Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Trypsin-treatment of isolated rat adipocytes abolishes the metabolic effects not only of insulin, but also of the insulin-like growth factors: in trypsin-treated cells, concentrations of these hormones that are otherwise maximally effective no longer stimulate 3-O-methylglucose transport and lipogenesis or inhibit epinephrine induced lipolysis. Concomitantly, the trypsin-treated adipocytes no longer display specific insulin binding. In contrast, the characteristics of the binding of the insulin-like growth factors are not grossly affected by prior trypsinization of the adipocytes. These findings add further support to the concept that the insulin-like growth factors act on glucose metabolism and antilipolysis via the insulin receptor of the adipocyte.
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PMID:Effect of trypsin treatment of rat adipocytes on biological effects and binding of insulin and insulin-like growth factors: further evidence for the action of insulin-like growth factors through the insulin receptor. 701 99

There is morphological and biochemical evidence that insulin is internalized in hepatocytes. The present study was designed to investigate the fate of the insulin receptor itself, subsequently to the initial binding step of the hormone to the hepatocyte plasma membrane. The insulin receptor was labeled with a 125I-photoreactive insulin analogue (B2[2-nitro,4-azidophenylacetyl]des-PheB1-insulin). This photoprobe was covalently coupled to the receptor by UV irradiation of hepatocytes after an initial binding step of 2-4 h at 15 degrees C. At this temperature, only limited (approximately 20%) internalization of the ligand occurred. In a second step, hepatocytes were resuspended in insulin-free buffer and further incubated for 2-4 h at 37 degrees C. After h at 37 degrees C, no significant radioactivity could be detected in non-UV-irradiated cells, whereas 12-15 % of the radioactivity initially bound remained associated to UV-irradiated cells. Morphological analysis after electron microscopy revealed that approximately 70% of this radioactivity was internalized and preferentially associated with lysosomal structures. SDS PAGE analysis under reducing conditions revealed that most of the radioactivity was associated with a 130,000-dalton band, previously identified as the major subunit of the insulin receptor in a variety of tissues. Internalization of the labeled insulin-receptor complex at the end of the 37 degrees C incubation was further demonstrated by its inaccessibility to trypsin. Conversely, at the end of the association step, the receptor (also characterized as a predominant 130,000-dalton species) was localized on the cell surface since it was cleaved by trypsin. We conclude that in hepatocytes the insulin receptor is internalized with insulin.
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PMID:Biochemical and morphological evidence that the insulin receptor is internalized with insulin in hepatocytes. 704 Apr 16

Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in total receptor level are due to changes in cell-surface receptor level is indicated by the fact that the level of trypsin-insensitive, presumably intracellular, insulin binding sites does not change appreciably upon down- and up-regulation. The effects of insulin-induced down-regulation and dexamethasone-induced up-regulation on the rates of insulin receptor synthesis and decay were assessed by the heavy-isotope density-shift technique. Cells were shifted to medium containing heavy (2H, 13C, 15N) amino acids and, at various times after the shift, light and heavy receptors solubilized from total cellular membranes were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that the insulin- and dexamethasone-induced alterations in insulin receptor level were due entirely to changes in the rate constant for receptor inactivation. The decrease in the first-order rate constant for receptor decay caused by dexamethasone is unexpected in view of the known action of steroid hormones in the induction of the synthesis of specific proteins.
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PMID:Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation. 704 73

The glucose transport activity associated with the plasma membrane-rich and Golgi-rich fractions of fat cells was determined after they were reconstituted into egg lecithin liposomes. When the two subcellular fractions were isolated under conditions that would minimize their cross-contamination, the transport activity in the plasma membrane-rich fraction was found to be increased 6.3- to 8.6-fold by insulin, which was added to cells before homogenization, and that the activity in the Golgi-rich fraction was reduced approximately to one-half. In this study, the glucose transport activity in the plasma membrane-rich fraction (either in the basal or plus insulin state) was solubilized, reconstituted, and assayed with an overall efficiency of 25-35%. Four agents known to have insulin-like effects on the glucose transport activity in intact fat cells (hydrogen peroxide, sodium vanadate, trypsin, and p-chloromercuriphenyl sulfonate) not only increased the transport activity in the plasma membrane-rich fraction, but also decreased the activity in the Golgi-rich fraction. The effect of hydrogen peroxide, unlike that of insulin, was not abolished when the insulin receptor was modified proteolytically. Upon administration of insulin to fat cells, and subsequent elimination of the hormone, the glucose transport activities associated with the plasma membrane-rich and Golgi-rich fractions were affected almost concomitantly towards opposite directions. It is proposed as a working hypothesis that translocation of the glucose transport system to the plasma membrane from the Golgi-rich fraction is the major, if not the sole, mechanism by which insulin stimulates glucose transport in fat cells.
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PMID:Evidence that translocation of the glucose transport activity is the major mechanism of insulin action on glucose transport in fat cells. 705 Jan 25

The turkey erythrocyte membrane insulin receptor was solubilized and reconstituted into vesicles composed of either soy or dimyristoyl phosphatidylcholine. Reconstitution with soy phosphatidylcholine provided a lipid environment containing 43% unsaturated fatty acids, as compared with 82% saturated fatty acids in the dimyristoyl phosphatidylcholine preparation. After reconstitution, both species of vesicles were isolated from a 2 to 30% continuous sucrose gradient at a density of 1.071 g/ml. Scatchard analysis of binding data obtained at 15 degrees C revealed that the reconstituted receptor had a greater affinity for [125I]iodoinsulin in the saturated lipid environment (Ke = 0.167 nM-1; K1 = 2.18 nm-1) than in the unsaturated lipid environment (Ke = 0.0162 nM-1; K1 = 0.479 nm-1). Low affinity binding also was increased in the saturated vesicles. These increases were paralleled by a reduction in the number of available insulin binding sites in the saturated lipid environment. There was no difference, however, in the relative affinity of the reconstituted receptor preparations for insulin or proinsulin. Electron microscopy and gel filtration indicated that the binding differences are not due to differences in vesicle size. They also are not due to differences in the orientation of the receptor within the lipid bilayer, for its sensitivity to trypsin digestion was similar in both types of vesicles. Solubilization studies with 1% beta-octylglucoside indicated, however, that the dimyristoyl phosphatidylcholine vesicles incorporated a slightly lesser amount of insulin receptor. Similar results were also observed at 37 degrees C. These results suggest that the membrane lipid environment, especially the degree of unsaturation of the phospholipid fatty acyl chains, can influence the binding properties of the insulin receptor.
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PMID:Lipid effects on the binding properties of a reconstituted insulin receptor. 705 82

Partially purified liver insulin receptors from full-term pregnant rats show decreased autophosphorylation rates if compared with receptors from virgins. We studied the molecular mechanism of this phenomenon, looking at possible structural and functional changes of several domains. The ATP-binding domain seems to be unaltered in receptors from pregnant rats since Km for ATP was similar to that observed in virgins. In contrast, the Vmax. is decreased some 45%, suggesting changes in the kinase domain. Truncation of a fragment of 10 kDa from the C-terminal tail does not normalize the kinase activity in receptors from pregnant rats, suggesting that this domain is not involved in the inhibitory regulation. Treatment with alkaline phosphatase increases the [32P]Pi incorporation into receptors from pregnant rats; however, the autophosphorylation remains lower than that observed in virgin rats. Tryptic phosphopeptide maps of phosphorylated receptors show that the same phosphopeptides are present in receptors from virgin and pregnant rats. However, the progression through the autoactivation cascade in the kinase domain is impaired in receptors from pregnant rats. Differences in the cleavage by trypsin at the two alternative sites in the kinase domain were observed, indicating possible structural changes in receptors from pregnant rats that could be related to the impairment of the autoactivation cascade. Integrity of the alpha- and beta-subunits, as well as differential expression of the two receptor isotypes, were shown to be unaltered. We conclude that (1) the decreased autophosphorylation rate of the liver insulin receptor from pregnant rats is associated with the impairment of its autoactivation cascade, probably as a consequence of the basal Ser/Thr phosphorylation; and (2) the inhibition of the autoactivation cascade does not account for the overall inhibition of autophosphorylation observed in receptors from pregnant rats.
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PMID:Impairment of the liver insulin receptor autoactivation cascade at full-term pregnancy in the rat. 748 90

1. Treatment of intact cultured H35 cells with trypsin (1 mg/ml) for 15 min at low temperature (4 degrees C) or for 30 sec at 37 degrees C causes activation of the insulin receptor subsequently isolated from the cells. 2. Receptor activation was assessed by increased phosphotyrosine content of the beta-subunit of the receptor, and increased autophosphorylation using [32P]-ATP. 3. Treatment of the cells for 15 min at 37 degrees C however completely abolished insulin binding and all insulin receptor kinase activity. 4. These data demonstrate that proteolytic damage of the extracellular domain of the insulin receptor can render the receptor kinase inactive and lead to a cell which is unresponsive to insulin.
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PMID:Activation and inhibition of insulin receptor autophosphorylation by trypsin treatment of intact H35 cells. 768

Previous work suggests the existence of different isoforms of the insulin-like-growth-factor-1 (IGF-1) receptor in various tissues. In the present study we provide support for the concept that heterogeneous IGF-1 receptors exist in the brain and that part of the heterogeneity is derived from IGF-1 receptor hybrids formed from different beta-subunits. IGF-1 receptors were extracted from adult-rat forebrain synaptosomes and partially purified by wheat-germ agglutinin (WGA) chromatography. Hormone-binding studies in this preparation demonstrate the presence of receptors for IGF-1 and insulin. An antibody, a-RIR, specific for the rat insulin receptor was used to remove insulin receptors from the WGA extract. Studies with the immunodepleted material demonstrated two proteins of 92 and 99 kDa that are phosphorylated on tyrosine during incubation with low concentrations of IGF-1. Both proteins bound with high affinity and specificity to IGF-1 immobilized on agarose, and each underwent phosphorylation when the agarose beads were incubated with [gamma-32P]ATP and MnCl2. Two-dimensional phosphopeptide maps after exhaustive trypsin treatment of the two proteins showed significant differences in their structure as well as differences from the phosphopeptide map for the beta-subunit of the insulin receptor. The relationship of the two proteins to the IGF-1 receptor was further probed by an antibody (a-HF) raised against a specific sequence in the beta-subunit of the human IGF-1 receptor, and a polyclonal antibody raised against the liver insulin receptor (L1) which cross-reacts with the IGF-1 receptor. Both antibodies immunoprecipitated the two phosphorylated proteins. However, reduction of the receptors to form receptor dimers or monomers showed that a-HF precipitated only the 99 kDa protein, whereas L1 precipitated primarily the 92 kDa protein. In conclusion, the brain IGF-1 receptor apparently has two structurally different beta-subunits, one of 92 kDa and a second of 99 kDa. Interestingly, at least a portion of the IGF-1 receptor population has both isoforms in the same receptor.
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PMID:Distinct beta-subunits are present in hybrid insulin-like-growth-factor-1 receptors in the central nervous system. 769 Oct 55

Data presented here show that there are significant differences in the insulin binding affinity and the tyrosine kinase activity of the insulin proreceptor isoforms which contain or lack exon 11. The exon 11(+) proreceptor does not show significant variations from the mature processed insulin receptor and has only a 3- to 4-fold reduced affinity for insulin. In contrast, the exon 11(-) proreceptor showed a markedly reduced insulin binding (25- to 50-fold less) when assayed on intact cells. Upon solubilization of the cells, exon 11(-) proreceptor bound insulin with somewhat higher affinity. Mild trypsin treatment of the cells expressing either isoform of the insulin proreceptor restored insulin binding to near-normal levels. Analysis of tyrosine kinase activity revealed that the exon 11(+) proreceptor required somewhat higher concentrations of insulin than the mature processed receptor to achieve maximal autophosphorylation. The exon 11(-) proreceptor failed to fully phosphorylate even at 10(-6) M insulin. Thus, the presence or absence of this short sequence of 12 amino acids affects the folding and/or conformation of the proreceptor so as to confer altered binding of insulin. We suggest that in the absence of exon 11 the proreceptor assumes a strained conformation that disrupts the insulin binding site. Cleavage of the proreceptor at the alpha-beta-subunit junction then allows the alpha-subunit to achieve its normal binding conformation.
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PMID:Exon 11 enhances insulin binding affinity and tyrosine kinase activity of the human insulin proreceptor. 779 70

Activation of the insulin receptor, like other tyrosine kinase receptors, appears to require dimerization. We have shown previously that, even in the absence of insulin, full receptor activation can be induced by changes in the receptor transmembrane domain (TMD), suggesting that TMD dimerization is sufficient for receptor activation. To further understand the importance of the TMD in insulin receptor activation, we have inverted the entire TMD sequence including flanking basic amino acids, residue-for-residue. This mutation was predicted to alter the ability of a TMD alpha-helix to form homodimers and higher level aggregates. Despite apparently normal protein folding on either side of the membrane, this mutation caused ER retention and, for those receptors that reached the cell surface, blockade of insulin-stimulated kinase signal transmission. However, the signaling blockade could be overcome by proteolytic activation with trypsin. In contrast, shifting only the basic cytoplasmic residues to the opposite side of the TMD or mutation to neutral residues had no detectable effect on assembly, biosynthesis, topology, or signaling. These findings extend our previous observations to suggest that TMD interactions within the membrane are not only sufficient for receptor activation, but may be required. TMD interactions also appear to be necessary for oligomeric assembly and biosynthetic maturation of the insulin receptor.
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PMID:Transmembrane domain inversion blocks ER release and insulin receptor signaling. 782 53


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