Insulin and rabbit anti-insulin receptor antibodies stimulate additively the intrinsic receptor kinase activity.

This paper describes the properties of rabbit polyclonal antibodies directed against purified human insulin receptor which strongly stimulate the intrinsic tyrosine kinase activity. The stimulatory effect of the antibodies on the kinase activity was obtained on the insulin receptor autophosphorylation as well as on the kinase activity towards a synthetic substrate. This stimulation is additive to that induced by insulin. Moreover, rabbit antibodies do not impair insulin binding. These data strongly suggest that antibodies and insulin act through separate pathways. This conclusion is reinforced by the differences observed on the phosphopeptide maps of the receptor's beta subunit whose phosphorylation was performed either in the presence of insulin or rabbit antibodies. Interestingly, these polyclonal antibodies can also induce an activation of the receptor autophosphorylation by interacting only with extracellular determinants. The anti-insulin receptor antibodies mimic insulin in their stimulatory effect on amino acid (AIB) uptake, but they have a different effect to that found on the kinase activity; the simultaneous addition of the antiserum and insulin failed to stimulate this amino acid transport over the level induced by a saturating concentration of hormone.     

Direct modulation of insulin receptor protein tyrosine kinase by vanadate and anti-insulin receptor monoclonal antibodies

Sodium vanadate activates "in vitro" insulin receptor autophosphorylation and protein tyrosine kinase in a dose-dependent manner. Insulin receptor protein tyrosine kinase is directly activated also by the anti-insulin receptor beta subunit monoclonal antibody 18-44. We previously demonstrated that the anti-insulin receptor monoclonal antibody MA-10 decreases insulin-stimulated receptor protein tyrosine kinase activity "in vitro", without inhibiting insulin receptor binding. In this report we show that insulin receptor protein tyrosine kinase, activated by sodium vanadate or by monoclonal antibody 18-44, is inhibited by MA-10 antibody. These data suggest that insulin receptor protein tyrosine kinase activity can be either activated and inhibited through mechanisms different from insulin binding.     

Insulin-mimetic effect of trypsin on the insulin receptor tyrosine kinase in intact adipocytes

It has previously been demonstrated that the insulin-mimetic agent trypsin stimulates autophosphorylation of purified insulin receptors and activates the insulin receptor tyrosine kinase in vitro. We now report the effects of trypsin on whole cell tyrosine kinase activation and insulin receptor autophosphorylation. Trypsin treatment of intact adipocytes produces a time-dependent stimulation of tyrosine kinase activity as measured in lectin extracts containing the insulin receptor, or specifically immunoprecipitated insulin receptor samples. Trypsin treatment of adipocytes also results in a loss of insulin binding capacity, and a linear correlation exists between loss of binding and stimulation of tyrosine kinase activity. Exposure of adipocytes to trypsin is known to result in a time- and dose-dependent activation of intracellular glycogen synthase. Examination of the time courses of stimulation of tyrosine kinase and glycogen synthase activation in our system indicates that the stimulation of tyrosine kinase activity by trypsin occurs with sufficient rapidity and magnitude to be consistent with a role of phosphorylation in the activation of glycogen synthase. Trypsin has further been demonstrated to stimulate autophosphorylation of the beta-subunit of the insulin receptor in intact adipocytes. Cells prelabeled with [32P]PO4 for 2 h were exposed to trypsin, and receptors were partially purified over wheat germ agglutinin-agarose columns. Receptors were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the beta-subunit was identified by autoradiography. The protein was extracted and hydrolyzed, and the phosphoamino acids were separated by electrophoresis and quantitated. Two- and five-fold increases in phosphotyrosine were observed with 3 and 10 min of trypsin treatment, respectively. We conclude that trypsin-induced cleavage of the insulin receptor alpha-subunit is relevant to the ability of trypsin to activate the insulin receptor tyrosine kinase in intact adipocytes. We further conclude that autophosphorylation of the insulin receptor and activation of its tyrosine kinase by trypsin may be important to the insulin-mimetic anabolic effects of trypsin.     

Monoclonal antibodies to the insulin receptor stimulate the intrinsic tyrosine kinase activity by cross-linking receptor molecules.

The effect of monoclonal anti-insulin receptor antibodies on the intrinsic kinase activity of solubilized receptor was investigated. Antibodies for six distinct epitopes stimulated receptor autophosphorylation and kinase activity towards exogenous substrates. This effect of antibodies was seen only within a narrow concentration range and monovalent antibody fragments were ineffective. Evidence was obtained by sucrose density-gradient centrifugation for the formation of antibody-receptor complexes which involved both inter- and intra-molecular cross-linking, although stimulation of autophosphorylation appeared to be preferentially associated with the latter. There was partial additivity between the effects of insulin and antibodies in stimulating autophosphorylation, although the sites of phosphorylation appeared identical on two-dimensional peptide maps. Antibodies for two further epitopes failed to activate receptor kinase, but inhibited its stimulation by insulin. The effects of antibodies on kinase activity paralleled their metabolic effects on adipocytes, except for one antibody which was potently insulin-like in its metabolic effects, but which antagonized insulin stimulation of kinase activity. It is concluded that antibodies activate the receptor by cross-linking subunits rather than by reacting at specific epitopes. The ability of some antibodies to activate receptor may depend on receptor environment as well as the disposition of epitopes.     

Effect of anti-insulin receptor antibodies on insulin-sensitive phosphodiesterase in rat fat cells

Effect of sera with anti-insulin receptor antibodies (AIRS) on insulin-sensitive phosphodiesterase in rat fat cells was examined. AIRS activated the enzyme when incubated with intact fat cells. AIRS (1:400 dilution) were less potent for activation of the phosphodiesterase than insulin (3 nM), but were more potent for inhibition of 125I-insulin binding to fat cells than insulin. When insulin receptor of fat cells was destroyed with trypsin-treatment, AIRS as well as insulin completely lost the ability to activate the phosphodiesterase. These findings suggest that AIRS bind to or very near the insulin receptor and exhibit insulin-like biological effect of the phosphodiesterase activation.     

Crystallization of intact monoclonal antibodies

Attempts were made to crystallize four monoclonal antibodies, one IgG_2ak and three IgG_1k. Using a PEG 3350 screen combined with detergents, and developed from our experiments with an IgG_2ak antibody specific for canine lymphoma cells,^1,2 crystals have now been obtained of two of these four immunoglobulins, an antiphenytoin and an antiphenobarbital antibody. A complex between the antiphenobarbital antibody and its drug antigen crystallized as well. The antibody for phenytoin has, to this point, produced only clustered microcrystals, marginally suitable for X-ray analysis. Single crystals of the IgG_1k antibody against phenobarbital, however, were characterized by X-ray diffraction to be primitive monoclinic, with unit cell dimensions a = 67 Å, b = 193 Å, c = 74 Å, and β = 110°. These crystals have an entire IgG_1k molecule as the asymmetric unit and they diffract to at least 3.2 Å resolution. © 1995 Wiley-Liss, Inc.     

Binding of monoclonal antibodies to the nuclear estrogen receptor in intact nuclei

A monoclonal antibody to calf uterus cytoplasmic estrogen receptor shows a specifically displaceable and saturable binding to intact nuclei of mouse uterus after estradiol stimulation. The binding is complete after 3 hr at 0 degree C. The binding of the antibody correlates with the exchangeable estradiol binding activity of the nuclei over a 4-hr time course following in vivo injection of 17 beta-estradiol.     

Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells.

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.     

Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.

Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted to be in a region between the catalytic domain and predicted beta-propeller structure in the C-terminal portion of GPI-PLD (T. A. Springer, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.     

Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.     

Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation.

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.     

Purification of an active EGF receptor kinase with monoclonal antireceptor antibodies

A method is described for a rapid two-step purification of the membrane receptor for epidermal growth factor (EGF) from cultured human A-431 cells. After solubilization of the cells with Triton X-100, the receptor is immobilized on an immunoaffinity column containing a monoclonal antibody directed against the receptor. In the second step of purification, the receptor, eluted from the antibody column, is adsorbed and specifically eluted from a lectin-agarose column. The molecular species obtained is mainly the 170,000-dalton EGF receptor polypeptide. The activity of the pure receptor depends on the conditions used for the desorption from the immunoaffinity beads. High-yield elution is obtained with acidic buffer and the receptor so purified specifically binds EGF, but is devoid of the kinase activity. When the elution is done with alkaline buffers or with buffer containing urea, a fully active receptor kinase is purified (yield of 10%). The pure receptor binds 125I-EGF with a Kd of 4 X 10(-8) M and retains EGF-sensitive protein kinase activity which phosphorylates tyrosine residues on the receptor itself. An additional protocol is described for large-scale purification (yield of 55%) of EGF receptor for the analysis of its primary structure. In this procedure, the EGF receptor is first purified by immunoaffinity chromatography which is followed by preparative gel electrophoresis of the 32P internally labeled receptor to remove minor protein contaminants.     

Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells.

We report a highly specific fluorescence lifetime imaging microscopy (FLIM) method for monitoring epidermal growth factor receptor (EGFR) phosphorylation in cells based on fluorescence resonance energy transfer (FRET). EGFR phosphorylation was monitored using a green fluorescent protein (GFP)-tagged EGFR and Cy3-conjugated anti-phosphotyrosine antibodies. In this FRET-based imaging method, the information about phosphorylation is contained only in the (donor) GFP fluorescence lifetime and is independent of the antibody-derived (acceptor) fluorescence signal. A pixel-by-pixel reference lifetime of the donor GFP in the absence of FRET was acquired from the same cell after photobleaching of the acceptor. We show that this calibration, by acceptor photobleaching, works for the GFP-Cy3 donor-acceptor pair and allows the full quantitation of FRET efficiencies, and therefore the degree of exposed phosphotyrosines, at each pixel. The hallmark of EGFR stimulation is receptor dimerisation [1] [2] [3] [4] and concomitant activation of its intracellular tyrosine kinase domain [5] [6] [7]. Trans-autophosphorylation of the receptor [8] [9] on specific tyrosine residues couples the activated dimer to the intracellular signal transduction machinery as these phosphorylated residues serve as docking sites for adaptor and effector molecules containing Src homology 2 (SH2; reviewed in [10]) and phosphotyrosine-binding (PTB) [11] domains. The time-course and extent of EGFR phosphorylation are therefore important determinants of the underlying pathway and resulting cellular response. Our results strongly suggest that secondary proteins are recruited by activated receptors in endosomes, indicating that these are active compartments in signal transduction.     

Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.     

Antiphosphotyrosine antibodies modulate insulin receptor kinase activity and insulin action

Insulin elicits the autophosphorylation of the beta-subunit of its receptor on tyrosine residues: this effect appears to be the earliest post-binding event involved in insulin action. In the present study we have raised highly specific antibodies to phosphotyrosine residues, and we have taken advantage of these antibodies to further evaluate the role of the insulin receptor tyrosine kinase in the generation of insulin's biological responses. Using a cell-free phosphorylation assay, we show here that these antibodies increase the tyrosine kinase activity of the receptor, and its phosphorylation on tyrosine residues. In contrast, the antibodies do not interfere with dephosphorylation of the insulin receptor. Introduction of the same antibodies in living Fao hepatoma cells enhances the effect of insulin on both glucose transport and aminoacid uptake. As a whole our data indicate that the insulin receptor kinase is involved in the generation of an early (glucose transport) and late (aminoacid uptake) response to insulin. Further, conformational changes in phosphotyrosine containing domains of the insulin receptor appear to modulate insulin's biological effects. Finally, the injection of antibodies in intact cells provides us with a novel and promising tool to search for cellular substrates for the insulin receptor tyrosine kinase.     

Generation and characterization of novel monoclonal antibodies to the Ret receptor tyrosine kinase

Ret is a tyrosine kinase receptor involved in several human diseases germ-line mutations are responsible for multiple endocrine neoplasia type 2 syndromes while somatic mutations of Ret are found in sporadic medullary thyroid carcinomas. In the present work, we describe the generation and characterization of a panel of novel monoclonal antibodies to Ret obtained by immunizing mice with a Ret-FC fusion protein. Fifty-five independent monoclonal antibodies recognize Ret-FC by enzyme linked immunosorbent assay but not a non-related FC fusion protein. Twenty antibodies further characterized recognize Ret expressing cells by flow cytometry. Finally, immunoprecipitation analysis showed that these antibodies recognize Ret mature glycosylated and immature forms. Thus, these monoclonal antibodies could be used as diagnostic tools to detect Ret expression, as well as therapeutic tools to downmodulate Ret or to deliver cytotoxic drugs to malignancies that overexpress Ret as neuroblastomas, medullary and papillary thyroid carcinomas, seminomas, and leukemia.     

Alanine scanning mutants of rat proinsulin I show functional diversity of anti-insulin monoclonal antibodies

In contrast to autoantibodies that are functionally silenced or deleted, IgG Abs that react with autologous insulin routinely follow hormone administration and arise spontaneously in autoimmune (type I) diabetes mellitus. To understand Ab interactions with autologous insulin, rat proinsulin I and 32 alanine substituted analogues were expressed as fusion proteins and used to examine 16 anti-insulin mAb in ELISA. The results identify several amino acid residues that contribute to binding by a large majority (>75%) of mAb, although no single residue is uniformly required for binding by all mAb. Replacements at charged or polar residues on the insulin surface including A4 (Asp), A5 (Gln), A9 (Ser) A12 (Ser), A17 (Gln), A18 (Asn), B13 (Glu), and B21 (Glu) consistently decreased mAb binding. Single alanine substitutions at positions A16 (Leu), A11 (Cys), B8 (Gly), and B15 (Leu) that are predicted to alter the core structure or chain folding vary widely in their impact on Ab binding. mAb that bind insulin preferentially on solid phase (i.e., ELISA) are highly sensitive to replacement of single residues, and substitutions that alter conformation abolish binding. In contrast, high affinity mAb that bind insulin in solution are relatively insensitive to substitutions at single residues, and they maintain binding to all mutants, including those with disrupted conformation. For such high affinity mAb, replacement of long hydrophobic side chains can augment binding, suggesting mAb interactions with insulin include an induced fit. Thus, the ability of insulin to function as a "molten globule" may contribute to the diversity and autoreactivity of the anti-insulin repertoire.     

Intrinsic kinase activity of the insulin receptor. The intact (alpha 2 beta 2) insulin receptor from rat liver contains a kinase domain with greater intrinsic activity than the intact insulin receptor from human placenta

We are interested in developing methods to rigorously characterize the intrinsic enzymatic activity of the insulin receptor. We have previously shown that the intact, kinase active form of the receptor can be separated from inactive forms isolated from human placenta. Therefore, the determination of kinase activity, when normalized to the number of receptors based on binding, is not complicated by the presence of insulin receptor forms which bind insulin normally, but are kinase inactive. We now have extended this separation technique to insulin receptor preparations from rat liver. Thus, the determination and comparison of the intrinsic kinase activity of insulin receptor from human placenta and rat liver was performed. When normalized to the same number of insulin receptors which are autophosphorylated to the same degree, the rat liver insulin receptor catalyzes the transfer of phosphate from ATP to three different substrates, on average, 2.8-fold quicker than receptor from human placenta. This probably represents an inherent difference in the intrinsic kinase activity (Vmax), since the values for KM of the substrates are essentially identical, for insulin receptors from both sources. Intrinsic kinase differences may reflect different biological roles and/or differential regulation by exogenous factors. We are now examining this hypothesis in light of reports that demonstrate regulation of intrinsic kinase activity of the insulin receptor in certain physiological and pathological states.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle. Effect of insulin, epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigated in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 mM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5-10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Protein kinase activity and substrates at the surface of intact HeLa cells

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.