Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

The ligand binding characteristics of a kinase-defective A/K1018 human insulin receptor expressed in Rat 1 fibroblasts

Expression of the cDNA encoding a human insulin receptor with replacement of alanine for lysine at residue 1018 in the ATP binding domain of the beta subunit results in a receptor that is not only kinase-defective, but also biologically inactive. Interestingly, this mutated receptor shows a decreased insulin binding affinity when expressed at high level. We, therefore, studied the binding property of this mutant receptor expressed in Rat 1 fibroblasts. The association rate (Ka) of insulin to the mutant receptor was comparable to normal, but the dissociation rate (Kd) was twice as fast. Furthermore, the Kd of the mutant receptor was also more sensitive to changes in pH, accelerating more rapidly with pH changes than did the Kd of normal receptors. Despite this difference, the mutant receptor still exhibited negative cooperativity. These results indicate that the loss of tyrosine kinase activity of the beta subunit of the insulin receptor leads to alteration of the ligand binding affinity of the alpha subunit.     

Intramolecular subunit interactions between insulin and insulin-like growth factor 1 alpha beta half-receptors induced by ligand and Mn/MgATP binding.

We have previously demonstrated that isolated insulin and IGF-1 alpha beta half-receptors can be reconstituted into a functional alpha 2 beta 2 hybrid receptor complex [Treadway et al. (1989) J. Biol. Chem. 264, 21450-21453]. In the present study, we have examined this assembly process by determining the effect of ligand occupancy and Mn/MgATP binding on the dimerization of mutant and wild-type insulin and IGF-1 alpha beta half-receptors. IGF-1 or Mn/MgAMPPCP binding to wild-type IGF-1 alpha beta half-receptors resulted in the specific assembly of the alpha beta half-receptors into an alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor complex. Similarly, insulin binding to the kinase-deficient mutant (A/K1018) insulin alpha beta half-receptor also resulted in the specific assembly into an alpha 2 beta 2 holoreceptor complex. In contrast, Mn/MgAMPPCP treatment of A/K1018 mutant insulin alpha beta half-receptors did not induce heterotetramer assembly, consistent with the inability of this mutant receptor to bind ATP. The ability of the insulin alpha beta receptors to assemble with the IGF-1 alpha beta half-receptors was used to examine the intermolecular subunit interactions responsible for dimerization. In the presence of Mn/MgAMPPCP, the wild-type insulin and wild-type IGF-1 alpha beta half-receptors were observed to assemble into an insulin/IGF-1 alpha 2 beta 2 hybrid receptor complex. Similarly, a combination of insulin and IGF-1 induced hybrid receptor formation between wild-type IGF-1 and A/K1018 mutant insulin alpha beta half-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the functional insulin binding epitopes of the A and B isoforms of the insulin receptor

The human insulin receptor is expressed as two isoforms that are generated by alternate splicing of its mRNA; the B isoform has 12 additional amino acids (718-729) encoded by exon 11 of the gene. The isoforms have been reported to have different ligand binding properties. To further characterize their insulin binding properties, we have performed structure-directed alanine-scanning mutagenesis of a major insulin binding site of the receptor, formed from the receptor L1 domain (amino acids 1-470) and amino acids 705-715 at the C terminus of the alpha subunit. Alanine mutants of each isoform were transiently expressed as recombinant secreted extracellular domain in 293 cells, and their insulin binding properties were evaluated by competitive binding assays. Mutation of Arg(86) and Phe(96) of each isoform resulted in receptors that were not secreted. The Kds of unmutated receptors were almost identical for both isoforms. Several new mutations compromising insulin binding were identified. In L1, mutation of Leu(37) decreased affinity 20- to 40-fold and mutations of Val(94), Glu(97), Glu(120), and Lys(121) 3 to 10-fold for each isoform. A number of mutations produced differential effects on the two isoforms. Mutation of Asn(15) in the L1 domain and Phe(714) at the C terminus of the alpha subunit inactivated the A isoform but only reduced the affinity of the B isoform 40- to 60-fold. At the C terminus of the alpha subunit, mutations of Asp(707), Val(713), and Val(715) produced 7- to 16-fold reductions in affinity of the A isoform but were without effect on the B isoform. In contrast, alanine mutations of Tyr(708) and Asn(711) inactivated the B isoform but only reduced the affinities of the A isoform 11- and 6-fold, respectively. In conclusion, alanine-scanning mutagenesis of the insulin receptor A and B isoforms has identified several new side chains contributing to insulin binding and indicates that the energetic contributions of certain side chains differ in each isoform, suggesting that different molecular mechanisms are used to obtain the same affinity.     

Binding of mutant insulins to a mutated insulin receptor.

We studied the binding of mutant insulins to both the normal human insulin receptor and an insulin receptor in which the sequence 240-250 of the receptor alpha subunit was mutated to provide an additional net positive charge. One mutant insulin (AspB10), which has an additional negative charge, bound to both types of receptors with a higher affinity than native insulin. Moreover, this mutant insulin was more effective in activating the tyrosine kinase activity of both types of receptors. This study suggests, therefore, that charge interactions between insulin and its receptor may play a role in insulin receptor binding and action.     

Influence of differential stability of G protein βγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-β

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.     

The insulin receptor. Structural basis for high affinity ligand binding

Treatment of the soluble insulin receptor from human placenta with 1.25 mM dithiothreitol and 75 mM Tris at pH 8.5 results in complete reduction of interhalf disulfide bonds (class 1 disulfides) and dissociation of the tetrameric receptor into the dimeric alpha beta form. The alpha beta receptor halves exhibit a reduced affinity for insulin binding (B?ni-Schnetzler, M., Rubin, J. B., and Pilch, P. F. (1986) J. Biol. Chem. 261, 15281-15287). Kinetic experiments reveal that reduction of class 1 disulfides is a faster process than the loss of affinity for ligand, indicating that events subsequent to reduction of interhalf disulfides are responsible for the affinity change. We show that a third class of alpha subunit intrachain disulfides is more susceptible to reduction at pH 7.6 than at pH 8.5 and appears to form part of the ligand binding domain. Reduction of the intrachain disulfide bonds in this part of the alpha subunit leads to a loss of insulin binding. Modification of this putative binding domain by dithiothreitol can be minimized if reduction is carried out at pH 8.5. When the insulin receptor in placental membranes is reduced at pH 8.5, the receptor's affinity for insulin is not changed when binding is measured in the membrane. However, the Kd for insulin binding is reduced 10-fold when alpha beta receptor halves are subsequently solubilized. Scatchard analysis of insulin binding to reduced or intact receptors in the membrane and in soluble form together with sucrose density gradient analysis of soluble receptors suggests that alpha beta receptor halves remain associated in the membrane after reduction, but they are dissociated upon solubilization. We interpret these results to mean that the association of two ligand binding domains, 2 alpha beta receptor halves, is required for the formation of an insulin receptor with high affinity for ligand.     

Structural analysis of normal and mutant insulin receptors in fibroblasts cultured from families with leprechaunism.

Leprechaunism is an inherited disorder characterized by insulin resistance and intrauterine growth restriction. In this study we analyze insulin binding and subunit structure of the insulin receptor in dermal fibroblasts cultured from three unrelated families whose probands (Ark-1, Atl, and Minn) were affected by leprechaunism. Cells cultured from all three probands had markedly reduced insulin binding at equilibrium. Fibroblasts cultured from the parents of Ark-1 and Atl had partial and differing degrees of impairment in insulin binding. The structure of the alpha subunit of insulin receptors was analyzed by cross-linking 125I-insulin to plasma membranes. A major band of 350 kilodaltons (kD) (corresponding to the heterotetrameric insulin receptor alpha 2 beta 2) was observed in control and leprechaun fibroblasts. The relative amount of radioactivity cross-linked to plasma membranes reflected the genetic variations seen in insulin binding to intact cells. In reducing gels, 125I-insulin was cross-linked equally to a 250-kD (alpha-alpha dimer) and a 125-kD (alpha monomer) protein in cells from controls, the parents of Ark-1 and Atl, and probands Atl and Minn. By contrast, cells from the Ark-1 proband had diminished cross-linking of alpha-alpha dimers. The ratio of dimer to monomer in cells from controls was 0.93 +/- 0.06, and that in cells from Ark-1 was 0.31 +/- 0.19 (P less than .01). Beta-subunit structure and function was analyzed by studying insulin-enhanced autophosphorylation. Although maximal stimulation of beta-subunit phosphorylation was reduced to 30% in proband Ark-1 fibroblasts, this reduction was quantitatively related to reduced insulin binding. These results indicate that mutations causing severe insulin resistance and defective insulin binding are transmitted with autosomal recessive patterns of inheritance and that heterogeneity exists for these mutations. The mutation in pedigree Ark-1 most likely produces conformational changes in alpha-subunit interaction.     

Dimeric fragment of the insulin receptor alpha-subunit binds insulin with full holoreceptor affinity

The insulin receptor (IR) is a dimeric receptor, and its activation is thought to involve cross-linking between monomers initiated by binding of a single insulin molecule to separate epitopes on each monomer. We have previously shown that a minimized insulin receptor consisting of the first three domains of the human IR fused to 16 amino acids from the C-terminal of the alpha-subunit was monomeric and bound insulin with nanomolar affinity (Kristensen, C., Wiberg, F. C., Sch?ffer, L., and Andersen, A. S. (1998) J. Biol. Chem. 273, 17780-17786). To investigate the insulin binding properties of dimerized alpha-subunits, we have reintroduced the domains containing alpha-alpha disulfide bonds into this minireceptor. When inserting either the first fibronectin type III domain or the full-length sequence of exon 10, the receptor fragments were predominantly secreted as disulfide-linked dimers that both had nanomolar affinity for insulin, similar to the affinity found for the minireceptor. However, when both these domains were included we obtained a soluble dimeric receptor that bound insulin with 1000-fold higher affinity (4-8 pm) similar to what was obtained for the solubilized holoreceptor (14-24 pm). Moreover, dissociation of labeled insulin from this receptor was accelerated in the presence of unlabeled insulin, demonstrating another characteristic feature of the holoreceptor. This is the first direct demonstration showing that the alpha-subunit of IR contains all the epitopes required for binding insulin with full holoreceptor affinity.     

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.     

Oxygen equilibrium and EPR studies on alpha1beta1 hemoglobin dimer

We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity. For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties. The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated. Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations. Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers. However, EPR results on the [alpha(N)(Fe-NO)beta(M)(Mg)] hybrid showed some alpha1beta1 interactions. These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.     

Tryptophan W207 in transducin T alpha is the fluorescence sensor of the G protein activation switch and is involved in the effector binding.

We have produced a recombinant transducin alpha subunit (rT alpha) in sf9 cells, using a baculovirus system. Deletion of the myristoylation site near the N-terminal increased the solubility and allowed the purification of rT alpha. When reconstituted with excess T beta gamma on retinal membrane, rT alpha displayed functional characteristics of wild-type T alpha vis à vis its coupled receptor, rhodopsin and its effector, cGMP phosphodiesterase (PDE). We further mutated a tryptophan, W207, which is conserved in all G proteins and is suspected to elicit the fluorescence change correlated to their activation upon GDP/GTP exchange or aluminofluoride (AlFx) binding. [W207F]T alpha mutant displayed high affinity receptor binding and underwent a conformational switch upon receptor-catalysed GTP gamma S binding or upon AlFx binding, but this did not elicit any fluorescence change. Thus W207 is the only fluorescence sensor of the switch. Upon the switch the mutant remained unable to activate the PDE. To characterize better its effector-activating interaction we measured the affinity of [W207F]T alpha GDP-AlFx for PDE gamma, the effector subunit that binds most tightly to T alpha. [W207F]T alpha still bound in an activation-dependent way to PDE gamma, but with a 100-fold lower affinity than rT alpha. This suggests that W207 contributes to the G protein effector binding.     

Relationship between the affinity and proteolysis of the insulin receptor. Evidence that higher affinity receptors are preferentially degraded

125I-Insulin binding to rat liver plasma membranes initiated two processes that occurred with similar time courses: an increase of receptor affinity for hormone and degradation of the Mr 135,000 alpha subunit of the insulin receptor to a fragment of Mr 120,000. Inhibitors of serine proteinases prevented alpha subunit degradation without affecting the affinity change. This shows that the change of affinity is not produced by receptor proteolysis and that the intact alpha subunit of the insulin receptor can exist as a higher or lower affinity species. Hormone binding was much more rapid than receptor proteolysis and the initial rate of alpha subunit degradation was independent of the concentration of occupied lower affinity receptors. Only persistent hormone binding and the accumulation of higher affinity insulin-receptor complexes led to significant receptor proteolysis. As the incubation time between 125I-insulin and membranes increased, the rate at which hormone dissociated from Mr 135,000 complexes diminished, whereas hormone dissociated from Mr 120,000 complexes slowly after brief or extended incubations. These observations suggest that 125I-insulin binds to membranes to form low affinity complexes that are not substrates for proteolysis. A slow conformational change produces higher affinity hormone-receptor complexes that are selectively degraded. Thus, the conversion between states of affinity may play a role in the regulation of receptor proteolysis and, consequently, insulin action in cells.     

Molecular genetics of severe insulin resistance

Leprechaunism and type A diabetes represent inborn errors of insulin resistance whose phenotypes suggested causation by mutations in the insulin receptor gene. Cells cultured from patients with leprechaunism specifically lacked high-affinity insulin binding. Partial but different degrees of impairment were observed in cells cultured from first-degree relatives. Different mutations in the insulin receptor's alpha subunit were proposed in different families (Ark-1, Atl, Minn, Mount Sinai) based on phenotype, cellular insulin binding, and insulin receptor structure. Molecular cloning and sequencing of mutant insulin receptor cDNA from family Ark-1 confirmed that the proband inherited a maternal missense and a paternal nonsense mutation in the alpha subunit and was a compound heterozygote. The insulin receptor was immunologically present on the plasma membrane of fibroblasts cultured from patients Ark-1 and Atl but was markedly reduced in cells from patients Minn and Mount Sinai. In cells from patient Minn, but not from patient Mount Sinai, the decreased number of insulin receptors was associated with reduced insulin receptor mRNA. In two families with the less severe form of insulin resistance, type A diabetes, mutations altered post-translational processing of the insulin receptor molecule. At a cellular level, these mutations of the alpha subunit of the insulin receptor shared defective binding and impaired stimulation of sugar transport by insulin. In family Atl, however, glucose uptake was constitutively increased. Thus, genetic variation in the insulin receptor gene causes a spectrum of inherited insulin-resistant syndromes and altered cellular signaling.     

A naturally occurring mutation of insulin receptor alanine 1134 impairs tyrosine kinase function and is associated with dominantly inherited insulin resistance

We have identified a previously undescribed genetic variant of the insulin receptor (Ala1134----Thr1134) in a family with the Type A syndrome of insulin resistance. Using the polymerase chain reaction to amplify insulin receptor cDNA and genomic DNA (exon 19), this mutation was detected in 1/2 alleles in the proband, her two affected sisters, and her affected father. Two normal alleles were present in the unaffected mother. No additional structural changes were encoded by the remainder of the proband's receptor cDNA. The Ala1134 mutant receptor was expressed in Chinese hamster ovary cells. The expressed mutant receptors were processed normally and displayed normal affinity of insulin binding but were markedly deficient in insulin-stimulated autophosphorylation. The mutant receptor was unable to catalyze the phosphorylation of the endogenous substrate, pp185, and insulin-stimulated kinase activity toward an exogenous substrate in vitro also was markedly impaired. Ala1134 is a highly conserved residue located in a consensus sequence found in most tyrosine kinases. It is likely that this previously uncharacterized residue and/or the immediate region surrounding it are important for normal kinase function in other members of this receptor family. This study also demonstrates that severe insulin resistance with dominant inheritance may be caused by a missense mutation in one allele of the insulin receptor gene.     

Evidence supporting a passive role for the insulin receptor transmembrane domain in insulin-dependent signal transduction

We previously have demonstrated that intramolecular interactions between alpha beta-alpha beta subunits are necessary for insulin-dependent activation of the protein kinase domain within a single alpha 2 beta 2 heterotetrameric insulin-receptor complex (Wilden, P. A., Morrison, B. D., and Pessin, J. E. (1989) Biochemistry 28, 785-792). To evaluate the role of the beta subunit transmembrane domain in the insulin-dependent signalling mechanism, mutant human insulin receptors containing a series of nested transmembrane domain deletions (amino acids 941-945) were generated and stable Chinese hamster ovary-transfected cell lines were obtained. In addition, a substitution of Val-938 for Glu (E/V938) similar to the oncogenic mutation found in the neu transmembrane domain was also introduced into the insulin receptor. Scatchard analysis of insulin binding to the stable Chinese hamster ovary cell lines expressing either wild type or mutant insulin receptors indicated equivalent receptor number (2-4 x 10(6)/cell) and similar high affinity binding constants (Kd 0.1-0.3 nM). 125I-Insulin affinity cross-linking demonstrated that all of the expressed insulin receptors were assembled and processed into alpha 2 beta 2 heterotetrameric complexes. Surprisingly, all the mutant insulin receptors retained insulin-stimulated autophosphorylation both in vivo and in vitro. Furthermore, endogenous substrate phosphorylation in vivo as well as insulin-stimulated thymidine incorporation into DNA were unaffected by the transmembrane domain mutations. These data demonstrate that marked structural alterations in the insulin receptor transmembrane domain do not interfere with insulin-dependent signal transduction.     

Insulin binding changes the interface region between alpha subunits of the insulin receptor

The homobifunctional cross-linking reagent disuccinimidyl suberate (DSS) was used to probe the interface region between the two alpha subunits of the alpha 2 beta 2 human insulin receptor. The two alpha subunits formed a covalent dimer when affinity-purified receptor or membrane-bound receptor was reacted with DSS. The alpha 2 species was detected on protein blots from SDS gels using an anti-alpha-subunit antibody or 125I-concanavalin A. Alternatively, iodinated receptor was reacted with DSS and the alpha 2 species measured directly in an SDS gel. As shown by all three assay systems, more alpha 2 was formed when insulin was bound to receptor than when insulin was absent. These data indicate that the conformational change which occurs in the alpha subunit in response to insulin binding results in a change in the alpha-alpha interaction within the receptor complex. The results are consistent with a kinase activation mechanism involving communication between the two alpha beta receptor halves.     

Adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) regulation of insulin receptor function: phosphorylation of insulin receptor with A kinase decreases the insulin binding activity.

The effect of phosphorylation of insulin receptor with adenosine 3',5'-cyclic monophosphate-dependent protein kinase (A kinase) on its insulin binding activity was investigated by using insulin receptors prepared from rat liver in vitro. A 95 KDa protein was phosphorylated by stimulation of insulin receptor kinase. This protein was also phosphorylated by A kinase. Analysis of phosphoamino acid showed that tyrosine residue(s) was phosphorylated by activation of insulin receptor kinase, whereas phosphoserine and phosphothreonine were dominantly generated by activation of A kinase. [125I] Iodoinsulin binding activity was decreased by prior phosphorylation of the receptor with A kinase. Scatchard analysis showed that the affinity for insulin was decreased by the phosphorylation with A kinase. Although the maximal activity of insulin receptor kinase was not affected by phosphorylation with A kinase, the insulin concentration which induced half maximal activity (ED50) of the receptor kinase was increased by the phosphorylation with A kinase. These results suggested that counter regulatory hormones whose actions are mediated by the generation of adenosine 3',5'-cyclic monophosphate regulate the insulin binding to the alpha subunit through phosphorylation of the beta subunit of insulin receptor.     

Secretion of soluble functional insulin receptors by transfected NIH3T3 cells

In order to determine whether the human insulin receptor ectodomain can be expressed as a functional protein, the coding regions for the transmembrane and cytoplasmic domain of a full-length human insulin receptor cDNA were deleted by site-directed mutagenesis, and the resultant construct was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH3T3 cells, a cell line secreting an insulin binding protein was isolated. The insulin binding alpha subunit had an Mr of 138,000 and a beta subunit of Mr 48,000 (compared to 147,000 and 105,000 for the full-length human insulin receptor expressed in NIH3T3 cells). This difference in size of the alpha subunit was due to a difference in glycosylation as N-glycanase digestion reduced the apparent size of the alpha subunits of secreted and normal membrane-bound receptors to identical values. The secreted receptor formed disulfide-linked heterotetrameric structures with an Mr of 280,000. It was synthesized as an Mr 160,000 precursor which was cleaved into mature subunits with a t1/2 of 3 h. Increasing expression of the cDNA by induction with sodium butyrate lead to the appearance of an Mr 180,000 protein in the medium as well as the mature alpha and beta subunits. A Scatchard plot of insulin binding to the secreted receptor was curvilinear with a Kd of 7 X 10(-10) M for the high affinity sites and 10(-7) M for the low affinity site (compared to Kd values of 1.1 X 10(-9) M and 10(-7) M, respectively, for human insulin receptors expressed in these cells.