Binding of insulin to solubilized insulin receptor from human placenta. Evidence for a single class of noninteracting binding sites

Binding of 125I-insulin to insulin receptor was studied at equilibrium using plasma membrane protein, from human placenta, solubilized in Triton X-100. Measured at 1 nM 125I-insulin, the amount of radioligand bound specifically was dependent upon pH, the optimal association at 4 degrees C occurring in the pH range 8.2-8.7. The Adair equation was employed for analysis of binding isotherms generated at several pH values for concentrations of radioligand varying from 0.01-8.0 nM. Association constants derived from these analyses showed the same pH dependence described above and were found to be independent of receptor concentration. Hill plots derived from these titrations consistently yielded Hill coefficients of 1 and Scatchard plots gave virtually straight lines. Close correspondence was found between theoretical analyses and observations under all experimental conditions and graphical methods employed. These results are consistent with a single class of noninteracting 125I-insulin binding sites on the solubilized insulin receptor.     

Human placenta contains a high affinity R1881 binding site that is not the androgen receptor

Human placental cytosol was shown to contain a species that binds the synthetic androgen, methyltrienolone (R1881) with high affinity (Kd 6.5 nM). Major differences were found between this placental androgen binding species and the classical androgen receptor found in human foreskin cytosol. Competitive binding assays in the placental cytosol using [3H]R1881 as tracer showed a 200-fold excess of testosterone to compete poorly, while dihydrotestosterone and the synthetic androgen mibolerone did not compete at all. The placental R1881 binding component was found not to bind to hydroxylapatite, although all classes of steroid receptors are reported to do so. Temperature studies showed that the placental binding site is stable at elevated temperatures with no loss of binding after 4 h at 45 degrees C. Ion exchange chromatography showed that the placental R1881 binding site eluted from DEAE cellulose at a lower salt concentration than foreskin androgen receptors. These results show that R1881 is not entirely specific for androgen receptors and that human placenta contains an androgen binding site that is not the classical androgen receptor.     

Evidence for phosphorylation of actin by the insulin receptor-associated protein kinase from human placenta

A purified preparation of rabbit muscle actin (43-kDa protein) is phosphorylated at tyrosine residues when incubated with solubilized insulin receptor from human placenta. Phosphorylation of the 95-kDa receptor subunit and of 43-kDa protein is stimulated by insulin and vanadate, respectively; however, the mode of action of the two agents is distinguishable.     

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.     

IGF-II binding on human lymphoid cells: demonstration of a common high affinity receptor for insulin like peptides

We have studied the binding of 125I-GF-II to the IM-9 human lymphoid cell line, and to human placental membranes. All of IGF-II radioligand binding to IM-9 cells, and half of the binding to human placental membranes is to a previously unrecognized common (Type-III) high affinity receptor site for insulin-like peptides, in which IGF-I and IGF-II are equipotent and insulin only slightly less potent. This common receptor represents another mechanism by which insulin, and the somatomedins can exert biological action.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system

Abstract: This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri . A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogenicity of Y. ruckeri for fish.     

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.     

Characteristics of the solubilized insulin receptor of human placenta

A soluble human insulin receptor can be obtained in high yield from placenta membranes, using the detergent Ammonyx-LO. In crude soluble preparations, the placenta receptor exhibits complex insulin binding kinetics (two binding plateaus, half-saturated at about 40 pM and 700 pM insulin) and an apparent chromatographic heterogeneity (Sepharose 6B) with two insulin binding components having apparent Stokes radii of 72Å and 38Å. However, subsequent to purification by affinity chromatography on insulin-Sepharose, the placenta receptor exhibits a simple insulin binding isotherm, without evidence for binding cooperativity (K_D about 830 pM), and upon chromatography behaves as a single component with a Stokes radius of 38Å_A. When combined with a previously described non-receptor glyco-protein preparation isolated from liver membranes, the affinity-purified plancenta receptor undergoes an increase in its Stokes radius from 38Å to 72Å. Because of the physicochemical similarities between the placenta receptor and the insulin receptor previously isolated from liver cell membranes, it is concluded that the placenta receptor is representative of the insulin receptor present in other traditional target tissues for insulin. The study underscores a possible role for insulin in placental physiology and provides for the large scale isolation of the human receptor from a readily available source.     

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.     

Evidence that Yersinia ruckeri possesses a high affinity iron uptake system.

This work represents the first evidence of the presence of an iron uptake system siderophore mediated in the bacterial fish pathogen Yersinia ruckeri. A group of 20 strains representative of this species, with different serotype and origin were examined. All of them were able to grow at high concentrations (from 0.7 to 1.1 mM) of the iron chelator EDDA. Although the Y. ruckeri isolates failed to cross-feed the indicator strains for enterobactin and aerobactin production, the chemical tests revealed the presence in the culture supernatants of phenolate siderophores. At least three outer membrane proteins were induced in iron limiting conditions. All the strains showed a similar pattern of induced membrane proteins regardless their serotype or origin, which suggests a similarity in the iron uptake system. This system could have an important role in the pathogeneicity of Y. ruckeri for fish.     

Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate

Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.     

Prorenin has high affinity multiple binding sites for (pro)renin receptor

An important role of the decoy peptide sequence has recently been suggested in vitro for the binding of prorenin to the (pro)renin receptor [(P)RR]. In this study, other prospective crucial regions in renin and prorenin responsible for their interaction with (P)RR were investigated using various kinds of peptides, e.g., the “hinge” S¹⁴⁹QGVLKEDVF¹⁵⁸ designed from the structure of renin also common to prorenin, L^1PPTDTTTF^8P, L^1PPTDTTTFKRIFLKR^15P and the decoy (R^10PIFLKRMPSI^19P) designed from the predicted structure of prorenin. For the kinetic analysis, the recombinant h(P)RR was immobilized on the biosensor surface through a specific anti-(P)RR antibody. In case of the equilibrium state analysis, the (P)RR was directly adsorbed on plastic wells for observing the bindings of renin/prorenin. The dissociation constants (K_D) for the bindings of renin and prorenin to the pre-adsorbed receptors were 4.5 and 1.0 nM, respectively, similar to those stated in the kinetic study by BIAcore assay. The “hinge” region peptide bound to (P)RR in a dose-dependent manner with a K_D estimated 17.0 nM which was five times higher than that of the decoy. The K_D values for L^1PPTDTTTF^8P and L^1PPTDTTTFKRIFLKR^15P were 52 and 7.6 nM, respectively. The “hinge” peptide, as the decoy, inhibited the bindings of renin and prorenin to (P)RR. The inhibition constant (K_i) for the binding of renin and prorenin by the decoy and “hinge” were 16.7 and 15.1, and 37.1 and 30.7 nM, respectively. These in vitro studies suggest that renin has a single and prorenin has at least two high affinity binding sites for the (P)RR.     

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant.

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.     

High affinity antibody from hen's eggs directed against the human insulin receptor and the human IGF-I receptor

Large quantities of high affinity antibodies directed against the human insulin receptor and the human insulin-like growth factor-I (IGF-I) receptor were obtained from hen's eggs. Hens were immunized with human placental membranes and human liver membranes by intramuscular injections. Specific antibodies to the receptors were demonstrated in serum and egg yolks at 5 weeks and these antibodies presisted for at least 6 months. Antibodies from egg yolks were purified by the polyethylene glycol precipitation technique of Polson et al. The eggs provided the equivalent of about 450 ml of immunized serum per month per bird. The purified antibodies were approximately equally reactive with receptors for insulin or IGF-I. Antibodies immunoprecipitated affinity-labeled receptors, inhibited binding of each ligand, and were capable of stimulating 2-deoxyglucose uptake in rat adipocytes and thymidine incorporation in cultured fibroblasts. The presence of antibodies directed against the IGF-I receptor in those hens immunized with human liver membranes was unexpected, since liver membranes possess little or no IGF-I binding activity. We conclude that antibodies against human antigens may be relatively easily obtained by immunization of hens and purification of those antibodies from eggs.     

Regulation of insulin receptor function by a small molecule insulin receptor activator

In type 2 diabetes mellitus, impaired insulin signaling leads to hyperglycemia and other metabolic abnormalities. TLK19780, a non-peptide small molecule, is a new member of a novel class of anti-diabetic agents that function as activators of the insulin receptor (IR) beta-subunit tyrosine kinase. In HTC-IR cells, 20 microm TLK19780 enhanced maximal insulin-stimulated IR autophosphorylation 2-fold and increased insulin sensitivity 2-3-fold. In contrast, TLK19780 did not potentiate the action of insulin-like growth factor-1, indicating the selectivity of TLK19780 toward the IR. The predominant effect of TLK19780 was to increase the number of IR that underwent autophosphorylation. Kinetic studies indicated that TLK19780 acted very rapidly, with a maximal effect observed 2 min after addition to insulin-stimulated cells. In 3T3-L1 adipocytes, 5 microm TLK19780 enhanced insulin-stimulated glucose transport, increasing both the sensitivity and maximal responsiveness to insulin. These studies indicate that at low micromolar levels small IR activator molecules can enhance insulin action in various cultured cells and suggest that this effect is mediated by increasing the number of IR that are tyrosine-phosphorylated in response to insulin. These studies suggest that these types of molecules could be developed to treat type 2 diabetes and other clinical conditions associated with insulin resistance.     

Rapidly growing rumen methanogenic organism that synthesizes coenzyme M and has a high affinity for formate.

Methanogenic bacteria with a coccobacillus morphology similar to Methanobrevibacter ruminantium were isolated from the bovine rumen. One isolate, 10-16B, represented a previously undescribed rumen population that, unlike M. ruminantium, synthesized coenzyme M, grew rapidly (mu = 0.24 h-1) on H2-CO2 in a complex medium, had simple nutritional requirements, and metabolized formate at reported rumen concentrations. H2 was metabolized to partial pressures 10-fold lower than those reported for the rumen. After H2 starvation for 26 h, strain 10-16B rapidly resumed growth when H2 was made available. The minimum concentrations of acetate (6 mM) and ammonia (less than 7 mM) that were required for optimal growth were lower than the reported acetate and ammonia concentrations in the rumen. Isoleucine and leucine stimulated growth, but only at concentrations (greater than 50 microM) higher than those reported for the rumen. Another coccobacillary methanogenic organism that synthesized coenzyme M was isolated from a different animal as were organisms that required an exogenous supply of coenzyme M. In general, methanogenic bacteria that required an exogenous supply of coenzyme M had lower maximum growth rates and more complex nutritional requirements than organisms that synthesized the cofactor. The ability of all isolates to metabolize formate below the detection limit of 10 microM indicated that, in contrast to previous reports, methanogenic bacteria have the potential to directly metabolize formate in the rumen. This study demonstrated that there are physiologically diverse populations of coccobacillary methanogenic bacteria in the rumen that can interact competitively and cooperatively.     

Evidence of high and low affinity binding sites for basic fibroblast growth factor in mouse placenta

The placenta has been shown to contain bFGF, but the presence of specific binding sites for this growth factor in this tissue remained to be established. In order to study the role of bFGF in the placenta growth, we looked for specific binding sites on mouse placental cell membranes at days 12, 14, 16, and 18 of pregnancy. At day 12, Scatchard analyses indicated that two classes of specific interaction sites for bFGF were detected. One class of high affinity binding sites was characterized by an apparent Kd of 10 pM and a binding capacity of 10 fmoles per mg of membrane protein. A second class of low affinity binding sites was detected with an apparent Kd of 60 nM and a binding capacity of 26 pmoles per mg of membrane protein. At days 14, 16 or 18, Scatchard analyses only showed low affinity binding sites with an apparent Kd of 24 nM and a binding capacity of 230 pmoles per mg of membrane protein. The characterization of these binding sites was performed by cross linking experiments that revealed two forms of specific complexes. This result suggested that the high affinity binding sites correspond to putative receptors with relative molecular masses equal to 65,000 and 85,000. The dramatic decrease of the high affinity receptor number after the 12th day of pregnancy, which is synchronous with the 9-fold increase of the low affinity binding site number, suggests that the biological activity of bFGF could be regulated by a balance between both the numbers of high and low affinity binding sites on placenta cell membranes. Thus, as it was shown for other growth factors, bFGF could only be involved at specific pregnancy stages.