The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics

A new method is described in which the cell surface insulin receptor can be radioactively tagged in a specific manner with a small insulin-free probe. After protecting the amino groups of insulin essential for binding and bio-activity, insulin is coupled to the heterobifunctional, cleavable cross-linking reagent SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate), via displacement of the N-hydroxysuccinimide moiety of SASD. Removal of the protecting groups results in the formation of 2-(p-azidosalicylamido)-1,3'-dithiopropionate (ASD)-insulin with insulin receptor binding activity equivalent to unmodified insulin. Iodination of ASD-insulin results in the incorporation of 125I into both the azidohydroxybenzoyl moiety of SASD and a tyrosine residue of insulin. Following binding of 125I-ASD-insulin to intact monolayers of 3T3-C2 cells, radiolabel is incorporated exclusively into a 135-kDa protein in a manner dependent upon the length of exposure of the cells to short wavelength ultraviolet light. This protein corresponds in molecular weight to the alpha subunit of the insulin receptor. Labeling of this protein can be inhibited by excess unlabeled insulin. Reduction of the disulfide bond of ASD with 10 mM glutathione causes the release of the 125I-insulin portion of the reagent from the receptor complex, with the iodinated photoactivated end of ASD covalently attached to the receptor. Insulin receptor labeled in this manner retains its ability to bind insulin. General metabolic processes of the intact cells do not appear to be perturbed by this labeling procedure, and the cellular processing of the insulin receptor does not appear to be modified by the covalent labeling of the receptor protein. This procedure therefore provides a way to specifically label the cell surface insulin receptor in a manner which does not perturb the normal functioning of the labeled cell and equally importantly, does not perturb the normal cellular processing of the insulin receptor itself.     

Characterization of the human placental receptor for basic somatomedin

We have determined optimal conditions for the solubilization of the basic somatomedin (SM) receptor from human placental membranes and for the measurement of the binding of basic SM to the solubilized receptor. Further, we have developed conditions under which the basic SM receptor, in the presence of equivalent amounts of insulin receptor, can be selectively and specifically affinity-labeled with 125I-labeled basic SM, using the cross-linking reagent disuccinimidyl suberate (DSS). Our results with these developed methods indicate that the properties of the soluble basic SM receptor (pH optimum for ligand binding, pH 7 to 9; adsorption to lectin-agarose derivatives; sedimentation coefficient in detergent-sucrose solutions, 11S) closely parallel data previously reported for the insulin receptor. Based on the sedimentation coefficient and the previously estimated Stokes radius of the soluble receptor (7.2 nm), a molecular weight of 402 000 can be calculated for the detergent-receptor complex. Electrophoretic analysis of the basic SM receptor, selectively cross-linked to 125I-labeled basic SM with DSS in the presence of excess unlabeled insulin revealed, under reducing conditions, a major labeled constituent of 140 kdaltons, substantiating our previous work employing a photoaffinity labeling reagent. DSS cross-linking also demonstrated the presence of less intensely labeled components with apparent molecular weights of 54 000, 43 000 and 35 000 but failed to reveal a distinct 90- to 100-kdalton species visualized in parallel experiments with insulin. The 53-kdalton species was not detected in similar experiments with insulin. A specifically labeled basic SM receptor component of 300 kdaltons was also observed under reducing conditions; in the absence of beta-mercaptoethanol, all labeled components migrated in the 300-kdalton range. In comparison, selective DSS labeling of the insulin receptor in the presence of excess basic SM revealed components which, upon electrophoresis under reducing conditions, exhibited apparent molecular weights of 300 000, 140 000, 90 000--100 000, 43 000 and 35 000. The major insulin-labeled component (140 000) comigrated with the major constituent (140 000) selectively labeled with basic SM. Chymotryptic digestion of the receptors selectively DSS labeled with either 125I-labeled insulin or 125I-labeled basic SM yielded quite similar, but distinctive, gel electrophoretic maps. We conclude that the receptors for basic SM and insulin are highly homologous structures, particularly with respect to their glycoprotein nature, their hydrodynamic properties, their disulphide cross-linked composition, and with respect to the size of the major constituent detected by selective affinity labeling. Nonetheless, the detection of electrophoretically distinct labeled receptor substituents upon analysis of specifically labeled material, both before and after chymotryptic cleavage, points to subtle differences between the polypeptide compositions of the two receptors.     

Structural differences between insulin receptors in the brain and peripheral target tissues

Insulin receptors in various brain regions (olfactory tubercle, hippocampus, and hypothalamus) were photoaffinity labeled using the photoreactive analogue of insulin B2(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). A protein with an apparent Mr of 400,000 was specifically labeled with 125I-NAPA-DP-insulin in all three brain regions. When radiolabeled proteins were reduced with dithiothreitol prior to electrophoresis, specific labeling occurred predominantly in a protein with an apparent Mr of 115,000 and to a much lesser extent in a protein with an apparent Mr of 83,000. The size of these receptor proteins, based on their electrophoretic mobilities, was consistently smaller than insulin receptor proteins in adipocytes. The covalent labeling of insulin receptors in brain by 125I-NAPA-DP-insulin was not blocked by anti-insulin receptor antiserum. Additionally, in contrast to effects observed in peripheral target tissues, this antisera did not inhibit the binding of 125I-insulin to brain membranes. Neuraminidase treatment resulted in an increase in the electrophoretic mobilities of insulin receptor subunits in adipocytes, but, had no effect on receptor subunits in brain. Solubilized insulin receptors from adipocytes were retained by wheat germ agglutinin columns and specifically eluted with N-acetylglucosamine. In contrast, solubilized insulin receptors from brain did not bind to these columns. The results from this study indicate that structural differences, including molecular weight, antigenicity, and carbohydrate composition exist between insulin receptors in brain and peripheral target tissues.     

Identification of liver cell membrane galactoglycoproteins involved in the process of insulin binding

The glycoproteinic nature of the insulin receptor was indicated using two different approaches: 1. [¹²⁵I]insulin binding to soluble receptors from mouse liver was inhibited by digestion with β-galactosidase or pretreatment with Ricinus communis I or concanavalin A. An other enzyme (neuraminidase) and lectins (wheat germ agglutinin, Dolichos biflorus) did not affect the binding reaction. These data confirmed that insulin directly interacts with the galactoglycoproteins of liver membranes. 2. The galactose oxidase-sodium boro[³H]hydride technique, previously used for labeling accessible membrane galactoglycoproteins, was again utilized to discern the components that interact with insulin. When liver membranes were equilibrated with 10^?7 M insulin prior to labeling, the SDS gel radioactive profiles were specifically modified within two galactoglycoproteins of apparent molecular sizes 195 000 and 145 000, compatible with their participation in the insulin binding interaction. Membrane pretreatment with β-galactosidase or Sophora japonica lectin reduced the labeling in most peaks, thus supporting the argument for labeling sensitivity. Preincubation of membranes with 10^?7 M proinsulin slightly hindered labeling while pretreatment with 10^?7 M glucagon was ineffective, suggesting a specificity of the insulin effect. These data indicate the glycoprotein nature of the insulin receptor for two reasons: alteration of insulin binding after modification of the galactoglycoproteins, and alteration of galactoglycoprotein labeling after insulin binding. Two galactoglycoproteins, with apparent molecular weights 145 000 and 195 000, respectively, were identified and they are suggested to have insulin binding properties.     

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)     

An insulin epidermal growth factor-binding protein from Drosophila has insulin-degrading activity

We have recently described the purification and characterization of an insulin-degrading enzyme (IDE) from Drosophila melanogaster that can cleave porcine insulin, is highly conserved through evolution and is developmentally regulated. We now report that the IDE is, in fact, an insulin EGF-binding protein (dp100) that we had isolated previously from Drosophila using an antihuman EGF receptor antiserum. This conclusion is based upon the following evidence. (a) dp100, identified by its ability to cross-link to labeled insulin, EGF, and transforming growth factor-alpha (TGF-alpha), and to be immunoprecipitated by anti-EGF receptor antisera, copurifies with the IDE activity. Thus, the purified IDE can be affinity labeled with either 125I-insulin, 125I-EGF, or 125I-TGF-alpha, and this labeling is specifically inhibited with unlabeled insulin, EGF, and the insulin B chain. (b) The antiserum to the human EGF receptor, which recognizes dp100, is able to specifically immunoprecipitate the insulin-degrading activity. (c) The purified IDE preparation contains a single protein of 110 kD which is recognized by both the anti-EGF receptor antiserum and anti-Drosophila IDE antiserum. (d) Polyclonal antiserum to the purified IDE, which specifically recognized only the 110-kD band in Drosophila Kc cells, immunoprecipitates dp100 cross-linked to 125I-TGF-alpha and dp100 cross-linked to 125I-insulin from the purified IDE preparation. (e) EGF, which competes with insulin for binding to dp100, also inhibits the degradation of insulin by the purified IDE. These results raise the possibility that a functional interaction between the insulin and EGF growth factor families can occur which is mediated by the insulin-degrading enzyme.     

Comparative studies on human placental insulin and basic somatomedin receptors

The disuccinimidy! suberate, affinity-labeling procedure, and proteolytic mapping techniques have been employed to characterize further the human placental receptors for insulin and basic somatomedin. Electrophoretic analysis of the basic somatomedin receptor, selectively crosslinked to ¹²⁵I basic somatomedin in the presence of excess native insulin revealed, under reducing conditions, major labeled constituents of 270-280 and 125-140 kd, substantiating our previous work employing a photoaffinity labeling reagent. Affinity labeling also demonstrated the presence of less intensely labeled components with apparent molecular weights of 40 and 45 kd but failed to reveal a distinct 90- to 100-kd species observed in parallel experiments with insulin. In the absence of β-mercaptoethanol, all components specifically labeled with ¹²⁵I basic somatomedin migrated in the 300- to 400-kd range. In comparison, selective affinity labeling of the insulin receptor in the presence of excess native basic somatomedin revealed components, upon electrophoresis under reducing conditions, with apparent molecular weights of 270-280, 125-140, 90-100, and 40 kd. The major insulin-labeled component (125-140 kd) comigrated with the major constituent (125-140 kd) selectively labeled with basic somatomedin. When digestion was performed prior to solubilization, chymotryptic and tryptic proteolysis of the membrane-localized selectively labeled insulin, and basic somatomedin receptors yielded quite similar gel electrophoretic maps. However, when digestion was done subsequent to solubilization, chymotryptic and tryptic proteolysis of selectively labeled insulin and basic somatomedin receptors solubilized in SDS yielded similar but not identical gel electrophoretic maps. We conclude that the receptors for basic somatomedin and insulin are highly homologous structures with respect to their disulfide crosslinked composition, and with respect to the size of the major components detected by selective affinity-labeling procedures. Nevertheless, the detection of electrophoretically distinct labeled receptor components upon analysis of specifically labeled intact or proteolytically digested receptors points to subtle differences between the polypeptide compositions of the two receptors.     

Evidence that insulin receptor from human placenta has a high affinity for only one molecule of insulin

Insulin receptor, partially purified from human placenta by chromatography on wheat germ agglutinin, was shown, by means of double probe labeling, to bind only one molecule of insulin with a high affinity. In the double probe labeling protocol used, 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess of unlabeled N epsilon B29-biotinylinsulin (probe 2). The ability of succinylavidin to bind to receptor-linked probe 2 and alter the electrophoretic mobility of the cross-linked complex (during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate) was used to determine the amount of receptor which was cross-linked to both probes relative to that which was cross-linked to only probe 1. The fraction of receptor bound to two molecules of insulin prior to cross-linking was estimated from the cross-linking efficiency and the yield of receptor cross-linked to both probes relative to the yield of receptor cross-linked only to probe 1. The low fraction of receptor bound to both probes in the presence of high concentrations of probe 2 indicated that the affinity of the receptor for a second molecule of insulin was approximately 100 times less than that for the first and that in the range of insulin concentrations (less than 20 nM) usually used to determine the stoichiometry for the interaction between receptor and insulin, more than 80% of the receptor molecules should be bound to only one molecule of insulin. This knowledge of how insulin receptor interacts with insulin was shown to be important for proper determination of receptor purity, interpretation of curvilinear Scatchard plots, and interpretation of the insulin-enhanced rate of dissociation of receptor-bound insulin.     

Covalent crosslinking of thyrotropin to thyroid plasma membrane receptors: subunit composition of the thyrotropin receptor

The subunit composition of the thyrotropin (TSH) receptor has been characterized using the bifunctional crosslinking agent, disuccinimidyl suberate (DSS), to covalently link [125I]TSH to its receptor. Purified thyroid membranes were labeled with [125I]TSH, and the hormone-receptor complex was crosslinked by incubation with 0.1 mM DSS. Analysis of this crosslinked complex by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the presence of a specifically labeled hormone-receptor complex, corresponding to a Mr of 68,000 +/- 3000 before correction for the relative molecular mass of TSH. When reducing agents were absent during SDS solubilization, the mobility of the band increased slightly, suggesting the presence of intramolecular disulfide bonds. The labeling of the 68,000 band was specifically inhibited by TSH, but not by other glycoprotein hormones. Specific labeling occurred only in thyroid, and not in liver or muscle plasma membranes. Protease-free immunoglobulin G, isolated from sera of patients with Graves' disease and capable of competing with TSH for binding to its receptor, inhibited the labeling of the 68,000 complex. When the hormone-receptor complex was crosslinked with higher concentrations of DSS (greater than 0.3 mM), a second specifically labeled band was observed, with a Mr of 80,000 +/- 5000. This complex exhibited hormone, tissue, and immunologic specificities similar to those of the 68,000 band. Continuous sucrose density gradient analysis indicated that the intact solubilized receptor possessed a sedimentation coefficient of 10.5 S prior to correction for detergent binding. However, this value increased to 16 S when determined under conditions which took into account the change in hydrodynamic properties attributable to bound Triton X-100. These data suggest that the 80,000 and 68,000 bands represent binding components of the TSH receptor and that the receptor molecule most likely contains multiple subunits, linked by noncovalent forces.     

Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins

Two distinct sequences of amino acids, RGDS and HHLGGAKQAGDV, each inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to the platelet membrane glycoprotein IIb-IIIa complex. We have employed radiolabeled, photoactivatable aryl azide derivatives of the two sequences to explore the relationship between the binding sites for these peptides on the glycoprotein IIb-IIIa complex. Each probe specifically labeled only the glycoprotein IIb-IIIa complex of intact platelets. Since each peptide inhibited labeling of the receptor complex by the other, the peptides compete for binding sites on the receptor complex. However, the binding sites do not appear to be identical. Whereas the RGDS probe specifically labeled both glycoproteins IIb and IIIa, the HHLGGAKQA-GDV probe specifically labeled only glycoprotein IIb.     

Characterization of insulin receptor subunits in brain and other tissues by photoaffinity labeling

Specific insulin receptor proteins of plasma membrane preparations from various tissues of the rat were identified using a photoreactive insulin derivative, N^εB29-mono(azidobenzoyl)insulin. Except for the brain, all tissues examined showed the specific photolabeling of two proteins of M_r～130K and ～90K. In brain tissue, only one protein, M_r～115K, was specifically labeled. Liver and adipocyte membranes of the genetic obese ($\text{ob}\text{ob}$) mice showed decreased labeling of both 130K and 90K proteins when compared to those of lean littermates. Labeling of these proteins in liver plasma membranes was abolished by trypsin, whereas neuraminidase increased their electrophoretic mobility in SDS-polyacrylamide gel. The labeling of these two proteins was inhibited by a human anti-receptor serum which also formed an immunocomplex with both proteins. The labeling of the 115K protein in brain tissue was, however, not affected by the antiserum.     

Subunit structure and dynamics of the insulin receptor

A model for the minimum subunit composition and stiochiometry of the physiologically relevant insulin receptor has been deduced based on results obtained by affinity labeling of this receptor in a variety of cell types and species. We propose that the receptor is a symmetrical disulfide-linked heterotetramer composed of two alpha (apparent Mr = 125,000) and two beta (apparent Mr = 90,000) glycoprotein subunits in the configuration (beta-S-S-alpha)-S-S-(alpha-S-S-beta). The disulfide or disulfides linking the two (alpha-S-S-beta) halves (class I disulfides) exhibit greater sensitivity to reduction by exogenous reductants than those linking the alpha and beta subunits (class II disulfides). When the class I disulfides are reduced by addition of diothiothreitol to intact cells, the receptor retains its ability to bind insulin and to effect a biological response. The beta subunit contains a site at about the center of its amino acid sequence that is extremely sensitive to proteolytic cleavage by elastaselike proteases, yielding a beta 1 fragment (Mr = 45,000) that remains disulfide linked to the receptor complex and a free beta 2 fragment. Binding of insulin to the receptor complex appears to result in the formation or stabilization of a new receptor conformation as evidenced by an altered susceptibility of the alpha subunit to exogenous trypsin. A receptor structure with high affinity for insulinlike growth factor (IGF) I and low affinity for insulin in fibroblast and placental membranes has also been affinity labeled. It exhibits the same structural features found for the insulin receptor, including two classes of disulfide bridges and beta subunits highly sensitive to proteolytic cleavage. These recent observations identifying the presence of distinct insulin and IGF-I receptors that share similar complex structures suggest that these hormones may also share common mechanisms of transmembrane signaling.     

Stoichiometry for the binding of insulin to insulin receptors in adipocyte membranes

Insulin receptor molecules in rat adipocyte plasma membranes were shown to be monovalent with respect to their capacity to bind insulin. The 1:1 stoichiometry for insulin binding was determined by a "double-probe labeling" procedure, wherein 125I-insulin (probe 1) was affinity cross-linked to its receptor in the presence of an excess saturating concentration of an unlabeled biotinylated insulin derivative (probe 2). If the receptor were competent to bind more than one insulin molecule, any receptor molecule that was cross-linked to probe 1 also should have been cross-linked to probe 2 in the double probe labeling procedure. The monovalent character of the insulin receptor was indicated by the failure of the probe 1-linked receptor to be cross-linked to probe 2. This was indicated by the failure of succinylavidin to increase the molecular weight of the probe 1-linked receptor. Control experiments indicated that succinylavidin increased the molecular weight of receptor that had been cross-linked to probe 2. The 1:1 stoichiometry for insulin binding demonstrated here indicates that if insulin receptors contain more than one insulin binding subunit, the binding of insulin to its receptor must be a highly negatively cooperative process.     

The different receptor species of liver have similar complex insulin binding properties

Liver plasma membranes bind insulin in a complex fashion via three prominent disulfide-linked insulin receptor structures of 360K, 300K, and 260K molecular weight. To determine if the complex binding is explained by different binding affinities among the different structures, 125I-insulin was specifically cross-linked to the binding sites and the amount of radiolabeled insulin was determined after SDS-gel electrophoresis. The insulin binding characteristics of each structure were similar to the binding properties of the intact membrane. The Scatchard plot for each structure was curvilinear and the Kd values for the high and low affinity components were similar to the membrane values. Thus, the curvilinear Scatchard plot of insulin binding to liver membranes is also a feature of each receptor structure and is not a function of different receptors with different binding properties.     

Characterization of a Novel Receptor in Toad Retina with Dual Specificity for Insulin and Insulin-Like Growth Factor I

The biochemical properties of insulin receptors from toad retinal membranes were examined in an effort to gain insight into the role this receptor plays in the retina. Competition binding assays revealed that toad retinal membranes contained binding sites that displayed an equal affinity for insulin and insulin-like growth factor I (IGF-I). Affinity labeling of toad retinal membrane proteins with 125I-insulin resulted in the specific labeling of insulin receptor alpha-subunits of approximately 105 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partially reduced (alpha beta-heterodimer) receptors affinity-labeled with 125I-insulin indicated the presence of a disulfide-linked beta-subunit of approximately 95 kDa. Endoglycosidase F digestion of the affinity-labeled alpha-subunits increased their mobility by reducing their apparent mass to approximately 83 kDa. This receptor was not detected by immunoblot analysis with a site-specific antipeptide antibody directed against residues 657-670 of the carboxy terminal of the human insulin receptor alpha-subunit, whereas this antibody did label insulin receptor alpha-subunits from pig, cow, rabbit, and chick retinas. In in vitro autophosphorylation assays insulin stimulated the tyrosine phosphorylation of toad retina insulin receptor beta-subunits. These data indicate that toad retinal insulin receptors have a heterotetrameric structure whose alpha-subunits are smaller than other previously reported neuronal insulin receptors. They further suggest that a single receptor may account for both the insulin and IGF-I binding activities associated with toad retinal membranes.     

Immunoaffinity-purified opiate receptor specifically binds the delta-class opiate receptor ligand, cis-(+)-3-methylfentanylisothiocyanate, SUPERFIT

Using an antibody generated against the opiate receptor on NG108-15 cells, we recently purified the putative receptor from this hybrid cell line. We herein report that the purified receptor complex specifically binds tritiated cis-(+)-3-methylfentanylisothiocyanate (SUPERFIT), with the predominant binding associated with a 58 kDa polypeptide chain. Consistent with these findings is the in situ labeling of a 58 kDa protein with [3H]SUPERFIT on NG108-15 cells.     

Subunit structure of the purified human placental insulin receptor. Intramolecular subunit dissociation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Insulin receptors purified from human placental membranes by gel-filtration and insulin-agarose affinity chromatography were found to be composed of eight different high molecular weight complexes as identified by nonreducing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The subunit stoichiometry of these different high molecular weight forms of the insulin receptor were determined by comparisons of silver-stained gel profiles with the autoradiograms of 125I-insulin specifically cross-linked to the alpha subunit and [gamma-32P]ATP specifically autophosphorylated beta subunit gel profiles. Two-dimensional SDS-polyacrylamide gel electrophoresis in the absence and presence of reductant confirmed the subunit stoichiometries as alpha 2 beta 2, alpha 2 beta beta 1, alpha 2 (beta 1)2, alpha 2 beta, alpha 2 beta 1, alpha 2, alpha beta, and beta, where alpha is the Mr = 130,000 subunit, beta is the Mr = 95,000 subunit, and beta 1 is the Mr = 45,000 subunit. Treatment of the insulin receptor preparations with oxidized glutathione or N-ethylmaleimide prior to SDS-polyacrylamide gel electrophoresis increased the relative amount of the alpha 2 beta 2 complex concomitant with a total disappearance of the alpha 2 beta, alpha 2 beta 1, alpha 2, and free beta forms. The effects of oxidized glutathione were found to be completely reversible upon extensive washing of the treated insulin receptors. In contrast, the effects of N-ethylmaleimide were totally irreversible by washing, consistent with known sulfhydryl alkylating properties of this reagent. The formation of these lower molecular weight insulin receptor subunit complexes was further demonstrated to be due to SDS/heat-dependent intramolecular sulfhydryl-disulfide exchange occurring within the alpha 2 beta 2 complex. These studies demonstrate that the largest disulfide-linked complex (alpha 2 beta 2) is the predominant insulin receptor form purified from the human placenta with the other complexes being generated by proteolysis and by internal subunit dissociation.     

Oligomeric states of the insulin receptor: binding and autophosphorylation properties

Properties of oligomeric states of the insulin receptor were analyzed by polyacrylamide gel electrophoresis in nondenaturing buffer conditions (ND-PAGE). Partially purified insulin receptors resolve in ND-PAGE as three distinct species: (i) the fast electrophoretic mobility, low molecular mass form manifests intense labeling by iodinated insulin and shows basal and insulin-stimulated autophosphorylation; (ii) the middle, intermediate mobility form exhibits strong labeling by iodinated ligand but does not possess the capacity to be autophosphorylated; (iii) the slow mobility, highest molecular mass form necessitates covalent binding with iodinated hormone to withstand electrophoresis and shows autophosphorylation enhanced by insulin. This receptor form is more heavily labeled by phosphorylation than the low form. At 22 degrees C, binding and autophosphorylation do not appear to be time dependent. At 37 degrees C, binding and autophosphorylation of low and high species attain a maximum after 15 min and then decrease as time of incubation with insulin is prolonged to 120 min; the middle species exhibits a much slower association rate, and its labeling by iodinated hormone becomes more intense with time. Our data show that in cell-free systems insulin receptors appear in various oligomeric states and that the highest molecular mass oligomer exhibits the most pronounced autophosphorylation. This is compatible with the concept that insulin receptor oligomerization provides a mechanism for transmembrane signaling.     

Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat heptocytes

Three derivatives of a triantennary glycopeptide, each containing a single uniquely located 6-amino-galactose residue at either position 6', 6, or 8, were modified at the 6-amino group by attachment of a photolyzable reagent and radiolabeled by iodination of tyrosine. These were allowed to bind to the asialoglycoprotein receptor of isolated rat hepatocytes and photolyzed for affinity labeling. (formula; see text) Each probe specifically labeled either the major (RHL1) or minor (RHL2/3) subunits which comprise the receptor. A photolyzable group attached to galactose residue 6 6' specifically radiolabeled RHL1, whereas a photolyzable group attached to galactose 8 specifically labeled RHL2/3. Photoaffinity labeling of a soluble rat hepatic lectin preparation demonstrated that the minor subunits (RHL2/3) were no longer labeled by the triantennary probe with a photolyzable group at galactose 8. The inhibitory potency of a variety of complex glycopeptides against radiolabeled ligand binding to both rat hepatocytes and soluble lectin are in agreement with photoaffinity results that galactose 8 of triantennary glycopeptide is of unique importance by binding solely to the minor subunits (RHL2/3) of the asialoglycoprotein receptor on hepatocytes. Conversely, galactose residues 6 and 6' bind specifically to the major subunit (RHL1), indicating a precise binding geometry between the trivalent ligand and lectin.