scholarly journals The endogenous functional turkey erythrocyte and rat liver insulin receptor is an α2β2 heterotetrameric complex

1990 ◽  
Vol 271 (1) ◽  
pp. 99-105 ◽  
Author(s):  
J L Treadway ◽  
B D Morrison ◽  
J A Wemmie ◽  
I Frias ◽  
T O'Hare ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Rat Liver ◽  
Human Placenta ◽  
Alkylating Agents ◽  
Insulin Receptors ◽  
Cross Linking ◽  
Protein Kinase Activity ◽  
Liver Membranes ◽  
Alpha Beta ◽  
Beta 2

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.

Insulin receptor binding and protein kinase activity in muscles of trained rats

1987 ◽  
Vol 252 (2) ◽  
pp. E170-E175
Author(s):  
G. L. Dohm ◽  
M. K. Sinha ◽  
J. F. Caro
Keyword(s):  
Insulin Sensitivity ◽  
Protein Kinase ◽  
Exercise Training ◽  
Insulin Receptor ◽  
Insulin Action ◽  
Kinase Activity ◽  
Insulin Receptors ◽  
Treadmill Running ◽  
Female Rats ◽  
Protein Kinase Activity

Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, we postulated that exercise training would change insulin receptors in muscle and in this study we have investigated this hypothesis. Female rats initially weighing approximately 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise. Insulin binding by the partially purified receptor preparation s was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training.

scholarly journals Delineation of atypical insulin receptors from classical insulin and type I insulin-like growth factor receptors in human placenta

1989 ◽  
Vol 257 (1) ◽  
pp. 101-107 ◽  
Author(s):  
H A Jonas ◽  
A J Cox ◽  
L C Harrison
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Human Placenta ◽  
Insulin Receptors ◽  
Type I ◽  
Insulin Like Growth Factor ◽  
Binding Properties ◽  
High Affinity ◽  
Igf Receptors ◽  
Placental Membranes

Insulin-like growth factor (IGF)-binding sites copurifying with human placental insulin receptors during insulin-affinity chromatography consist of two immunologically distinct populations. One reacts with monoclonal antibody alpha IR-3, but not with antibodies to the insulin receptor, and represents Type I IGF receptors; the other reacts only with antibodies to the insulin receptor and is precipitated with a polyclonal receptor antibody (B-10) after labelling with 125I-multiplication-stimulating activity (MSA, rat IGF-II). The latter is a unique sub-population of atypical insulin receptors which differ from classical insulin receptors by their unusually high affinity for MSA (Ka = 2 x 10(9) M-1 compared with 5 x 10(7) M-1) and relative potencies for insulin, MSA and IGF-I (40:5:1 compared with 150:4:1). They represent 10-20% of the total insulin receptor population and account for 25-50% of the 125I-MSA binding activity in Triton-solubilized placental membranes. Although atypical and classical insulin receptors are distinct, their immunological properties are very similar, as are their binding properties in response to dithiothreitol, storage at -20 degrees C and neuraminidase digestion. We conclude that atypical insulin receptors with moderately high affinity for IGFs co-exist with classical insulin receptors and Type I IGF receptors in human placenta. They provide an explanation for the unusual IGF-II binding properties of human placental membranes and may have a specific role in placental growth and/or function.

scholarly journals A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques

1988 ◽  
Vol 250 (1) ◽  
pp. 215-219 ◽  
Author(s):  
C F Webb ◽  
M Wallis
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Growth ◽  
Cell Receptor ◽  
Receptor Protein ◽  
Cross Linking ◽  
Pregnant Rats ◽  
Reducing Conditions ◽  
Nb2 Cells ◽  
Liver Membranes

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.

Angiotensin II modulates tyr-phosphorylation of IRS-4, an insulin receptor substrate, in rat liver membranes

2006 ◽  
Vol 293 (1-2) ◽  
pp. 35-46 ◽  
Author(s):  
Rodrigo S. Villarreal ◽  
Sergio E. Alvarez ◽  
Maximiliano Juri Ayub ◽  
Gladys M. Ciuffo
Keyword(s):  
Angiotensin Ii ◽  
Insulin Receptor ◽  
Rat Liver ◽  
Insulin Receptor Substrate ◽  
Liver Membranes

Purification and properties of insulin receptors from rat liver membranes

1977 ◽  
Vol 77 (3) ◽  
pp. 981-988 ◽  
Author(s):  
Steven Jacobs ◽  
Yoram Shechter ◽  
Karen Bissell ◽  
Pedro Cuatrecasas
Keyword(s):  
Rat Liver ◽  
Insulin Receptors ◽  
Purification And Properties ◽  
Liver Membranes

scholarly journals Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro

1987 ◽  
Vol 248 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Y C Kwok ◽  
C C Yip
Keyword(s):  
Insulin Receptor ◽  
Rat Liver ◽  
Growth Factor Receptor ◽  
Insulin Receptors ◽  
Artificial Substrates ◽  
Receptor Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Cytosolic Proteins ◽  
Insulin Receptor Kinase

Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase.

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