Full Receptor Occupancy Research Articles

Quantitative dissociation of glucose transport stimulation and insulin receptor tyrosine kinase activation in isolated adipocytes with a covalent insulin dimer (B29,B29′-sunberoyl-insulin)

The covalent insulin dimer B29,B29′-suberoyl-insulin was investigated for its effects on insulin receptor binding, insulin receptor tyrosine kinase activity and glucose transport in isolated adipose cells. The dimer stimulated glucose transport (initial 3- O-methylglucose uptake rate) to the same extent as insulin did (basal rate, 35 ± 3 pmol/sec/ μl lipid; insulin, 380 ± 27; B29,B29′-suberoyl-insulin, 369 ± 24, means ± S.E.), although at higher concentrations ( ec 50 1.94 ± 0.64nM versus 0.1 ± 0.02 with insulin). In contrast, the dimer only partially (23%) mimicked insulin's effect on phosphate incorporation into insulin receptors immunoprecipitated after equilibration of cells with [ 32P]phosphate. Similarly, insulin receptor tyrosine kinase as assessed by receptor autophosphorylation and phosphorylation of the substrate poly-(Glu/Tyr) was not fully activated by treatment of cells with the insulin dimer (31 and 42% of the effect of insulin, respectively) in concentrations which maximally activate glucose transport and give rise to full insulin receptor occupancy (5 × 10 −7M). Further, the dimer activated the receptor tyrosine kinase in solubilized purified insulin receptor preparations from adipose cells to only 25% of the effect of insulin ( ec 50) 32.0 ± 16 versus 1.9 ± 1.0 nM with insulin) in spite of full receptor occupancy. Binding of the dimer to insulin receptors followed single site binding kinetics, indicating that the derivative is unable to induce negative cooperativity of the insulin receptor. It is concluded that a partial phosphorylation of insulin receptors and a submaximal tyrosine kinase activation are sufficient for full stimulation of glucose transport in the adipocyte. Further, it is suggested that negative cooperativity of the insulin receptor and activation of its tyrosine kinase require a similar conformational change of the receptor protein.

Monoclonal antibodies to the insulin receptor mimic metabolic effects of insulin but do not stimulate receptor autophosphorylation in transfected NIH 3T3 fibroblasts.

The metabolic actions of insulin and anti-insulin receptor monoclonal antibodies were compared with their effects on insulin receptor phosphorylation in mouse NIH 3T3 fibroblasts transfected with human insulin receptor cDNA. In serum-starved NIH 3T3 HIR3.5 cells, uptake of 2-deoxy-[3H]glucose was stimulated up to 2-fold after 30 min with insulin, with a half-maximal effect at 0.1 nM insulin. Incorporation of [3H]thymidine was stimulated approximately 12-fold after a 16-hr preincubation with insulin, with a half-maximal effect at 2 nM insulin. Phosphorylation of insulin receptor beta-subunit in cells prelabeled with [32P]phosphate was increased 10- to 20-fold within 5 min of adding insulin, with a half-maximal effect at approximately 3 nM insulin. Monoclonal antibodies reacting with four different epitopes on the insulin receptor mimicked the effect of insulin on 2-deoxyglucose uptake. These antibodies also stimulated thymidine incorporation, although the maximum stimulation was only approximately 30% that of insulin. Two antibodies (25-49 and 83-14) showed a similar concentration dependence to insulin in their metabolic effects and in the inhibition of 125I-labeled insulin binding to cells. The other two antibodies (83-7 and 18-44) were somewhat less potent and did not inhibit insulin binding. None of the antibodies significantly increased insulin receptor phosphorylation at concentrations up to 100 nM, which at least in the case of 25-49 and 83-14 was sufficient for full receptor occupancy. It is concluded that the insulin-like metabolic effects of antibodies involve a mechanism of receptor activation that is independent of autophosphorylation and hence that receptor autophosphorylation is not an essential step in triggering at least some events in the insulin signaling pathway.

Internalization and degradation of human recombinant interferon-γ in the human histiocytic lymphoma cell line, U937: Relationship to Fc receptor enhancement and antiproliferation

This report describes the association between the intracellular fate of human recombinant interferon-γ (rIFN-γ) and the induction of enhanced numbers of Fc receptors and an antiproliferative effect in the human monocyte-like cell line, U937. Full receptor occupancy of Bolton-Hunter labeled 125I-rIFN-γ occurred within 10 min at 37°C. However, only 50% of those molecules bound were internalized within 30 min. Residency of rIFN-γ within the cell was 60–120 min. Eventually, 60–70% of those molecules initially bound to the cell were completely degraded within monensin-sensitive compartments of the cell as measured by the presence of trichloroacetic acid-soluble radioactivity in the medium. Exposure of rIFN-γ to cell extracts resulted in a shift in its p I from 8 to 6, presumably due to proteolytic cleavage of carboxy-terminal basic amino acids. A single brief exposure (5–15 min) of U937 cells to rIFN-γ resulted in enhanced numbers of receptors for the Fc portion of 125I-IgG1 as measured 24 hr later. Eighty percent of a maximal Fc receptor response occurred at only 30% receptor occupancy (50 U/ml). In contrast, repeated daily exposure of U937 cells to moderate concentrations of rIFN-γ (125–250 U/ml) was necessary to induce an antiproliferative effect. These data suggest that a given response of the cell to rIFN-γ may require different intensities of the signal. This may reflect the overall complexity of the response generated.

Cellular Processing of Murine Colony-Stimulating Factor (Multi-CSF, GM-CSF, G-CSF) Receptors by Normal Hemopoietic Cells and Cell Lines

The binding, internalization and degradation rates of three different murine colony-stimulating factors (Multi-CSF or interleukin-3, GM-CSF and G-CSF) and their receptor turnover rates were determined for normal bone marrow cells and a number of different cell lines at 37 degrees C. The kinetic parameters were extracted from a curve-fitting analysis of the approach to steady-state of surface-bound and internalized CSFs by methods described by Myers et al. (1987). The primary binding kinetic constants (association and dissociation) for each CSF on different cell types were similar, suggesting a single type of receptor for each CSF. In all cases, CSF binding induced a faster rate of internalization of occupied receptors than unoccupied receptors and resulted in significant accumulation of CSF inside the cell under steady-state conditions. The steady-state constant, determining the relationship between CSF concentration and receptor occupancy, indicated that, in all cases, more receptors were occupied at a given CSF concentration under steady-state conditions than would be under equilibrium conditions. Nevertheless, the data predicted that maximal biological effects of the CSFs were exerted at concentrations that did not result in full receptor occupancy. Comparison of the kinetic constants derived for the same CSF interacting with different types of cells or different CSFs interacting with the same cell type indicated that CSF and receptor processing resulted from a dynamic interplay of receptor-determined and cell-determined events. This resulted in a flexibility of the kinetic parameters that matched the variety of biological responses elicited by CSFs in different cell types.

Nuclear 3,5,3'-triiodothyronine receptor occupancy, phosphoenolpyruvate carboxykinase (PEPck) messenger ribonucleic acid levels and PEPck enzyme activity in rat liver.

The expression of the gene coding for phosphoenolpyruvate carboxykinase (PEPck) is under complex multi-hormonal control. A stimulatory effect of T3 on the expression of this gene has been described, but it has not been related to nuclear T3 receptor occupancy. We therefore studied simultaneously nuclear T3 receptor occupancy, PEPck enzyme activities, and PEPck messenger RNA (mRNA) levels in the liver of thyroidectomized, T3-treated rats. A dose-dependent increase in receptor occupancy and in enzyme activities was observed 24 h after a single ip injection of T3. However, no increase in the levels of PEPck mRNA was observed, using a specific complementary DNA (pPCK10). To evaluate this further, time-course experiments were performed over a 24 h period after a single ip injection of a receptor saturating dose of T3. These experiments showed simultaneous increases of PEPck enzyme activities and PEPck mRNA levels. Receptor occupancy had increased to 95% 3 h after injection of T3, whereas maximal levels of enzyme activity and mRNA were obtained 6-12 h after T3 administration. Translational efficiency did not change during induction. Twenty-four hours after T3 administration, mRNA levels and enzyme activity had declined, but receptor occupancy was still maximal. We conclude that T3 influences PEPck gene expression primarily at a pretranslational (transcriptional) level. The transient increase in PEPck mRNA despite persisting full receptor occupancy suggests a modification in time of the interaction between the T3-receptor complex and the regulatory region of the PEPck gene.

Obligatory separation of hormone binding and biological response curves in systems dependent upon secondary mediators of hormone action.

A mathematical model is presented that describes the effects of hormone concentration on receptor saturation and biological response in systems dependent upon the generation of a secondary mediator such as cyclic AMP. The analysis makes the following assumptions: (i) the binding of hormone to its receptor is a reversible, second-order reaction; (ii) the concentration of mediator that is generated is directly proportional to the number of membrane binding sites occupied by hormone; and (iii) the binding of the mediator with its intracellular receptor to generate an effector complex is also second-order and results in a proportionate biological response. It follows from this treatment that the hormone concentration required for half-maximal biological response is formally lower than that required for half-maximal receptor saturation and that the difference between these two concentrations will depend upon the ratio of total mediator generated at full receptor occupancy to the dissociation constant of the mediator with its receptor. Without invoking concepts of negative cooperativity, this model offers a simple explanation for discrepancies between receptor occupancy and biological response curves that are often observed. Moreover, the mathematical form of the predicted biological response curves conforms to the shape of the response curves observed experimentally in a wide variety of systems.