Timing-dependent modulation of insulin mitogenic versus metabolic signalling.

This chapter will not deal sensu stricto with the mechanisms and biological significance of pulsatile hormone secretion, the general theme of this book. Rather, we will attempt to demonstrate that timing events at the receiving end of the hormonal signal, i.e. the kinetics and duration of receptor activation in target cells and subsequent downstream signalling, can play an equally important role as that of the timing aspects of secretion, in determining the qualitative and quantitative aspects of hormonal responses. We will focus on the mechanisms that determine signalling specificity by the receptor tyrosine kinases, especially the insulin receptor and the type I insulin-like growth factor receptors (IGF-I receptor). We will be succinct and refer the reader to our recent reviews and publications on this topic and references therein.

Timing-dependence of insulin-receptor mitogenic versus metabolic signalling: a plausible model based on coincidence of hormone and effector binding.

Mitogenic signalling through the insulin receptor is enhanced compared with metabolic signalling for insulin analogues having slower dissociation kinetics than insulin itself. A plausible explanation in molecular terms of this timing-dependent specificity is lacking. We show here that if signalling is transmitted through a single effector, binding coincidentally with hormone to the insulin receptor and whose association and dissociation kinetics are slow relative to the hormone dissociation rate, the resulting biological effect is predicted to be dependent on hormone-binding kinetics. However, known primary effector molecules associating with the insulin receptor bind and interact rapidly with the receptor, contrary to the assumptions of the single-effector model. A model with two effectors which must bind coincidentally with hormone for signalling to occur also gives the required dependence of signalling on hormone-binding kinetics, provided that at least one of the effectors has slow binding kinetics relative to hormone binding. In this case, the other effector can have rapid kinetics, which is consistent with the properties of the major known substrates of the insulin receptor, such as the insulin receptor substrate (IRS) molecules.

Logical analysis of timing-dependent receptor signalling specificity: application to the insulin receptor metabolic and mitogenic signalling pathways.

We present a method for logical analysis of signal-transduction networks, focusing on metabolic and mitogenic signalling by the insulin receptor, with specific emphasis on dependence of the signalling properties on the timing of binding events. We discuss a basic model which demonstrates this dependence (hormone binding leads to activation of the receptor which can lead to a commitment to mitogenic signalling), and show how residence time of the hormone on the receptor can determine the specificity of signalling between the alternative metabolic or mitogenic pathways. The method gives conditions for the selection of specific branches in the signalling pathway expressed in terms of inequalities among the characteristic activation or deactivation times of components of that pathway. In this way, the conditions for mitogenic signalling can be given in terms of a required range of values of the hormone residence time on the receptor, which is directly related to the kinetic dissociation rate.

Mitogenic properties of insulin and insulin analogues mediated by the insulin receptor.

Insulin has traditionally been considered as a hormone essential for metabolic regulation, while the insulin-like growth factors (IGF-I and IGF-II) are postulated to be more specifically involved in growth regulation. The conventional wisdom is that they share each other's effects only at high concentrations, due to their weak affinity for the heterologous receptor. We discuss here the evidence that in the proper cellular context, insulin can be mitogenic at physiologic concentrations through its own receptor. We studied the insulin and IGF-I binding characteristics of a new model suitable for analysing insulin receptor mediated mitogenesis; that is, a T-cell lymphoma line that depends on insulin for growth, but is unresponsive to IGFs. The cells showed no specific binding of 125I-IGF-I and furthermore, no IGF-I receptor mRNA was detected by RNAse protection assay in the LB cells, in contrast with mouse brain and thymus. The cells bound at saturation about 3000 insulin molecules to receptors that had normal characteristics in terms of affinity, kinetics, pH dependence and negative co-operativity. A series of insulin analogues competed for 125I-insulin binding with relative potencies comparable to those observed in other insulin target cells. The full sequence of the insulin receptor cDNA was determined and found to be identical to the published sequence of the murine insulin receptor cDNA. The LB cell line is therefore an ideal model with which to investigate insulin mitogenic signalling without interference from the IGF-I receptor. Using this model, we have started approaching the molecular basis of insulin-induced mitogenesis, in particular the role of signalling kinetics in choosing between mitogenic and metabolic pathways.

Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3.

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcoretrade mark instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1-3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I.

Biological effects of human growth hormone in rat adipocyte precursor cells and newly differentiated adipocytes in primary culture.

The effects of human growth hormone (hGH) on proliferation and differentiation of primary adipocyte precursor cells isolated from rat epididymal fat pads were studied under serum-free culture conditions. hGH markedly reduced the formation of new fat cells and the expression of glycerophosphate dehydrogenase activity, a marker enzyme of adipose differentiation, in a dose-dependent manner. To find an explanation for this inhibitory effect, we investigated the action of GH on (1) cell proliferation and on (2) lipid accumulation, the latter in the absence and presence of corticosterone. In undifferentiated cells, 5 nmol/L hGH increased both cell number and [3H]-thymidine incorporation (1.3- and 2.6-fold over basal, respectively). This effect was mediated by insulin-like growth factor-I (IGF-I), since hGH stimulated IGF-I production in undifferentiated cells by 12-fold and addition of an anti-IGF-I monoclonal antibody (IGF-I MAb) abolished the mitogenic effect of hGH but did not prevent hGH-induced suppression of adipose differentiation. In developing fat cells, hGH significantly reduced cellular 2-deoxyglucose uptake and glucose incorporation into lipids. In addition, hGH exhibited a lipolytic action in the presence of insulin and triiodothyronine. These effects were not prevented by IGF-I MAb. Specific binding of [125I]-hGH to precursor cells increased significantly during adipose conversion. In differentiated cells Scatchard analysis yielded linear plots with an apparent Kd of 0.16 nmol/L and 8,400 sites per cell. Taken together, these data show that hGH reduces adipose conversion in primary cultures of rat adipocyte precursor cells while promoting cell proliferation through an increase in IGF-I production.

Kinetics of the interaction between the human factor VIIIa subunits: effects of pH, ionic strength, Ca2+ concentration, heparin, and activated protein C-catalyzed proteolysis.

Coagulation factor VIIIa consists of a heterotrimer in which the A2 subunit is bound to the A1/A3C1C2 dimer. The dissociation of this complex causes the spontaneous and reversible decay of factor VIIIa activity. In order to characterize the kinetics and affinity of the interaction between A2 and A1/A3C1C2, as well as the influence of different parameters on the interaction, the subunits were chromatographically separated and reassembled in a BIAcore instrument (Pharmacia Biosensor). In the binding experiments, A2 was free in solution, whereas A1/A3C1C2 was immobilized on the dextran surface by direct coupling or captured on an immobilized monoclonal anti-C2 antibody. At our chosen standard condition (pH = 6.0, I = 0.12, and [Ca2+] = 2 mM), the association rate constant, dissociation rate constant, and resulting equilibrium dissociation constant were ca. 1.4 x 10(4) M-1s-1, 2.1 x 10(-4)s-1, and 16 nM, respectively. Increasing the ionic strength or Ca2+ concentration resulted in both slower association and faster dissociation. At 0.3 M NaCl or 25 mM Ca2+, the dissociation constant was > 1 microM. This implies that electrostatic forces involved in the interaction contribute at least one-fourth of the total binding energy. Increasing pH caused a similar effect, yielding a dissociation constant of ca. 0.9 microM at pH 7.5. In those cases where the equilibrium dissociation constants had been determined from solution phase experiments [Fay, P. J., & Smudzin, T. M. (1992) J. Biol. Chem. 267, 13246-13250; Lollar, P., Parker, E. T., & Fay, P. J. (1992) J. Biol. Chem. 267, 23652-23657], these constants agreed well with our results.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of the time factor in signaling specificity: application to mitogenic and metabolic signaling by the insulin and insulin-like growth factor-I receptor tyrosine kinases.

The signal transduction pathways activated by hormones, growth factors, and cytokines show an extraordinary degree of cross-talk and redundancy. This review addresses the question of how the specificity conferred at the binding step is maintained through the signaling network despite the convergence of multiple signals on common efferent pathways such as mitogen-activated protein (MAP) kinase. The mechanism of receptor activation by ligand-induced dimerization provides a signaling device with both a switch and a timer. The role of the time factor, ie, of signaling kinetics, as a determinant of selectivity is discussed with emphasis on the receptor tyrosine kinases and cytokine receptors, and especially mitogenic versus metabolic signaling by insulin and insulin-like growth factor-I (IGF-I).

Negative cooperativity in the insulin-like growth factor-I receptor and a chimeric IGF-I/insulin receptor.

Insulin and insulin-like growth factor-I (IGF-I) share a spectrum of metabolic and growth-promoting effects, mediated through homologous receptors that belong to the tyrosine kinase family. The dissociation rate of insulin from its receptor is affected by negative cooperativity, i.e. accelerates with increased receptor occupancy. The dose-response curve for the acceleration of tracer dissociation by unlabeled insulin has a distinct bell-shaped curve, with a progressive slowing down at insulin concentrations greater than 100 nM. The kinetics of the IGF-I interaction with its receptor has not been studied in such detail. In the present work, we report that while the IGF-I receptor exhibits negative cooperativity like the insulin receptor, the concentration dependence of the dissociation kinetics is distinct from that of native human insulin by not being bell-shaped, but monophasic like that of insulin analogues mutated at the hexamer-forming surface; it is changed to an insulin-type curve by substitution of IGF-I receptor's sequence including residues 382-565 with the homologous insulin receptor domain. The data suggest that like insulin, IGF-I has a bivalent binding mode and crosslinks two distinct areas of the two alpha subunits that are close, but distinct from the equivalent insulin receptor binding sites.

Receptor dimerization determines the effects of growth hormone in primary rat adipocytes and cultured human IM-9 lymphocytes.

Binding of GH to its cell surface receptors is thought to result in the formation of a complex comprised of one molecule of hormone per two molecules of receptor. It has been proposed that this hormone-induced receptor dimerization is important for the mechanism of signal transduction. We have developed a mathematical model for quantitative evaluation of the biological responses associated with sequential receptor dimerization. Based on these predictions, we have investigated whether GH-induced receptor dimerization plays a role in two classical effects of GH, i.e. stimulation of lipogenesis in primary rat adipocytes and GH receptor down-regulation in cultured human IM-9 lymphocytes. Model predictions of biological responses linked to dimer formation yielded a bell-shaped pattern, with self-antagonism at high GH concentrations when monomeric GH-receptor complexes become predominant. The GH lipogenic bioactivity curve was indeed biphasic and first increased in a concentration-dependent manner between 10(-10)-10(-8) M GH (ED50, 0.5 nM), up to a maximum of 1.7-fold stimulation above basal. Then, the response decreased continuously above 5 x 10(-8) M GH, returning to basal levels around 10(-5) M GH. Incubation of IM-9 cells with wild-type human GH resulted in a dose-dependent loss of their surface receptors. In contrast, a human GH analog (G120R), mutated in the second binding surface of the hormone and, therefore, unable to induce GH receptor dimerization, failed to induce receptor down-regulation in the IM-9 cells. Furthermore, when added together with wild-type human GH, human GH(G120R) inhibited, in a concentration-dependent manner, the down-regulation induced by wild-type human GH. Taken together, these data support the hypothesis that receptor dimerization is critical for the stimulation by GH of both lipogenesis in primary rat adipocytes and receptor down-regulation in cultured human IM-9 lymphocytes.

The insulin-like growth factor-I receptor. Structure, ligand-binding mechanism and signal transduction.

The nonclassical binding kinetics of IGF-I and insulin to their respective receptors, suggestive of negative cooperativity, can be readily explained by our recently proposed novel binding mechanism whereby the bivalent ligand bridges the two receptor alpha-subunits alternatively at opposite sites in a symmetrical receptor structure. The bivalent binding mechanism also explains bell-shaped bioactivity curves. The possible role of different binding modes versus differences in downstream signaling by insulin and IGF-I in producing specific mitogenic or metabolic responses is discussed.

Mutation of arginine 86 to proline in the insulin receptor alpha subunit causes lack of transport of the receptor to the plasma membrane, loss of binding affinity and a constitutively activated tyrosine kinase in transfected cells.

We have investigated the role of Ser 85 and Arg 86 of the human insulin receptor (HIR) in insulin binding and tyrosine kinase activity by mutational analysis. Four mutant cDNAs were created (R86P, R86N, S85T+R86N, S85W+R86K) and stably transfected into BHK cells. R86P-HIR was also transiently expressed in 293 cells. Only the R86P receptor had substantially altered properties: lack of transport to the plasma membrane, loss of insulin binding, a constitutively activated autophosphorylation and tyrosine kinase, and an incomplete processing. Some of these alterations mimic those reported for the insulin receptor of the leprechaun Atl, which has a homozygous R86P mutation (Longo, N., et al, Biochem. Biophys. Res. Commun., 167, 1229, 1990; Clin. Res., 40, 2, 329, 1992).

Identification of a ligand-binding region of the human insulin receptor encoded by the second exon of the gene.

Structure-function studies of the insulin molecule indicate that an insulin B chain domain comprising residues 22-26 is involved both in binding to the insulin receptor (INSR) and in insulin dimer formation, suggesting that this domain might also interact with a structure resembling the insulin dimer interface in the INSR. Expression of a mutant INSR cDNA with a deletion of the region corresponding to exon 2 of the INSR gene produces a protein devoid of insulin-binding activity, although the mutant protein is processed appropriately to alpha- and beta-subunits, suggesting that the insulin-binding domain is encoded at least in part by exon 2. Within this region of the INSR molecule, the sequence 83-103 fulfills the structural criteria for a dimer interface. Studies of mutant INSRs with substitutions for phenylalanine 88 or 89 show that the presence of phenylalanine at position 89 is essential for full binding affinity.

Binding kinetics of mutated insulin receptors in transfected cells grown in suspension culture: application to the Tyr----Phe 960 insulin receptor mutant.

Site-directed mutagenesis of the insulin receptor cDNA is now widely used to elucidate the role of various domains and residues of the receptor, particularly in order to examine the functional importance of the beta chain-associated tyrosine kinase. However, little has been done to correlate the functional repercussions of such mutations with alterations in the complex insulin binding kinetics. This is due in part to the difficulty of conducting large scale experiments using transfected cells on culture dishes. In an effort to overcome this problem, we have developed a method for culturing Chinese hamster ovary (CHO) cells in suspension culture, which provides a large number of cells and obviates the need for enzymatic or mechanical detachment of cells. The feasibility of this approach is demonstrated in a detailed study of the kinetics of insulin binding to the Tyr----Phe 960 insulin receptor mutant.

Swine basal cell proliferation during a course of daily irradiation, five days a week for six weeks (6000 rad).

In swine skin irradiated with 200 rad per day, 5 days per week for 6 weeks, basal cell density remained at control levels for the first 2 weeks and then decreased to a nadir of 50% at 38 days. Thereafter it began increasing and returned to near control levels within 1 day after the end of irradiation on day forty-three. The mitotic index increased progressively to a maximum value three times the controls at day forty-two and then decreased as the cell density returned to control levels. The pattern strongly suggests that cell proliferation occurred during the period of irradiation. The cell density changes are simulated by a model in which doubling time switches from 12 days to 2.5 days at the 50% cell density level.

Tissue population configuration as a modifier of organ dose response.

Organ tissue populations exist in different spatial configurations. There are the idealized "volume" configuration (bone marrow), the "monolayer" (skin basal cell or large vessel endothelial populations), the "linear" (microvascular endothelial populations) and the "isolated unit" (capillaries, intestinal crypt). This defines the size of the irradiated population (No) and influences the absolute number of cells (N) that survive per unit of tissue dimension to permit repopulation. A proliferative cell that survives irradiation replaces cells lost from distant locations according to its spatial dimension. However, in the isolated unit configuration, a proliferative cell can replace those cells lost from the unit, but an anatomical barrier prevents cell replacement extending to adjacent units. Recovery of a tissue following irradiation depends not only on the number of surviving proliferative cells, but also on the organization of those cells into sub-populations. A regenerative unit is a subpopulation capable of being regenerated from the survival of a single cell. A tissue "functional unit" is a subcomponent of the tissue which contributes independently to overall tissue function, and whose loss results in an irreversible incremental loss of function. Survival of the functional unit depends critically on the degree of interdependence among component regenerative units, and such interdependence is strongly influenced by the tissue's spatial organization. Using these concepts, a mathematical formulation expresses a tissue's functional response to irradiation in terms of cell survival parameters, the spatially-related parameters of regenerative unit size, and the number of interdependent regenerative units comprising a functional unit. It is argued that this provides a more realistic approach toward simulating the radiation response of an organ than one based on tissue bulk cell survival parameters alone.

Circadian gating of S phase in human ovarian cancer.

Cell proliferation in 30 patients with ovarian cancer was analyzed using flow cytometry to determine changes in the percentage of cells in S phase. By this measure, proliferation in tumor cells appears to follow a cyclical pattern of peaks and troughs that is out of phase with the circadian rhythm in proliferation of normal tissues. In round-the-clock monitoring of replication stages in tumor cells recovered from i.p. lavage fluid in postsurgery patients, peaks of tumor and nontumor cell DNA synthesis commonly occurred at different times of day. When patients were grouped so that only tumor cell proliferation was being measured, a highly significant 24-h rhythm nearly 12 h out of phase with nontumor cell proliferation was found. This peak in the percentage of S-phase cells occurs most commonly in mid- to late morning and appears to offer an opportunity for timing chemotherapy to coincide with high tumor cell vulnerability and low toxicity to normal tissue.

Field size dependence of radiation sensitivity and dose fractionation response in skin.

Four sets of data from the literature were analyzed to assess the effects of field size on dose tolerance and dose fraction size dependence in irradiated skin. The data consisted of combinations of total dose and dose per exposure (or number of fractions) required to yield a given degree of visible damage to the skin, for fields of different sizes. Putative cell survival curves were constructed, under the assumptions that the isoeffect represents a fixed cell survival, and that each exposure during a course of fractionated irradiation has equal effect on cell survival. The analysis showed that overall sensitivity to radiation, and dependence on dose per exposure, both increase with field size. To account for these results we describe a model that can be qualitatively related to the geometric properties of the dermal vascular network. First, vascular function after irradiation should depend on the length of the vessels exposed to the radiation. This directly predicts an increasing sensitivity in large irradiated fields. Furthermore, if vascular function determines radiation response, the shape of the shoulder (low-dose) region of the effective survival curve will depend on the average number of vessels nourishing each cell, with a more pronounced shoulder for a high multiplicity of vessels. The model predicts a greater fractionation sensitivity in large than in small fields, in agreement with our analysis of the isoeffect data. It is therefore possible that the advantages of hyperfractionation in reducing late effects in normal tissues may be related to vascular architecture, and not to inherent differences between late and acutely responding cell populations.