The Biological Potency of Covalent Insulin-Receptor Complexes. Dependence on Site of Cross-Linkage

1984 ◽  
Vol 365 (1) ◽  
pp. 493-498 ◽  
Author(s):  
Gernot KLOTZ ◽  
Derek J. SAUNDERS ◽  
Dietrich BRANDENBURG
Keyword(s):  
Insulin Receptor ◽  
Receptor Complexes ◽  
Cross Linkage ◽  
Biological Potency

scholarly journals Time-dependence of biological activity induced by covalent insulin-receptor complexes in rat adipocytes

1985 ◽  
Vol 232 (1) ◽  
pp. 49-53 ◽  
Author(s):  
A Schüttler ◽  
C Diaconescu ◽  
D J Saunders ◽  
D Brandenburg
Keyword(s):  
Insulin Receptor ◽  
Inhibitory Effect ◽  
Receptor Complex ◽  
Half Time ◽  
Simple Method ◽  
Receptor Complexes ◽  
Maximal Stimulation ◽  
Monocomponent Insulin ◽  
Specific Receptors ◽  
Stimulation Of

Lipogenesis in isolated adipocyte preparations is stimulated when photosensitive insulin derivatives are attached covalently to specific receptors. This response was compared quantitatively with that to reversibly associated insulin, and it was shown that both covalent and reversible insulin-receptor complexes behave very similarly. The extent of stimulation of lipogenesis was studied as a function of time. Cells were incubated in buffer for various times before addition to vials containing 0 (basal) or 10 ng of monocomponent insulin/ml (maximal) and [U-3H]glucose. After 60 min, the toluene-soluble [3H]lipids were measured. The maximal stimulation induced by reversibly bound insulin was virtually constant over a period of 4 h. In contrast, adipocytes to which N alpha B2-(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin had been covalently attached at the start of the experiment showed a loss of stimulation with time when incubated at 37 degrees C. This loss was decreased in the presence of lysosomotropic agents such as chloroquine at concentrations (approx. 200 microM) that had very little or no effect on the basal and maximal lipogenesis rates. A simple method was used to transform the measured rate of loss of stimulation into a rate of loss of effective units. A half-time of 80 min was calculated for the effective covalent insulin-receptor units in adipocytes at 37 degrees C at pH 7.4. This is very close to values reported by others for the internalization of covalent complexes in these cells, suggesting that this may be the causative event for the deactivation of the insulin-receptor unit. The inhibitory effect of chloroquine on the deactivation may indicate that the insulin-receptor complex can function even after internalization.

Biological action and fate of photoaffinity-labelled insulin-receptor complexes

Biochimie ◽  
1985 ◽  
Vol 67 (10-11) ◽  
pp. 1111-1117 ◽  
Author(s):  
D. Brandenburg ◽  
C. Diaconescu ◽  
G. Klotz ◽  
P. Mucke ◽  
J. Neffe ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Biological Action ◽  
Receptor Complexes

Recognition of Covalent Insulin-Receptor Complexes on Viable Adipocytes by Anti-Insulin Antibodies

1987 ◽  
Vol 368 (1) ◽  
pp. 85-92 ◽  
Author(s):  
Peter MUCKE ◽  
Cornelia DIACONESCU ◽  
Gernot KLOTZ ◽  
Peer JØRGENSEN ◽  
Derek SAUNDERS ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Insulin Antibodies ◽  
Receptor Complexes

The interaction of sialidase-treated hCG with ovarian cellular membranes

1981 ◽  
Vol 97 (2) ◽  
pp. 270-280 ◽  
Author(s):  
E. C. Brand ◽  
J. Odink ◽  
G. Klok ◽  
E. V. van Hall
Keyword(s):  
Binding Sites ◽  
Association Constant ◽  
Apparent Difference ◽  
Receptor Complexes ◽  
Number Of Binding Sites ◽  
Biological Potency ◽  
The Difference ◽  
The Stability ◽  
Dissociation Curves ◽  
Hcg Receptor

Abstract. The potency of human chorionic gonadotrophin (hCG) in competition for binding to a gonadal membrane fraction is remarkably enhanced by sialidase treatment. The present study was undertaken to investigate the specificity and characteristics of the binding of sialidase-treated hCG (asialo-hCG) in a particulate hCG-binding system from luteinized rat ovaries. The competitive potency of asialo-hCG relative to hCG was 2.5, irrespective of whether [125I]hCG or [125I]asialo-hCG was used for tracer. This was due to a 2.1 times higher equilibrium association constant for asialo-hCG, whereas the estimated number of binding sites did not differ. There was no apparent difference in the stability of hCG and asialo-hCG, or in the stability of the respective hormone-receptor complexes. The effect of variation of the incubation conditions on the binding of both tracers was similar. In accordance with the difference in the equilibrium association constant, the association velocity of asialo-hCG was more than double that of hCG. With all of the tracers used the dissociation curves were biphasic, the size of the initial fast-dissociating fraction being inversely related to the pre-incubation time. Under identical conditions, the fast-dissociating fraction was smaller for the [125I]asialo-hCG complex than for the [125I]hCG complex. The dissociation velocities of these fractions appeared to be similar. The results indicate that asialo-hCG binds to the hCG receptor in a way similar to the binding of the unmodified hormone, but with a higher affinity. The smaller size of the fast-dissociation form of the asialo-hCG-receptor complex may be related to the lower biological potency of the hormone derivative.

scholarly journals Internalization and recycling of insulin receptors in hepatoma cells. Absence of regulation by receptor occupancy

1984 ◽  
Vol 222 (1) ◽  
pp. 111-117 ◽  
Author(s):  
Y Chvatchko ◽  
E Van Obberghen ◽  
M Fehlmann
Keyword(s):  
Insulin Receptor ◽  
Cellular Localization ◽  
Insulin Analogue ◽  
Insulin Receptors ◽  
Receptor Occupancy ◽  
Hepatoma Cells ◽  
Down Regulation ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Before And After

Insulin receptors of Fao hepatoma cells were labelled with a 125I-labelled photoreactive insulin analogue or by surface iodination catalysed by lactoperoxidase. Cells were then incubated at 37 degrees C, and the cellular localization of the labelled receptors was assessed by limited exposure of intact cells to trypsin. The results show that: (1) photolabelled insulin-receptor complexes are internalized and recycled in Fao hepatoma cells; (2) the dynamics of photolabelled insulin receptors (internalization and recycling) is similar before and after down-regulation; (3) the unoccupied receptors labelled by surface iodination are internalized and recycled similarly to covalent insulin-receptor complexes; (4) insulin does not induce internalization of surface-iodinated insulin receptors. We conclude that internalization and recycling of insulin receptors are independent of receptor occupancy by insulin in Fao hepatoma cells.

scholarly journals Protein kinase activity of the insulin receptor

1986 ◽  
Vol 235 (1) ◽  
pp. 1-11 ◽  
Author(s):  
S Gammeltoft ◽  
E Van Obberghen
Keyword(s):  
Insulin Receptor ◽  
Molecular Mechanisms ◽  
Kinase Activity ◽  
Beta Subunit ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Protein Kinase Activity ◽  
Intact Cells ◽  
Receptor Complexes ◽  
Alpha Subunits

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)

An evaluation of the cross-linking model for the interaction of insulin with its receptor

1997 ◽  
Vol 272 (6) ◽  
pp. E1136-E1144 ◽  
Author(s):  
B. J. Hammond ◽  
J. Tikerpae ◽  
G. D. Smith
Keyword(s):  
Insulin Receptor ◽  
Insulin Binding ◽  
Cross Linking ◽  
Cross Link ◽  
Binding Studies ◽  
Systematic Analysis ◽  
Concentration Difference ◽  
Receptor Complexes ◽  
Insulin Molecule ◽  
The Cross

The cross-linking model for insulin receptor interactions, in which a single insulin molecule may form a cross-link between an insulin receptor's alpha-subunits, has been expressed as a formal compartmental model and subjected to a systematic analysis, examining a number of predictions that have been made for this model. The kinetic parameters for the model were obtained by matching data from insulin receptor equilibrium binding studies and rates of formation of the insulin receptor complex. This analytical study has allowed a clear description of the kinetics of the ligand receptor complexes involved in such a mechanism. We conclude that the cross-linking model accounts for the anomaly of the 10-fold concentration difference in high- and low-affinity binding sites found when insulin binding is analyzed by conventional means. However, the phenomenon of acceleration of dissociation of labeled ligand by unlabeled ligand cannot be accounted for as an intrinsic part of the model. We suggest that this phenomenon arises from the destabilization of cross-link formation when a second insulin molecule binds.

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