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.     

Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form

Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form.     

Insulin stimulates proteolysis of the alpha-subunit, but not the beta-subunit, of its receptor at the cell surface in rat liver.

Insulin receptors in rat liver plasma membranes contain two alpha- and two beta-subunits held together by interchain disulphide bonds ([alpha beta]2 receptors). Affinity-labelled receptors were digested with chymotrypsin or elastase and then exposed to dithiothreitol before solubilization from membranes and SDS/polyacrylamide-gel electrophoresis. This resulted in partial reduction and isolation of Mr-225,000 alpha beta, Mr-200,000 alpha 1 beta, Mr-165,000 alpha beta 1 and Mr-145,000 alpha 1 beta 1 receptor halves containing intact (alpha, beta) or degraded (alpha 1, beta 1) subunits. The ability to identify half-receptor complexes containing intact or degraded subunits made it possible to assay each subunit simultaneously for insulin-induced proteolysis in isolated plasma membranes or during perfusion of rat liver in situ with insulin. In liver membranes, insulin binding increased the fraction of receptors containing degraded alpha-subunits to about one-third of the total population during 2 h of incubation at 23 degrees C. beta-Subunit proteolysis increased only minimally during this time. Plasma membranes isolated from livers perfused with insulin at 37 degrees C contained degraded alpha-subunits but only intact beta-subunits, showing that insulin induced cell-surface proteolysis of the binding, but not the kinase, domain of its receptor. Since previous observations [Lipson, Kolhatkar & Donner (1988) J. Biol. Chem 263, 10495-10501] have shown that receptors containing degraded alpha-subunits are internalized but do not recycle, it is possible that cell-surface degradation may play a role in the regulation of insulin-receptor number in hepatic tissue. Proteolysis of the beta-subunit is not a likely mechanism by which receptor-kinase activity may be attenuated under physiological conditions.     

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)     

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.     

The saxitoxin receptor of the sodium channel from rat brain. Evidence for two nonidentical beta subunits

The saxitoxin receptor of the sodium channel purified from rat bran contains three types of subunits: alpha with Mr approximately 270,000, beta 1 with Mr approximately 39,000, and beta 2 with Mr approximately 37,000. These are the only polypeptides which quantitatively co-migrate with the purified saxitoxin receptor during velocity sedimentation through sucrose gradients. beta 1 and beta 2 are often poorly resolved by gel electrophoresis in sodium dodecyl sulfate (SDS), but analysis of the effect of beta-mercaptoethanol on the migration is covalently attached to the alpha subunit by disulfide bonds while the beta 1 subunit is not. The alpha and beta subunits of the sodium channel were covalently labeled in situ in synaptosomes using a photoreactive derivative of scorpion toxin. Treatment of SDS-solubilized synaptosomes with beta-mercaptoethanol decreases the apparent molecular weight of the alpha subunit band without change in the amount of 125I-labeled scorpion toxin associated with either the alpha or beta subunit bands. These results indicate that the alpha and beta 1 subunits are labeled by scorpion toxin whereas beta 1 is not and that the beta 2 subunit is covalently attached to alpha by disulfide bonds in situ as well as in purified preparations.     

Comparison of insulin-like growth factor I receptor and insulin receptor purified from human placental membranes

Insulin-like growth factor (IGF)-I receptor purified from human placental membranes as previously described (LeBon, T. R., Jacobs, S., Cuatrecasas, P., Kathuria, S., and Fujita-Yamaguchi, Y. (1986) J. Biol. Chem. 261, 7685-7689) was characterized. The IGF-I receptor was similar to the insulin receptor with respect to subunit structure (beta-alpha-alpha-beta), apparent sizes of deglycosylated alpha (Mr = approximately 88,000) and beta (Mr = approximately 67,000) subunits, and amino acid composition of the subunits. Monoclonal antibody specific to each receptor recognized its own receptor whereas polyclonal anti-human insulin receptor antibody cross-reacted with the IGF-I receptor, indicating that the receptors share one or more antigenic sites. Further characterization of the purified IGF-I receptor tyrosine-protein kinase activity indicated that by analogy with the insulin receptor the monomeric alpha beta form of the IGF-I receptor appears to have higher kinase activity than the intact receptor in the alpha 2 beta 2 form. The most significant difference between the two receptors was found in the N-terminal amino acid sequences of their alpha subunits, which apparently show 60% identity. The IGF-I receptor alpha subunit lacks residues corresponding to the N-terminal 4 amino acids of the insulin receptor alpha subunit. These results provide the first direct proof that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.     

Internalization of insulin receptors in the isolated rat adipose cell. Demonstration of the vectorial disposition of receptor subunits

The internalization of the insulin receptor in the isolated rat adipose cell and the spatial orientation of the alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor in the plasma membrane have been examined. The receptor subunits were labeled by lactoperoxidase/Na125I iodination, a technique which side-specifically labels membrane proteins in intact cells and impermeable membrane vesicles. Internalization was induced by incubating cells for 30 min at 37 degrees C in the presence of saturating insulin. Plasma, high density microsomal (endoplasmic reticulum-enriched), and low density microsomal (Golgi-enriched) membrane fractions were prepared by differential ultracentrifugation. Receptor subunit iodination was analyzed by immunoprecipitation with anti-receptor antibodies, sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and autoradiography. When intact cells were surface-labeled and incubated in the absence of insulin, the alpha and beta receptor subunits were clearly observed in the plasma membrane fraction and their quantities in the microsomal membrane fractions paralleled plasma membrane contamination. Following receptor internalization, however, both subunits were decreased in the plasma membrane fraction by 20-30% and concomitantly and stoichiometrically increased in the high and low density microsomal membrane fractions, without alterations in either their apparent molecular size or proportion. In contrast, when the isolated particulate membrane fractions were directly iodinated, both subunits were labeled in the plasma membrane fraction whereas only the beta subunit was prominently labeled in the two microsomal membrane fractions. Iodination of the subcellular fractions following their solubilization in Triton X-100 again clearly labeled both subunits in all three membrane fractions in identical proportions. These results suggest that 1) insulin receptor internalization comprises the translocation of both major receptor subunits from the plasma membrane into at least two different intracellular membrane compartments associated, respectively, with the endoplasmic reticulum and Golgi-enriched membrane fractions, 2) this translocation occurs without receptor loss or alterations in receptor subunit structure, and 3) the alpha receptor subunit is primarily, if not exclusively, exposed on the extracellular surface of the plasma membrane while the beta receptor subunit traverses the membrane, and this vectorial disposition is inverted during internalization.     

Insulin/IGF-1 hybrid receptors: implications for the dominant-negative phenotype in syndromes of insulin resistance.

Classical insulin and IGF-1 receptors are alpha 2 beta 2 heterotetrameric complexes synthesized from two identical alpha beta half-receptor precursors. Recent data strongly suggests, however, that nonidentical alpha beta half-receptor precursors can assemble to generate hybrid holoreceptor species both in vivo and in vitro. This review focuses primarily on two types of hybrid receptors. The first type is an insulin/IGF-1 hybrid receptor generated by the association of an alpha beta insulin half-receptor with an alpha beta IGF-1 half-receptor. The second type is one formed from a wildtype (kinase-active) insulin or IGF-1 alpha beta half-receptor and a mutant (kinase-inactive) insulin alpha beta half-receptor. Although the functional properties of insulin/IGF-1 hybrid receptors have not yet been completely defined, wildtype/mutant hybrid receptors are essentially substrate kinase inactive. These data indicate that the mutant alpha beta half-receptor exerts a transdominant inhibition upon the wildtype alpha beta half-receptor within the alpha 2 beta 2 holoreceptor complex. This defect in substrate kinase activity may contribute to the molecular defect underlying some syndromes of severe insulin resistance and diabetes. Heterozygous individuals expressing both wildtype and mutant tyrosine kinase-defective insulin receptor precursors demonstrate varying degrees of insulin resistance and diabetes. In addition, cell lines which express both endogenous wildtype and transfected kinase-defective insulin receptors display markedly decreased insulin and IGF-1 sensitivity and responsiveness. Formation of hybrid receptors which results in premature termination of insulin signal transduction may be one mechanism underlying the observation that kinase-inactive receptors inhibit the function of native receptors.     

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.     

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.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.

The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).     

Insulin/IGF-I receptor hybrids: a mechanism for increasing receptor diversity.

Insulin and IGF-I receptors are homologous disulfide linked alpha 2 beta 2 tetramers. These tetramers are formed biosynthetically when proreceptors containing alpha and beta subunits in a single uninterrupted linear peptide form disulfide linked homodimers and are subsequently proteolytically cleaved at the alpha-beta junctions. Cells expressing both receptors also express hybrid receptors that contain one insulin receptor alpha and beta subunit, and one IGF-I receptor alpha and beta subunit. These presumably form by the association of mixed proreceptors. Hybrid receptors greatly expand the possible repertoire of cellular responses to hormonal stimulation. Although not yet examined in detail, both the hormone binding and the signaling properties of the hybrid receptor appear to be different from that of either insulin or IGF-I receptor. Regulatory mechanisms that involve either insulin or IGF-I receptor, at the level of expression or subsequently, could alter the expression or function of the hybrid receptor or the other receptor. Similarly, pathology in one receptor could affect both the hybrid and other receptor, or perhaps be partially compensated for by a hybrid receptor. The magnitude of these effects could vary greatly in different tissues depending upon the relative level of expression of the different receptor forms. These postulated responses might explain some of the complex heterogeneity and linkage of these receptors that have been observed previously.     

Functional differences between TRPC4 splice variants.

Functional characterizations of heterologously expressed TRPC4 have revealed diverse regulatory mechanisms and permeation properties. We aimed to clarify whether these differences result from different species and splice variants used for heterologous expression. Like the murine beta splice variant, rat and human TRPC4beta both formed receptor-regulated cation channels when expressed in HEK293 cells. In contrast, human TRPC4alpha was poorly activated by stimulation of an H(1) histamine receptor. This was not due to reduced expression or plasma membrane targeting, because fluorescent TRPC4alpha fusion proteins were correctly inserted in the plasma membrane. Furthermore, currents through both human TRPC4alpha and TRPC4beta had similar current-voltage relationships and single channel conductances. To analyze the assembly of transient receptor potential channel subunits in functional pore complexes in living cells, a fluorescence resonance energy transfer (FRET) approach was used. TRPC4alpha and TRPC4beta homomultimers exhibited robust FRET signals. Furthermore, coexpressed TRPC4alpha and TRPC4beta subunits formed heteromultimers exhibiting comparable FRET signals. To promote variable heteromultimer assemblies, TRPC4alpha/TRPC4beta were coexpressed at different molar ratios. TRPC4beta was inhibited in the presence of TRPC4alpha with a cooperativity higher than 2, indicating a dominant negative effect of TRPC4alpha subunits in heteromultimeric TRPC4 channel complexes. Finally, C-terminal truncation of human TRPC4alpha fully restored the channel activity. Thus, TRPC4beta subunits form a receptor-dependently regulated homomultimeric channel across various species, whereas TRPC4alpha contains a C-terminal autoinhibitory domain that may require additional regulatory mechanisms.     

Interaction of assembly protein AP-2 and its isolated subunits with clathrin

The clathrin assembly protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with clathrin as evidenced by its ability to bind to preassembled clathrin cages. It also reacted with dissociated clathrin trimers under conditions that favor assembly of coat structures, but did not yield discrete clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coassembly assay with clathrin, the clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with clathrin, are required for expression of complete lattice assembly activity.     

The nonclassical insulin binding of insulin receptors from rat liver is due to the presence of two interacting alpha-subunits in the receptor complex

The binding characteristics of the insulin receptor tetramer (alpha 2 beta 2) and dimer (alpha beta) were examined. Unlabelled insulin enhanced the dilution-induced dissociation only of the receptor tetramer-bound 125I-insulin. Furthermore, when both the receptor forms had been preincubated with anti-receptor-antibodies (B9-antiserum), insulin binding only to the receptor tetramer but not to the dimer was inhibited. However, both oligomers are not immunologically distinct since more than 80% of the two forms were immunoprecipitated by the antiserum. These results suggest that both insulin and anti-receptor-antibodies induce cooperative interactions between the two linked alpha-subunits of the receptor tetramer leading to a decrease in insulin binding of this receptor form.     

Phosphorylation of the insulin receptor by casein kinase I

Insulin receptor was examined as a substrate for the multipotential protein kinase casein kinase I. Casein kinase I phosphorylated partially purified insulin receptor from human placenta as shown by immunoprecipitation of the complex with antiserum to the insulin receptor. Analysis of the phosphorylated complex by polyacrylamide gel electrophoresis under nonreducing conditions showed a major phosphorylated band at the position of the alpha 2 beta 2 complex. When the phosphorylated receptor was analyzed on polyacrylamide gels under reducing conditions, two phosphorylated bands, Mr 95,000 and Mr 135,000, were observed which corresponded to the alpha and beta subunits. The majority of the phosphate was associated with the beta subunit with minor phosphorylation of the alpha subunit. Phosphoamino acid analysis revealed that casein kinase I phosphorylated only seryl residues. The autophosphorylated alpha 2 beta 2 receptor purified by affinity chromatography on immobilized O-phosphotyrosyl binding antibody was also a substrate for casein kinase I. Reduction of the phosphorylated alpha 2 beta 2 receptor indicated that casein kinase I incorporated phosphate into seryl residues only in the beta subunit.     

A study on the quaternary structure change of hemoglobin in the ligation process

In order to inquire into the molecular mechanism underlying the cooperative ligand binding to hemoglobin (Hb), conformational interaction at the interfaces between subunits are investigated on the basis of the atomic coordinates of human deoxy and human carbonmonoxy Hbs. Hypothetical intermediate structures are used, each of which is obtained from the procedure where one or more subunits in deoxy Hb are replaced by the corresponding CO-liganded subunits in carbonmonoxy Hb using the method of superimposition of two sets of atomic coordinates. When either alpha or beta subunit is substituted with the corresponding subunit in carbonmonoxy Hb, serious steric hindrances are produced between alpha 1FG4(92)Arg and beta 2C3(37)Trp or between alpha 1C6(41)Thr and beta 2FG4(97)His, all of which belong to the allosteric core affected directly by ligand binding. These steric hindrances become more serious when both alpha 1(alpha 2) and beta 2(beta 1) subunits are substituted. Therefore the change in the relative distance between iron atom and porphyrin by ligation results in strain in the C-terminal residues as an effect of the steric hindrance between the FG and C segments. However, no steric hindrance can be seen between subunits when the subunits in carbonmonoxy Hb are substituted with the corresponding subunits in deoxy Hb. The nature of the quaternary structural change from liganded to deoxy Hb seems to be different from that from deoxy to liganded Hb.     

Substructural analysis of the insulin receptor by microsequence analyses of limited tryptic fragments isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence or presence of dithiothreitol

Human placental insulin receptor contains 47 Cys per an alpha beta dimer. Most of the 94 Cys in an intact alpha 2 beta 2 receptor are expected to form interchain or intrachain disulfide bonds, since there appears to be only one free cysteine residue in each beta subunit. In order to gain more insight into the three-dimensional organization of the insulin receptor, we have used limited trypsin digestion, SDS-PAGE, and protein microsequencing. The present study revealed the following; major tryptic cleavages occurred at alpha 164, alpha 270, alpha 582, and beta 1115, generating Mr 175,000, 130,000, 100,000, 70,000, and 55,000 disulfide-linked complexes. Under reducing conditions, tryptic fragments of Mr values = 30,000, 70,000, 20,000, 55,000, and 20,000 were identified to be alpha(1-164), alpha(165-582), alpha(165-270), alpha(271-582), and alpha(583-C-terminal), respectively. The major beta subunit tryptic fragment of Mr = 55,000 was assumed to have beta(724-1115) or beta(N-terminal-392). The Mr 175,000 complex appeared to contain two alpha(1-164) and two alpha(165-582), whereas the Mr 70,000 complex contained alpha(583-C-terminal) and beta(724-1115). Tryptic cleavage at alpha 582 apparently produced one Mr 175,000 and two Mr 70,000 complexes, suggesting that the alpha(583-C-terminal) domain interacts with the extracellular domain of the beta subunit by disulfide bonds. Tryptic cleavage at alpha 270 resulting in a formation of one Mr 100,000 complex consisting of two alpha(1-270) and two Mr 130,000 complexes consisting of alpha(271-C-terminal) and beta(724-1115) suggests that Cys residues involved with disulfide bonds between the two alpha subunits are located in the alpha(1-270) domain. The identification of the Mr 55,000 complex consisting of small tryptic fragments between alpha(122-270) indicates that 40 Cys residues in the two alpha(122-270) domains are inter- and intramolecularly associated by disulfide bonds. The alpha(1-121) domain does not appear to be linked to any other domains by disulfide bonds. These results are consistent with the structural model that the N-terminal domains of alpha subunits (122-270) are disulfide-linked together while the C-terminal domain (583-C-terminal) of the alpha subunit is linked to the N-terminal domain of the beta subunit by disulfide bonds.