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Background: An interaction between tumor cells and the microenvironment, as well as the development of angiogenesis, are required to form liver metastases (LMs)., Material and Methods: Immunofluorescence detection of α-smooth muscle actin, desmin, Ki67, laminin, and CD31 was used to analyze the kinetics of tumor angiogenesis determinants, especially the contribution of hepatic stellate cells (HSCs) to angiogenesis in hepatic metastasis produced by intrasplenically injected LS174 colorectal cancer cells. Immunostaining was performed at various times (days 9, 14, 28, and 39)., Results: At the earliest stage, micrometastases consisted of proliferating cancer cells, a well-organized network of activated HSCs and laminin deposits. No vascular network was observed. As the LMs grew in size, an organized vascular network appeared; the laminin network colocalized with CD31 immunostaining. At the later stages, all the immunostained markers became peripheral as a central necrosis developed. Purified activated HSCs isolated from transgenic mice livers developing hepatocellular carcinoma secreted laminin and showed enhanced human umbilical vein EC network formation in a Matrigel assay. In a coinjection LM experiment, activated HSCs enhanced the metastatic process. Moreover, colorectal LMs from six patients were analyzed, and a pattern of marker distribution similar to the coinjection experiment was found in human LMs., Conclusions: For the first time, our results show that HSCs play a crucial role in organizing and accelerating the progression of metastasis in modulating the prometastatic niche, interacting with colorectal cancer cell recruitment, and the organization of angiogenesis during colorectal LM development. Therefore, HSCs may be an early therapeutic target in colorectal cancer therapies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Tumor angiogenesis is closely associated with clinical staging and has been proposed to correlate with clinical response in terms of subsequent metastases following primary resection. Netrin-4 (NT-4) regulates angiogenic responses. Therefore, we sought to examine the effects of NT-4 on the primary tumor growth of colon cancer cells, liver and lung metastases of colon cancer cells, and responses following primary tumor resection. We used 3 different mouse models of orthotopic primary tumor and liver and lung metastases, comparing 2 human colon cancer cells lines: wild-type (low expression of NT-4) and NT-4 (overexpression of NT-4) LS174 cells. NT-4 overexpression inhibited the primary tumor growth of colorectal LS174 xenografts in nude mice (144.3±12.9 vs. 62.4±4.5 mm3; p<0.0001) as well as its related local and systemic recurrence (38 vs. 0%; p<0.01). NT-4 overexpression also markedly decreased colorectal cancer progression in terms of tumor number and volume of liver metastases in the NT-4 group of the orthotopic liver metastasis model (25 vs. 90% and 4±1 vs. 709±190 mm3, p<0.001 and p<0.05). Collectively, our findings indicate that NT-4 overexpression decreases colorectal lung metastasis and its associated lymph node involvement. NT-4 overexpression decreases tumor recurrence and metastasis after surgical resection, likely via an anti-angiogenic effect. These observations suggest that NT-4 may hold therapeutic potential in the treatment of colorectal cancer growth and major metastatic sites.

Our findings that PlGF is a cancer target and anti-PlGF is useful for anticancer treatment have been challenged by Bais et al. Here we take advantage of carcinogen-induced and transgenic tumor models as well as ocular neovascularization to report further evidence in support of our original findings of PlGF as a promising target for anticancer therapies. We present evidence for the efficacy of additional anti-PlGF antibodies and their ability to phenocopy genetic deficiency or silencing of PlGF in cancer and ocular disease but also show that not all anti-PlGF antibodies are effective. We also provide additional evidence for the specificity of our anti-PlGF antibody and experiments to suggest that anti-PlGF treatment will not be effective for all tumors and why. Further, we show that PlGF blockage inhibits vessel abnormalization rather than density in certain tumors while enhancing VEGF-targeted inhibition in ocular disease. Our findings warrant further testing of anti-PlGF therapies., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background and Aims. An arterial blood supply and phenotypic changes of the sinusoids characterise the liver vasculature in human hepatocellular carcinoma (HCC). We investigated the effects of rosuvastatin on liver vessel anomalies, tumour growth and survival in HCC. Methods. We treated transgenic mice developing HCC, characterized by vessel anomalies similar to those of human HCC, with rosuvastatin. Results. In the rosuvastatin group, the survival time was longer (P < .001), and liver weight (P < .01) and nodule surface (P < .01) were reduced. Rosuvastatin decreased the number of smooth muscle actin-positive arteries (P < .05) and prevented the sinusoid anomalies, with decreased laminin expression (P < .001), activated hepatic stellate cells (P < .001), and active Notch4 expression. Furthermore, rosuvastatin inhibited endothelial cell but not tumour hepatocyte functions. Conclusions. Rosuvastatin reduced the vessel anomalies and tumour growth and prolonged survival in HCC. These results represent new mechanisms of the effects of statin on tumour angiogenesis and a potential target therapy in HCC.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development. We postulated that activation of the EphB4/ephrin-B2 system may enhance EPC proangiogenic potential. In this report, we demonstrate in a nude mouse model of hind limb ischemia that EphB4 activation with an ephrin-B2-Fc chimeric protein increases the angiogenic potential of human EPCs. This effect was abolished by EphB4 siRNA, confirming that it is mediated by EphB4. EphB4 activation enhanced P selectin glycoprotein ligand-1 (PSGL-1) expression and EPC adhesion. Inhibition of PSGL-1 by siRNA reversed the proangiogenic and adhesive effects of EphB4 activation. Moreover, neutralizing antibodies to E selectin and P selectin blocked ephrin-B2-Fc-stimulated EPC adhesion properties. Thus, activation of EphB4 enhances EPC proangiogenic capacity through induction of PSGL-1 expression and adhesion to E selectin and P selectin. Therefore, activation of EphB4 is an innovative and potentially valuable therapeutic strategy for improving the recruitment of EPCs to sites of neovascularization and thereby the efficiency of cell-based proangiogenic therapy.

Two-dimensional color-coded pulsed Doppler ultrasonography (US) with a 12-MHz linear transducer was used to follow tumor growth and neoangiogenesis development in 12 transgenic mice developing a whole liver hepatocellular carcinoma (HCC) induced by the expression of SV40-T antigen. In this model, male mice developed HCC at various temporal and histologic stages (hyperplastic, four-eight wk; nodular, 12 wk; diffuse carcinoma, 16-20 wk), whereas female mice remained tumor free. Seven age-matched tumor-free mice were used as controls. Liver volume was calculated from B-mode images of the abdomen. Blood flow waveforms were recorded from the hepatic tumor-feeding artery upstream from the tumor vessels, allowing quantitative blood flow velocity measurements. Measurements were performed every four weeks from four to 20 weeks. As early as the hyperplastic stage (eight weeks), liver volume was increased by 2.7-fold, hepatic artery peak-systolic blood flow velocities (BFV) by 1.5-fold, end-diastolic BFV by 1.6-fold and mean BFV by 2.0-fold compared with control values (p < 0.001). Differences increased until 20 weeks and peak-systolic reached 90 +/- 6, end-diastolic 54 +/- 5 and mean BFV 48 +/- 5 cm s(-1). Successive measurements of BFV were reproducible and intraobserver repeatability coefficient values were <3 cm s(-1). In contrast, mesenteric artery BFV, which did not supply tumor region, did not show any significant difference with respect to control values. Thus, an increase in BFV constitutes a functional evaluation of tumor vascularity. In preclinical studies in small animals, measurements of liver volume and blood flow velocities in hepatic tumor-feeding artery provide a useful, reproducible, noninvasive, easy-to-repeat tool to monitor tumor growth and neoangiogenesis in hepatocellular carcinoma in mice.

The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF(165)) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(121), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(121)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(121), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(121)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(121), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF(121) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.

Objectives: Recent studies have provided increasing evidence that postnatal neovascularization does not rely exclusively on sprouting of preexisting vessels, but also involves bone marrow-derived circulating endothelial precursors (BM-EPCs). Animal studies revealed that neovascularization of ischemic tissue can be enhanced by BM-EPCs transplantation. But a possible limitation to the use of vascular precursors for therapeutic angiogenesis is the relatively low number of these cells. In this study, we demonstrate that ex vivo expanded differentiated endothelial cells (ECs) and smooth muscle cells (SMCs), may home to the tumor vasculature allowing targeting of transgene expression to the neoangiogenic site., Methods: Mononuclear cells (MNCs) or CD34+ -enriched cells were purified from cord blood. We have defined culture conditions in which we observed two types of clones easily differentiated according to their morphology: cobblestone or spindle-shaped. Phenotypic characterization was assessed by immunocytochemistry, flow cytometry analysis and polymerase reaction with reverse transcription. Formation of capillary-like network in vitro was studied in three-dimensional collagen culture. And recruitment of these cells to a tumoral neoangiogenic site was assessed into tumor-bearing Severe Combined Immunodeficient (SCID) mouse model., Results: The cobblestone cells uniformly positive for CD31, VE-cadherin, vWF, VEGF R1 and R2, ecNOS and incorporating acetylated LDL were ECs. Spindle-shaped cells expressed alpha-smooth muscle actin (alpha-SMA), Smooth Muscle Heavy Chain (SMHC), SM22 and calponin. They also displayed a carbachol-induced contractility in a medium containing IGF1. So we concluded that spindle-shaped cells were SMCs. ECs and SMCs interacted with each other to form a capillary like network in three-dimensional type I collagen culture. Moreover, these ex vivo differentiated cells are able to home to the tumor vasculature., Conclusion: We provide evidence that progenitors for ECs and SMCs circulate in human cord blood and differentiate into functional ECs and SMCs. These differentiated cells could provide a biomaterial for vascular cell therapy, because of their homing capacity to the neovascularization site.

Unlabelled: The action of clopidogrel on platelet receptors was analysed using platelets obtained from 11 healthy volunteers given 75 mg of clopidogrel daily for 8 d. Samples of blood were taken before treatment and after 8 d of medication. Determination of 2-methylthioadenosine diphosphate trisodium (2MesADP)-induced platelet aggregation, serine/threonine and tyrosine phosphorylations were performed in the absence or presence of the P2Y1-receptor-specific antagonist: adenosine 3'-phosphate 5'-phosphate (A3P5P) or the strong inhibitor of GPIIb/IIIa activation: SR121566., Major Conclusions: 1). Serine and threonine phosphorylations of the myosin light chain (P20) and pleckstrin (P47) do not behave similarly, although they are both recognized as the result of phospholipase C pathway stimulation triggered by the P2Y1 receptor. P47 is strongly affected by the A3P5P, and this appears to be highly dependent on P2Y12. However, P20 phosphorylation occurs in the presence of A3P5P, suggesting that the P2Y12 receptor signal contributes to P20 phosphorylation mediated by a calcium-independent pathway. The results suggest that P2Y1 and P2Y12 receptors interact to modulate the phosphorylation of P20 and P47. 2). The inside-out signalling dependent on both P2Y12 and P2Y1 is necessary for GPIIb/IIIa activation. 3). Clopidogrel and SR121566 inhibited the increase in tyrosine phosphorylation induced by 2MesADP and concomitantly inhibited platelet aggregation, indicating that most of the phosphorylations are GPIIb/IIIa dependent. However, neither clopidogrel nor SR121566 inhibited the first wave of 80 kDa substrate (cortactin) which is involved in the reorganization of the cytoskeleton necessary for shape change and which appeared to be essentially P2Y1 dependent.

Platelet factor 4 (PF-4) is a member of the chemokine family with powerful antiangiogenic properties. The mechanism by which PF-4 inhibits endothelial cell proliferation is unclear. We investigated the effects of PF-4 on the intracellular signal transduction induced by basic fibroblast growth factor (FGF2). We found that PF-4 (10 microg/mL) inhibited the FGF2-induced proliferation of adrenal cortex capillary endothelial (ACE) cells. The inhibition of MEK1/2 (mitogen-activated protein kinase kinase) by PD98059 or of PI3K (phosphatidylinositol 3-kinase) by Ly294002 abolished the proliferation induced by FGF2, suggesting that ACE cell proliferation required dual signaling through both the extracellular signal-regulated kinase (ERK) and PI3K pathways. Ly294002 had no significant effect on ERK phosphorylation, whereas PD98059 had a weak effect on the phosphorylation of Akt, suggesting that 2 separate cascades are required for ACE cell proliferation. The addition of PF-4 (10 microg/mL) significantly inhibited ERK phosphorylation (95%), showing that PF-4 acted directly on or upstream from this kinase. Surprisingly, PF-4 did not affect FGF2-induced Akt phosphorylation. This suggests that PF-4 disrupts FGF2 signaling via an intracellular mechanism of inhibition. To exclude the possibility that PF-4 inhibited the binding of FGF2 to only one FGF receptor, preferentially activating the ERK pathway, we investigated the effect of PF-4 on FGF2-induced ERK and Akt phosphorylation, using mutant heparan sulfate-deficient Chinese hamster ovary cells transfected with the FGF-R1 cDNA. The addition of PF-4 (1 microg/mL) significantly inhibited ERK phosphorylation (90%), with no effect on Akt phosphorylation, suggesting that PF-4 acts downstream from the FGF-R1 receptor. In conclusion, this is the first report showing that PF-4 inhibits FGF2 activity downstream from its receptor.

Incubating endosomes with ATP decreased binding of 125I-insulin but not 125I-labeled human growth hormone. Increasing ATP concentrations from 0.1 to 1 mM increased beta-subunit tyrosine phosphorylation and insulin receptor kinase (IRK) activity assayed after partial purification. At higher (5 mM) ATP concentrations beta-subunit tyrosine phosphorylation and IRK activity were markedly decreased. This was not observed with nonhydrolyzable analogs of ATP, nor with plasma membrane IRK, nor with endosomal epidermal growth factor receptor kinase autophosphorylation. The inhibition of endosomal IRK tyrosine phosphorylation and activity was completely reversed by bafilomycin A1, indicating a role for endosomal proton pump(s). The inhibition of IRK was not due to serine/threonine phosphorylation nor was it influenced by the inhibition of phosphotyrosyl phosphatase using bisperoxo(1,10-phenanthroline)oxovanadate anion. Prior phosphorylation of the beta-subunit with 1 mM ATP did not prevent the inhibition of IRK activity on incubating with 5 mM ATP. To evaluate conformational change we incubated endosomes with dithiothreitol (DTT) followed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Without DTT the predominant species of IRK observed was alpha2 beta2. With DTT the alpha beta dimer predominated but on co-incubation with 5 mM ATP the alpha2 beta2 form predominated. Thus, ATP-dependent endosomal acidification contributes to the termination of transmembrane signaling by, among other processes, effecting a deactivating conformational change of the IRK.

Peroxovanadiums (pVs) are potent protein tyrosine phosphatase (PTP) inhibitors with insulin-mimetic properties in vivo and in vitro. We have established the existence of an insulin receptor kinase (IRK)-associated PTP whose inhibition by pVs correlates closely with IRK tyrosine phosphorylation, activation, and downstream signaling. pVs have also been shown to activate various tyrosine kinases (TKs) that could participate in activation of the insulin-signaling pathway. In the present study we have sought to determine whether pV-induced IRK tyrosine phosphorylation requires the intrinsic kinase activity of the IRK, and whether IRK activation is necessary to realize the early steps in the insulin-signaling cascade. To address this we evaluated the effect of a pure pV compound, bis peroxovanadium 1,10-phenanthroline [bpV(phen)], in HTC rat hepatoma cells overexpressing normal (HTC-IR) or kinase-deficient (HTC-M1030) mutant IRKs. We showed that at a dose of 0.1 mM, but not 1 mM, bpV(phen) induced IRK-dependent events. Thus, 0.1 mM bpV(phen) increased tyrosine phosphorylation and IRK activity in HTC-IR but not HTC-M1030 cells. Tyrosine phosphorylation of insulin signal-transducing molecules was promoted in HTC-IR but not HTC-M1030 cells by bpV(phen). The association of p185 and p60 with the src homology-2 (SH2) domains of Syp and the p85-regulatory subunit of phosphatidylinositol 3'-kinase was induced by bpV(phen) in HTC-IR, but not in HTC-M1030 cells, as was insulin receptor substrate-1-associated phosphatidylinositol 3'-kinase activity. Thus autophosphorylation and activation of the IRK by bpV(phen) is effected by the IRK itself, and the early events in the insulin- signaling cascade follow from this activation event. This establishes a critical role for PTP(s) in the regulation of IRK activity. bpV(phen) could be distinguished from insulin only in its ability to activate ERK1 in HTC-M1030 cells, thus indicating that this event is IRK independent, consistent with our previous hypothesis that bpV(phen) inhibits a PTP involved in the negative regulation of mitogen-activated protein kinases.

We provide evidence that both covalent and non-covalent inhibitors of chymotrypsin-like activities inhibit the insulin-induced DNA replication, while the hormonal metabolic effects such as induction of tyrosine aminotransferase activity or increase of amino-acid transport remain unchanged. Besides, the protease inhibitors that we tested were without any effect on both the autocatalytic phosphorylation of insulin receptors and the tyrosine kinase activity towards poly(glutamate/tyrosine). The inhibitory effect of protease inhibitors on DNA synthesis was also visible when fibroblast growth factor (FGF) was used to commit cells in the proliferative cycle. This observation proves that the involvement of a putative protease is not restricted to the insulin mitogenic pathway. Finally, we observed that Fao cells totally escaped the inhibitory action of a covalent inhibitor of chymotrypsin after having been exposed to insulin for 10 h. We propose that a chymotrypsin-like activity is involved in the intracellular signalling leading to the proliferation of rat hepatoma cells up to a non-return point situated in the middle of G1 (6-8 h).

Amongst the proteins that are subjected to variation during the cell division cycle few are under hormonal regulation. The variation in amount of tyrosine aminotransferase (TAT) in the hepatic tissue is under the control of glucagon, glucocorticoids and insulin. It has been reported that the inducibility of TAT activity by dexamethasone in rat hepatoma (HTC) is limited to the late G1 and the S portions of the cell cycle. Evidence is presented in this report that in the rat hepatoma Fao, insulin (which has the capability to promote both cell growth and hormonal effects via its own receptors) modulates the TAT activity during the cell cycle. The maximal insulin-stimulated induction of TAT activity was observed at the end of the G1 phase and then decreased as cells progressed through their mitotic cycle. The number of insulin binding sites per cell was decreased by only 30% during the same period of time. Furthermore, the extent of receptor autophosphorylation decreased in the same proportion, suggesting that insulin receptors remained functional through the whole cell cycle. In fact, another insulin-stimulated cellular function, neutral amino-acid transport, was not modified as cells progressed into the S phase. Hydroxyurea, which is known to prevent cell progression into the S phase, stabilized the insulin-induced TAT activity at its maximal level for several hours. Reciprocally, removal of hydroxyurea resulted in a concomitant decrease in TAT activity and reinitiation of DNA synthesis.

Expression of insulin metabolic effects can be obtained by anti-receptor antibodies without activation of the tyrosine kinase activity [O'Brien R. M., Soos M. A. and Siddle K. (1987) EMBO J. 6, 4003-4010; Forsayeth J. R., Caro J. F., Sinha M. K., Maddux B. A. and Goldfine I. D. (1987) Proc. natn. Acad. Sci. U.S.A. 84, 34,448-34,514; Ponzio G., Contreres J. O., Debant A., Baron V., Gautier N., Dolais-Kitabgi J. and Rossi B. (1988) EMBO J. 7, 4111-4117; Hawley D. M., Maddux B. A., Patel R. G., Wong K. Y., Mamula P. W., Firestone G. L., Brunetti A., Verspohl E. and Goldfine I. D. (1989) J. biol. Chem. 264, 2438-2444; Soos M. A., O'Brien R. M., Brindle N. P. J., Stigter J. M., Okamoto A. K., Whittaker J. and Siddle K. (1989) Proc. natn. Acad. Sci. U.S.A. 86, 5217-5221.]. Recently, we have proposed that receptor cross-linking is sufficient in itself to stimulate glycogen synthesis, even if aggregation was performed on receptors mutated on Tyr 1162 and Tyr 1163 and thus devoid of tyrosine kinase activity [Debant A., Ponzio G., Clauser E., Contreres J. O. and Rossi B. (1989) Biochemistry 28, 14-17]. The aim of this study was to gain information on the involvement of receptor clustering in the expression of the different insulin biological effects. To this end, we studied the mimetic effects of wheat-germ agglutinin, which is likely to induce receptor aggregation without interacting with the receptor protein moiety. Wheat-germ agglutinin failed to promote DNA synthesis, whereas the lectin behaved as a potent mimicker of insulin on tyrosine aminotransferase activity and amino-acid transport. However, this stimulatory effect did not parallel the activation of receptor autophosphorylation. Our data reinforce the idea that the expression of the metabolic effects of insulin are not strictly dependent on a general tyrosine kinase activation.

In a previous report we described the properties of a rabbit anti-insulin receptor antibody (RAIR-IgG) and its effects on the autophosphorylation and kinase activity of human insulin receptors. The present study was carried out on the hepatoma cell line Fao. We tested the mimetic effects of RAIR-IgG on different biological parameters known to be stimulated by insulin, receptor autophosphorylation and kinase activity. RAIR-IgG stimulated the metabolic effects (glucose and amino acid transport) but, unlike insulin, was unable to promote cell proliferation. These data clearly demonstrated the existence of two distinctly controlled pathways in the mediation of the hormonal response. When we investigated the effects of this antibody at the molecular level we found that in a cell-free system RAIR-IgG weakly stimulated receptor autophosphorylation on non-regulatory sites and failed to stimulate tyrosine kinase activity toward exogenous substrates. Accordingly, RAIR-IgG did not stimulate receptor autophosphorylation in 32P-labelled intact cells. Interestingly, under similar conditions RAIR-IgG elicited ribosomal S6 protein phosphorylation, as did insulin. The possibility that RAIR-IgG activated a cryptic tyrosine kinase activity is discussed.

Amino acid sequence of the precursor of the phosphorylated N-glycoprotein (pp63) secreted by rat hepatocytes was deduced from the cDNA sequence. This polypeptide (Mr = 40,586) was rich in both cysteine and proline and contained three potential N-glycosylation sites. A single pp63 mRNA species (approximately 2000 bp), found in normal hepatocytes but not in FaO hepatoma cells, appeared to result from transcription of a single gene. pp63 purified by affinity chromatography inhibited insulin receptor tyrosine kinase and receptor autophosphorylation. Only the phosphorylated form of the protein was active. In additon, pp63 antagonized the growth-promoting action of insulin in FaO cells but did not affect hormone-mediated increase in amino acid transport capacity or tyrosine aminotransferase induction in these cells.

The pivotal role that the tyrosine residues in positions 1162 and 1163 play in the control of the insulin action has been clearly established by substitution of these tyrosine residues for phenylalanine [Ellis, L. (1986) Cell 45, 721-732]. We have recently found that this type of mutation, which abolishes the effects of insulin on glucose metabolism, was without any effect on the mitogenic effect of the hormone [Debant, A. (1988) Proc. Natl. Acad. Sci. U.S.A. (in press)]. Here, we provide evidence that a polyclonal antibody, raised against the human insulin receptor, can restore the receptor-mediated stimulation of glycogen synthesis that was abolished by the mutation. Stimulation of the biological effect by the anti-receptor antibody did not necessitate, whatsoever, the activation of the tyrosine kinase activity and/or receptor autophosphorylation. Furthermore, the antibody-induced reversal of the mutation was not observed when we used Fab fragments alone, but addition of anti-(Fab')2 IgG in a second step resulted in a similar effect as that observed with intact IgG. We propose that Tyr 1162 and Tyr 1163 exert their control on the metabolic effects of insulin through the modulation of receptor aggregation.

Chinese hamster ovary transfectants that express insulin receptors in which tyrosine residues 1162 and 1163 were replaced by phenylalanine exhibit a total inhibition of the insulin-mediated tyrosine kinase activity toward exogenous substrates [histone, casein, and poly(Glu/Tyr)]; this latter activity is associated with total inhibition of the hypersensitivity reported for insulin in promoting 2-deoxyglucose uptake. We now present evidence that the twin tyrosines also control the insulin-mediated stimulation of glycogen synthesis. Surprisingly, this type of Chinese hamster ovary transfectant is as hypersensitive to insulin for its mitogenic effect as are Chinese hamster ovary cells expressing many intact insulin receptors. Such data suggest that (i) the insulin mitogenic effect routes through a different pathway than insulin uses to activate the transport and metabolism of glucose and (ii) the mitogenic effect of insulin is not controlled by the twin tyrosines. At the molecular level, the solubilized mutated receptor has no insulin-dependent tyrosine kinase activity, whereas this receptor displays measurable insulin-stimulated phosphorylation of its beta subunit in 32P-labeled cells. We therefore propose that the autocatalytic phosphorylating activity of the receptor reports a cryptic tyrosine kinase activity that cannot be visualized by the use of classical exogenous substrates.