The structural basis for insulin-like growth factor I receptor high affinity binding

We have recently identified high and low affinity insulin-like growth factor I (IGF I) binding sites in solubilized human placental membranes and purified the high affinity IGF I receptor by IGF I affinity chromatography (Tollefsen, S. E., Thompson, K., and Petersen, D. J. (1987) J. Biol. Chem. 262, 16461-16469). To define the structural basis for high affinity IGF I binding, we have examined the effect of disulfide bond reduction on the binding parameters of the high affinity IGF I receptor. We find that the disulfide bonds linking the two alpha beta dimers of the IGF I receptor heterotetramer are reduced by incubation at pH 8.75 with 2 mM dithiothreitol (DTT) for 5 min at room temperature. Gel filtration chromatography on a Superose 12 fast protein liquid chromatography column indicates that the alpha beta dimers do not remain associated by noncovalent interactions after reduction. Scatchard plots of IGF I binding to the IGF I receptor incubated at pH 8.75 with or without DTT indicate that the IGF I receptor alpha beta dimers have a 6.1 +/- 1.6 (mean +/- S.D.) times lower affinity than the heterotetramer for IGF I. The total binding capacity of the IGF I receptor treated with DTT is 1.6 +/- 0.3 (mean +/- S.D.) times higher than that of an equal amount of receptor treated without DTT. These results are consistent with a model in which the heterotetramer binds a single IGF I molecule with high affinity, whereas each of the two alpha beta dimers binds an IGF I molecule with lower affinity after dissociation. We conclude that association of two alpha beta dimers is required for formation of an IGF I receptor with high affinity for its ligand.     

Structural requirements for the transmembrane activation of the insulin receptor kinase

Tetrameric insulin holoreceptor (alpha 2 beta 2) was reduced with dithiothreitol into alpha beta dimers such that they maintain up to 50% of insulin binding at tracer ligand concentrations. Scatchard analysis of insulin binding to dimers revealed that they had a reduced affinity for ligand by a factor of 3-6 compared to holoreceptor, whereas the maximum number of high affinity binding sites was not affected. The alpha beta dimers can be separated from holoreceptor by sucrose density gradient centrifugation, and hence, they are not associated by noncovalent interactions. Insulin-dependent autophosphorylation of alpha beta dimers isolated from low ionic strength sucrose density gradients was minimal and was always accompanied by reoxidation of dimers to the tetrameric holoreceptor. The reformed tetramer exhibited a strong insulin-dependent autophosphorylation reaction. Reoxidation was prevented by isolating alpha beta dimers in sucrose density gradients containing 0.15 M NaCl. Under these conditions, no insulin-dependent autophosphorylation was observed. When insulin receptor was first autophosphorylated and then reduced, receptor kinase activity, as assayed by histone phosphorylation, was not affected. Also, the insulin-independent, basal autophosphorylation was maintained after reduction into alpha beta dimers. We conclude that alpha beta-alpha beta interaction is not necessary for the maintenance of basal kinase activity or for insulin-activated kinase activity once autophosphorylation occurs. However, dimer-dimer interaction appears critical for the insulin-dependent activation of the receptor's intrinsic kinase activity.     

Alteration of intramolecular disulfides in insulin receptor/kinase by insulin and dithiothreitol: insulin potentiates the apparent dithiothreitol-dependent subunit reduction of insulin receptor

Dithiothreitol (DTT) was observed to increase both beta-subunit autophosphorylation and exogenous substrate phosphorylation of the insulin receptor in the absence of insulin. The natural protein reducing agent thioredoxin was also observed to increase the insulin receptor beta-subunit autophosphorylation. The activation of the insulin receptor/kinase by both DTT and thioredoxin was found to be additive with that of insulin. Further, the increase in the insulin receptor beta-subunit autophosphorylation in the presence of DTT and insulin was demonstrated to be due to an increase in the initial rate of autophosphorylation without alteration in the extent of phosphorylation. Similarly, the increase in the exogenous substrate phosphorylation was due to an increase in the Vmax of phosphorylation without significant effect on the apparent Km of substrate binding. In the presence of relatively low concentrations of DTT, insulin was found to potentiate the apparent insulin receptor subunit reduction of the native alpha 2 beta 2 heterotetrameric complex into alpha beta heterodimers, when observed by silver staining of sodium dodecyl sulfate-polyacrylamide gels. N-[3H]Ethylmaleimide ([3H]NEM) labeling in the absence of DTT pretreatment demonstrated that only the beta subunit had accessible sulfhydryl group(s). However, treatment of insulin receptors with DTT increased the amount of [3H]NEM labeling in the beta subunit as well as exposing sites on the alpha subunit. Further, incubation of the insulin receptors with the combination of DTT and insulin also demonstrated the apparent insulin-potentiated subunit reduction without any increase in the total amount of [3H]NEM labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin-dependent intermolecular subunit communication between isolated alpha beta heterodimeric insulin receptor complexes

The dissociation of the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex into an alpha beta heterodimeric state was found to occur in a pH- and dithiothreitol (DTT)-dependent manner. Formation of the alpha beta heterodimeric complex, under conditions which preserved tracer insulin binding and protein kinase activities (pH 8.75 for 25 min followed by 2.0 mM DTT for 5 min) occurred with an approximate 50% efficiency. The resulting nondissociated alpha 2 beta 2 heterotetrameric complexes could then be separated effectively by Bio-Gel A-1.5m gel filtration chromatography at neutral pH. The isolated DTT-treated but nondissociated alpha 2 beta 2 heterotetrameric complex was resistant to any further dissociation by a second round of DTT and alkaline pH treatment, whereas the isolated alpha beta heterodimeric complex was stable to spontaneous reassociation for at least 72 h at pH 7.60. Kinetic analyses of the insulin receptor protein kinase activity demonstrated that the insulin stimulation of glutamic acid:tyrosine (4:1) synthetic polymer phosphorylation for both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes occurred via an increase in Vmax without any significant change in Km. Examination of beta subunit autophosphorylation of the alpha beta heterodimeric complex, in the presence but not in the absence of insulin, demonstrated the appearance of the covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Further, the initial rate of insulin-stimulated beta subunit autophosphorylation in the isolated alpha beta heterodimeric complex occurred in a dilution-dependent (intermolecular) manner. These data demonstrate that the isolated alpha beta heterodimeric insulin receptor complex is fully capable of expressing insulin-dependent activation of the beta subunit protein kinase domain with the covalent reassociation of the alpha beta heterodimeric complex into an alpha 2 beta 2 heterotetrameric disulfide-linked state.     

Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native alpha 2 beta 2 insulin receptor subunit complex

The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing (0.1% SDS) or nondenaturing (0.1% Triton X-100) conditions. Pretreatment of 32P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the alpha 2 beta 2 insulin receptor complex (Mr 400,000) into the monomeric 95,000 beta subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the alpha 2 beta 2 heterotetrameric complex with essentially no alpha beta heterodimeric or free monomeric beta subunit species present. This suggests that the insulin receptor can reoxidize into the Mr 400,000 complex after the removal of DTT by gel filtration chromatography. Surprisingly, these apparently reoxidized insulin receptors were also observed to be functional with respect to insulin binding, albeit with a 50% decrease in affinity for insulin and insulin stimulation of the beta subunit autophosphorylation. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT followed by incubation with excess N-ethylmaleimide prior to gel filtration chromatography in 0.1% Triton X-100. Under these conditions the insulin receptors migrated as the Mr 400,000 alpha 2 beta 2 complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of insulin-like growth factor I receptor from human placental membranes

Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.     

Functional and immunological distinction between insulin-like growth factor I receptor subtypes in KB cells.

Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.     

Separation of the high affinity insulin-like growth factor I receptor from low affinity binding sites by affinity chromatography

We have identified high and low affinity insulin-like growth factor I (IGF I)-binding sites with mean dissociation constants of 0.37 and 6.25 nM, respectively, in solubilized placental membranes. We have separated these sites and purified the high affinity IGF I receptor 1,300-fold, with an overall yield of 9.9%, using wheat germ agglutinin-Sepharose chromatography, insulin affinity chromatography, and IGF I affinity chromatography. The Scatchard plot of IGF I binding to the high affinity receptor is linear, suggesting the purification of a single homogeneous class of binding sites. Insulin is two orders of magnitude less effective than IGF I in competitively inhibiting IGF I binding to this receptor. The high affinity IGF I receptor is composed of alpha and beta subunits with apparent molecular weights of 135,500 and 96,200, respectively. IGF I at concentrations of greater than or equal to 50 ng/ml stimulates autophosphorylation of the beta subunit of the purified high affinity receptor 4.6-fold. Low affinity IGF I-binding sites run through the IGF I affinity column or are eluted from the insulin affinity column. The separation of IGF I receptors with different binding affinities by sequential affinity chromatography will make it possible to examine directly the determinants of receptor affinity.     

Characterization of affinity-purified insulin receptor/kinase. Effects of dithiothreitol on receptor/kinase function

The insulin receptor/kinase was purified to near homogeneity from human placenta. The purified kinase exhibited a specific activity of 300 nmol/min/mg of protein at 30 degrees C using the synthetic peptide, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly, as substrate in the presence of insulin. Treatment of the receptor/kinase with dithiothreitol (DTT) reduced insulin binding by 40-50% and also inhibited tyrosine kinase activity. Phosphorylation and activation of the receptor/kinase did not prevent the DTT-induced loss of binding but completely protected it from the deleterious effects of reducing agent on enzymic activity. Analyses of the structure of the receptor/kinase following phosphorylation and treatment with DTT indicated that the class I disulfide bonds were reduced under the conditions employed, but the tetrameric structure of the receptor/kinase was essentially unaltered. These findings indicate that intact class I disulfides are required for insulin binding but are not necessary for maintenance of the preactivated kinase. DTT was also found to enhance the autoactivation of the insulin receptor/kinase and to promote the reversal of the autophosphorylation reaction. Thus disulfide bonds appear to have multiple roles in the function of the insulin receptor/kinase.     

Functional properties of an isolated alpha beta heterodimeric human placenta insulin-like growth factor 1 receptor complex

Treatment of human placenta membranes at pH 8.5 in the presence of 2.0 mM dithiothreitol (DTT) for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, resulted in the formation of a functional alpha beta heterodimeric insulin-like growth factor 1 (IGF-1) receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The membrane-bound alpha beta heterodimeric complex displayed similar curvilinear 125I-IGF-1 equilibrium binding compared to the alpha 2 beta 2 heterotetrameric complex. Triton X-100 solubilization of the alkaline pH and DTT-pretreated placenta membranes, followed by Bio-Gel A-1.5m gel filtration chromatography, was found to effectively separate the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric IGF-1 receptor species, 125I-IGF-1 binding to both the isolated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes demonstrated a marked straightening of the Scatchard plots, compared to the placenta membrane-bound IGF-1 receptors, with a 2-fold increase in the high-affinity binding component. Similar to the membrane-bound IGF-1 receptor species, the 125I-IGF-1 binding properties between the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes were not significantly different. IGF-1 stimulation of IGF-1 receptor autophosphorylation indicated that the ligand-dependent activation of alpha beta heterodimeric protein kinase activity occurred concomitant with the reassociation into a covalent alpha 2 beta 2 heterotetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)     

The insulin receptor protein kinase. Physicochemical requirements for activity

We determined that the rate of insulin-stimulated autophosphorylation of the insulin receptor is independent of receptor concentration and thus proceeds via an intramolecular process. This result is consistent with the possibility that ligand-dependent autophosphorylation may be a means by which cells can distinguish occupied from unoccupied receptors. We employed dithiothreitol to dissociate tetrameric receptor into alpha beta halves in order to further elucidate the structural requirements for the receptor-mediated kinase activity. Dithiothreitol had a complex biphasic effect on insulin-stimulated receptor kinase activity. Marked stimulation of kinase activity was observed at 1-2 mM dithiothreitol when the receptor was predominantly tetrameric and kinase activity diminished when dimeric alpha beta receptor halves predominate (greater than 2 mM dithiothreitol). N-Ethylmaleimide inhibits insulin-stimulated receptor kinase activity. We suggest that the tetrameric holoreceptor is the most active kinase structure and this structure requires for maximal activity, a reduced sulfhydryl group at or near the active site. We treated receptor preparations with elastase to generate receptor proteolytically "nicked" in the beta subunit. This treatment completely abolishes insulin-dependent autophosphorylation and histone phosphorylation with essentially no effects on insulin binding as determined by affinity labeling of the receptor alpha subunit. We suggest such treatment functionally uncouples insulin binding from insulin-stimulated receptor kinase activity. The possible physiological significance of these findings is discussed.     

Blue native PAGE as a useful method for the analysis of the assembly of distinct combinations of nicotinic acetylcholine receptor subunits

Oligomerization of complete and incomplete combinations of rat muscle-type nicotinic acetylcholine receptor (nAChR) subunits in Xenopus oocytes was studied by blue native PAGE and compared with acetylcholine-activated current in these cells. The rank order of expression level judged by current was alpha 1 beta 1 gamma delta > alpha 1 beta 1 gamma > alpha 1 beta 1 delta > alpha 1 gamma delta > alpha 1 delta > alpha 1 gamma. alpha 1 and alpha 1 beta 1 were not functional. Protein complexes incorporating a heptahistidyl-tagged alpha 1 subunit were chromatographically purified from digitonin extracts of oocytes and resolved by blue native PAGE. In the absence of any co-expressed nAChR subunit, the majority of alpha 1 formed aggregates. Co-expression of beta 1 had no effect on alpha 1 aggregation, whereas both gamma and delta diminished alpha 1 aggregation in favor of discrete oligomers: alpha 1 formed tetramers together with gamma and dimers, trimers, and tetramers together with delta. When alpha 1 gamma was complemented with beta 1 to form a functional alpha 1 beta 1 gamma receptor, a small amount of a pentamer was found besides a prominent alpha 1-His7 beta 1 gamma trimer. Expression of the functional alpha 1 beta 1 delta receptor yielded marked amounts of a pentamer besides dimers and trimers. These results are discussed in terms of the assembly model of Green and Claudio (Cell 74, 57-69, 1994), substantiating that blue native PAGE is suited for the investigation of ion channel assembly.     

src kinase catalyzes the phosphorylation and activation of the insulin receptor kinase

When a partially purified insulin receptor preparation immobilized on insulin-agarose is incubated with [gamma-32P]ATP, Mn2+, and Mg2+ ions, the receptor beta subunit becomes 32P-labeled. The 32P-labeling of the insulin receptor beta subunit is increased by 2-3-fold when src kinase is included in the phosphorylation reaction. In addition, the presence of src kinase results in the phosphorylation of a Mr = 125,000 species. The Mr = 93,000 receptor beta subunit and the Mr = 125,000 32P-labeled bands are absent when an insulin receptor-deficient sample, prepared by the inclusion of excess free insulin to inhibit the adsorption of the receptor to the insulin-agarose, is phosphorylated in the presence of the src kinase. These results indicate that the insulin receptor alpha and beta subunits are phosphorylated by the src kinase. The src kinase-catalyzed phosphorylation of the insulin receptor is not due to the activation of receptor autophosphorylation because a N-ethylmaleimide-treated receptor preparation devoid of receptor kinase activity is also phosphorylated by the src kinase. Conversely, the insulin receptor kinase does not catalyze phosphorylation of the active or N-ethylmaleimide-inactivated src kinase. Subsequent to src kinase-mediated tyrosine phosphorylation, the insulin receptor, either immobilized on insulin-agarose or in detergent extracts, exhibits a 2-fold increase in associated kinase activity using histone as substrate. src kinase mediates phosphorylation of predominantly tyrosine residues on both alpha and beta subunits of the insulin receptor. Tryptic peptide mapping of the 32P-labeled receptor alpha and beta subunits by high pressure liquid chromatography reveals that the src kinase-mediated phosphorylation sites on both receptor subunits exhibit elution profiles identical with those phosphorylated by the receptor kinase. Furthermore, the HPLC elution profile of the receptor auto- or src kinase-catalyzed phosphorylation sites on the receptor alpha subunit are also identical with that on the receptor beta subunit. These results indicate that: the src kinase catalyzes tyrosine phosphorylation of the insulin receptor alpha and beta subunits; and src kinase-catalyzed phosphorylation of insulin receptor can mimic the action of autophosphorylation to activate the insulin receptor kinase in vitro, although whether this occurs in intact cells remains to be determined.     

Autophosphorylation within insulin receptor beta-subunits can occur as an intramolecular process.

The insulin receptor is a complex membrane-spanning glycoprotein composed of two alpha-subunits and two beta-subunits connected to form an alpha 2 beta 2 holoreceptor. Insulin binding to the extracellular alpha-subunits activates intracellular beta-subunit autophosphorylation and substrate kinase activity. The current study was designed to differentiate mechanisms of transmembrane signaling by the insulin receptor, specifically whether individual beta-subunits undergo cis- or trans-phosphorylation. We compared relative kinase activities of trypsin-truncated receptors, alpha beta-half receptors, and alpha 2 beta 2 holoreceptors under conditions that allowed us to differentiate intermolecular and intramolecular events. Compared to the insulin-stimulated holoreceptors, the trypsin-truncated receptor undergoes autophosphorylation at similar tyrosine residues and catalyzes substrate phosphorylation in the absence of insulin at a comparable rate. The truncated receptor sediments on a sucrose gradient at a position consistent with a structure comprising a single beta-subunit attached to a fragment of the alpha-subunit and undergoes autophosphorylation in this form in the absence of insulin. Autophosphorylation of the truncated insulin receptor is independent of receptor concentration, and immobilization of the truncated receptor on a matrix composed of an anti-receptor antibody bound to protein A-Sepharose diminishes neither autophosphorylation nor receptor-catalyzed substrate phosphorylation. Therefore, true intramolecular (cis) phosphorylations, which occur within individual beta-subunits derived from trypsin-truncated receptors, lead to kinase activation. However, insulin-stimulated autophosphorylation of insulin receptor alpha beta heterodimers is concentration-dependent, and both autophosphorylation and kinase activity are markedly reduced following immobilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural requirements for signal transduction of the insulin receptor

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico-chemical properties of (alpha beta)2 receptors solubilized with Triton X-100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X-100 in the buffer (Triton X-100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s20,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor-hormone complex, the alterations are reversed. After autophosphorylation of hormone-bound (alpha beta)2-insulin receptors, phosphate incorporation was found for 7.9-nm receptor forms when receptor-insulin complexes were crosslinked with disuccinimide suberate prior to Triton X-100/PAGE. However, phosphate incorporation was demonstrated for the 9.5-nm receptor forms when receptor-insulin complexes were not prevented from dissociation. This strongly indicates that the (alpha beta)2 receptor is autophosphorylated after assuming its 7.9-nm form upon insulin binding. Moreover, the insulin-dependent structural alterations are not affected by autophosphorylation. In contrast to (alpha beta)2 receptors, the diffusion and the sedimentation behaviour of alpha beta receptors, which carry a dormant tyrosine kinase even in the hormone-laden state, has been found to be insensitive to insulin binding. Different molecular properties of alpha beta and (alpha beta)2 receptors have also been detected by hormone binding studies. Insulin binding to (alpha beta)2 and alpha beta receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of alpha beta receptor changes on association into the (alpha beta)2 species. Alternatively, distinct hormone-induced conformational alterations at the molecular level of alpha beta and (alpha beta)2 receptor species may lead to the different binding properties. Our data demonstrate that the (alpha beta)2-insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone-inducable enhancement of tyrosine autophosphorylation of the 7.9-nm insulin-bound receptor form. In contrast, alpha beta receptors appear to be locked in an inactive nonconvertable state. Thus, interaction between two alpha beta receptor units is required to allow extended conformational alterations, which are assumed to be the triggering event for augmented auto-phosphorylation.     

Ligand-dependent intersubunit association within the insulin receptor complex activates its intrinsic kinase activity

Insulin receptor halves (alpha beta) were obtained upon selective reduction of the holoreceptor (alpha 2 beta 2) and were isolated in concentrated form. Autophosphorylation of concentrated alpha beta receptor halves can be stimulated by insulin an average of 4.0-fold, whereas nonreduced holoreceptor can be stimulated 5.4-fold. If alpha beta half-receptors are immobilized on wheat germ agglutinin-agarose, no insulin-stimulated autophosphorylation is observed, whereas immobilized holoreceptor retains insulin responsiveness. Treatment of alpha beta half-receptors with glutathione in the presence of insulin results in reoxidation to the holoreceptor form (alpha 2 beta 2) with an efficiency of 60-70% as visualized by immunoblotting, thus providing evidence that two alpha beta halves are in close physical proximity. This reoxidation reaction, which is evident prior to autophosphorylation, is rapid and strictly dependent on the presence of insulin, consistent with the hypothesis that insulin promotes the association of two alpha beta halves. Furthermore, the insulin-induced reoxidation reaction and the insulin-induced autophosphorylation show the same dose dependence ED50 3-4 x 10(-8) M insulin), suggesting that the noncovalent association of alpha beta half-receptors upon insulin binding is a prerequisite for insulin-stimulated autophosphorylation in concentrated alpha beta half-receptor preparations. If the alpha beta half-receptor forms are phosphorylated in the presence of an anti-phosphotyrosine antibody and separated from nonphosphorylated alpha beta receptors, we observed that the phosphorylated alpha beta receptor halves contain bound insulin. This excludes the possibility that alpha beta half-receptors that bind insulin, preferentially phosphorylate alpha beta halves that have no insulin bound.     

Separation and characterization of three insulin receptor species that differ in subunit composition

Partially purified human placental insulin receptor preparations give rise to three distinct insulin-binding peaks when eluted from a Mono Q high-performance liquid chromatography anion-exchange column. We analyzed the basis for this phenomenon by affinity cross-linking of insulin to each peak, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We find that the three insulin-binding peaks represent different molecular weight complexes with the following subunit composition: (alpha beta)2, (alpha beta)(alpha beta'), and (alpha beta')2, where beta' represents a proteolytically derived fragment of the beta subunit. This analysis of subunit composition was confirmed by silver staining of affinity-purified insulin receptor following resolution of the forms on a Mono Q column as described previously. We have characterized the three isolated insulin receptor forms with regard to ligand binding by LIGAND and Scatchard analysis. We also measured insulin-stimulatable autophosphorylation and exogenous kinase activity directed toward poly(Glu/Tyr) (4:1). The three forms of the insulin receptor exhibit similar KD's for insulin binding to the high- and low-affinity sites. The (alpha beta)2 and (alpha beta)(alpha beta') forms of the insulin receptor display superimposable curvilinear Scatchard plots. In contrast, only the intact holoreceptor (alpha beta)2 form demonstrates insulin-stimulatable autophosphorylation and exogenous kinase activity. The (alpha beta)(alpha beta') form has reduced basal kinase activity which was not increased by prior incubation with insulin. The (alpha beta')2 form lacks a kinase domain and consequently demonstrated no kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoinhibition of the insulin-like growth factor I receptor by the juxtamembrane region

The juxtamembrane (JM) regions of several receptor tyrosine kinases are involved in autoinhibitory interactions that maintain the low basal activity of the receptors; mutations can give rise to constitutive kinase activity and signaling. In this report, we show that the JM region of the human insulin-like growth factor I receptor (IGF1R) plays a role in kinase regulation. We mutated JM residues that were conserved in this subfamily of receptor tyrosine kinases, and expressed and purified the cytoplasmic domains using the Sf9/baculovirus system. We show that a kinase-proximal mutation (Y957F) and (to a lesser extent) a mutation in the central part of the JM region (N947A) increase the autophosphorylation activity of the kinase. Steady-state kinetic measurements show the mutations cause an increase in V(max) for phosphorylation of peptide substrates. When the holoreceptors were expressed in fibroblasts derived from IGF1R-deficient mice, the Y957F mutation led to a large increase in basal and in IGF1-stimulated receptor autophosphorylation. Together, these data demonstrate that the JM region of IGF1R plays an important role in limiting the basal activity of the receptor.     

Tyrosine kinase-defective insulin receptors undergo insulin-induced microaggregation but do not concentrate in coated pits

Biologically active colloid-gold complexes were used to compare ligand-induced microaggregation, redistribution, and internalization of insulin receptors on Rat 1 fibroblasts expressing wild type (HIRc) or tyrosine kinase-defective (HIR A/K1018) human insulin receptors. Insulin-like growth factor I (IGF I) and alpha 2-macroglobulin receptors also were compared. On both cell types, all four unoccupied receptor types occurred predominantly as single receptors. Ligand binding caused receptor microaggregation. Microaggregation of wild type or kinase-defective insulin receptors or IGF I receptors was not different. alpha 2-Macroglobulin receptors formed larger microaggregates. Compared to wild type insulin or IGF I receptors, accumulation of kinase-defective insulin receptor microaggregates in endocytic structures was decreased, and the size of microaggregates in coated pits was significantly smaller. As a result, receptor-mediated internalization of gold-insulin by HIR A/K1018 cells was less than 6% of the cell-associated particles compared to approximately 60% of the particles in HIRc cells. On HIR A/K1018 cells, alpha 2-macroglobulin and IGF I were internalized via coated pits demonstrating that those structures were functional. These results suggest that: 1) ATP binding, receptor autophosphorylation, and activation of receptor kinase activity are not required for receptor microaggregation; 2) receptor microaggregation per se is not sufficient to cause ligand-induced receptor-mediated internalization or the biological effects of insulin; and 3) autophosphorylation of the beta-subunit or activation of the receptor kinase activity is required for the insulin-induced concentration of occupied receptors in coated pits.     

Hydrogen peroxide inhibits activity of the IGF-1 receptor kinase

IGF-1 receptor (IGF1R) is a transmembrane tyrosine kinase, which is indispensable for cellular growth and differentiation. Using a recombinant GST-tagged cytosolic fragment of IGF1R (GST-IGFK), we now show that oxidation by low doses (50 muM) of hydrogen peroxide markedly inhibits maximum phosphate incorporation in autophosphorylation and substrate phosphorylation assays. A similar inhibition was observed on the activity of intact IGF1R after treatment of T-47D cells. These results are in sharp contrast to the positive influence of hydrogen peroxide on the highly homologous insulin receptor kinase, which was assayed for comparison. This reciprocal influence of physiologically relevant doses of hydrogen peroxide may have important implications on signal transduction of the closely related receptors for insulin and IGF-1.