduction of the rat liver receptor with dithiothreitol increases its insulin binding affinity whereas the binding affinity of the placental receptor is unchanged; (4) at optimal insulin concentration, rat liver receptor autophosphorylation is stimulated 25-50-fold whereas the placental receptor is stimulated only 4-6-fold. Conversion of the beta-subunit to beta' by proteolysis is a major problem that occurs during exposure of the receptor to the pH 5.0 buffer used to elute the insulin affinity column. The rat receptor is particularly subject to destruction. Frequently, we have obtained receptor preparations that did not contain intact beta-subunit. These preparations failed to undergo autophosphorylation, but their insulin binding capacity and binding isotherms were identical with those of receptor containing beta-subunit. Proteolytic destruction and the accompanying loss of insulin-dependent autophosphorylation can be substantially reduced by proteolysis inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties and partial purification of the detergent-solubilized insulin receptor A demonstration of negative cooperativity in micellar solution

Turkey erythrocytes possess insulin receptors with binding properties very similar to those of mammalian insulin receptors. In the present study, the insulin receptor of the avian erythrocyte has been solubilized in Triton X-100, extensively characterized and partially purified, and its properties compared to those of the membrane-bound receptor.The solubilized insulin receptor has a Stokes radius of 70 Å and an apparent molecular weight of 300 000 in 0.05% Triton. The binding of insulin to the soluble receptor was very similar to the binding observed with the membrane-bound receptor. Thus, binding was markedly temperature dependent for both the soluble and membrane-bound forms, although the kinetics of binding were slower with the soluble receptor. Both forms of the receptor also showed a sharp pH optimum; however, solubilization produced a shift from maximal binding at pH 7.8 to pH 7.3. The soluble receptor also retained insulin analog specificity, ion sensitivity and negative cooperativity. The soluble receptor did not appear to degrade either bound or free insulin.On DEAE-cellulose chromatography the receptor eluted as a single peak. The specific activity of this partially purified preparation was 25–30 pmol/mg protein (about 500-fold enrichment over crude extract and 5-fold over highly purified membranes). Extensive attempts to purify further the receptor by gel filtration, carboxymethyl-cellulose chromatography and affinity chromatography resulted in either a very low yield or only modest enrichment. Purification was also complicated because the receptor was easily denatured; about 40% of the activity was lost after a 90-min exposure to 3 M urea or pH 4.5.