Alpha Beta Heterodimeric Complex Research Articles

Expression and Characterization of the Catalytic Domains of Soluble Guanylate Cyclase: Interaction with the Heme Domain

The catalytic domains (alpha(cat) and beta(cat)) of alpha1beta1 soluble guanylate cyclase (sGC) were expressed in Escherichia coli and purified to homogeneity. alpha(cat), beta(cat), and the alpha(cat)beta(cat) heterodimeric complex were characterized by analytical gel filtration and circular dichroism spectroscopy, and activity was assessed in the absence and presence of two different N-terminal regulatory heme-binding domain constructs. Alpha(cat) and beta(cat) were inactive separately, but together the domains exhibited guanylate cyclase activity. Analysis by gel filtration chromatography demonstrated that each of the approximately 25-kDa domains form homodimers. Heterodimers were formed when alpha(cat) and beta(cat) were combined. Results from circular dichroism spectroscopy indicated that no major structural changes occur upon heterodimer formation. Like the full-length enzyme, the alpha(cat)beta(cat) complex was more active in the presence of Mn(2+) as compared to the physiological cofactor Mg(2+), although the magnitude of the difference was much larger for the catalytic domains than for the full-length enzyme. The K(M) for Mn(2+)-GTP was measured to be 85 +/- 18 microM, and in the presence of Mn(2+)-GTP, the K(D) for the alpha(cat)beta(cat) complex was 450 +/- 70 nM. The N-terminal heme-bound regulatory domain of the beta1 subunit of sGC inhibited the activity of the alpha(cat)beta(cat) complex in trans, suggesting a domain-scale mechanism of regulation by NO. A model in which binding of NO to sGC causes relief of an autoinhibitory interaction between the regulatory heme-binding domain and the catalytic domains of sGC is proposed.

Structural basis for the recognition of hydroxyproline in HIF-1 alpha by pVHL.

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that controls cellular and systemic homeostatic responses to oxygen availability. HIF-1 alpha is the oxygen-regulated subunit of HIF-1, an alpha beta heterodimeric complex. HIF-1 alpha is stable in hypoxia, but in the presence of oxygen it is targeted for proteasomal degradation by the ubiquitination complex pVHL, the protein of the von Hippel Lindau (VHL) tumour suppressor gene and a component of an E3 ubiquitin ligase complex. Capture of HIF-1 alpha by pVHL is regulated by hydroxylation of specific prolyl residues in two functionally independent regions of HIF-1 alpha. The crystal structure of a hydroxylated HIF-1 alpha peptide bound to VCB (pVHL, elongins C and B) and solution binding assays reveal a single, conserved hydroxyproline-binding pocket in pVHL. Optimized hydrogen bonding to the buried hydroxyprolyl group confers precise discrimination between hydroxylated and unmodified prolyl residues. This mechanism provides a new focus for development of therapeutic agents to modulate cellular responses to hypoxia.

The endogenous functional turkey erythrocyte and rat liver insulin receptor is an α2β2 heterotetrameric complex

Previous studies have indicated that turkey erythrocyte and rat liver membranes contain endogenous alpha beta heterodimeric insulin receptors in addition to the disulphide-linked alpha 2 beta 2 heterotetrameric complexes characteristic of most cell types. We utilized 125I-insulin affinity cross-linking to examine the structural properties of insulin receptors from rat liver and turkey erythrocyte membranes prepared in the absence and presence of sulphydryl alkylating agents. Rat liver membranes prepared in the absence of sulphydryl alkylating agents displayed specific labelling of Mr 400,000 and 200,000 bands, corresponding to the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes respectively. In contrast, affinity cross-linking of membranes prepared with iodoacetamide (IAN) or N-ethylmaleimide identified predominantly the alpha 2 beta 2 heterotetrameric insulin receptor complex. Similarly, affinity cross-linking and solubilization of intact turkey erythrocytes in the presence of IAN resulted in exclusive labelling of the alpha 2 beta 2 heterotetrameric insulin receptor complex, whereas in the absence of IAN both alpha 2 beta 2 and alpha beta species were observed. Turkey erythrocyte alpha 2 beta 2 heterotetrameric insulin receptors from IAN-protected membranes displayed a 3-4-fold stimulation of beta subunit autophosphorylation and substrate phosphorylation by insulin, equivalent to that observed in intact human placenta insulin receptors. Turkey erythrocyte alpha beta heterodimeric insulin receptors, prepared by defined pH/dithiothreitol treatment of IAN-protected membranes, were also fully competent in insulin-stimulated protein kinase activity compared with alpha beta heterodimeric human placenta receptors. In contrast, endogenous turkey erythrocyte alpha beta heterodimeric insulin receptors displayed basal protein kinase activity which was insulin-insensitive. These data indicate that native turkey erythrocyte and rat liver insulin receptors are structurally and functionally similar to alpha 2 beta 2 heterotetrameric human placenta insulin receptors. The alpha beta heterodimeric insulin receptors previously identified in these tissues most likely resulted from disulphide bond reduction and denaturation of the alpha 2 beta 2 holoreceptor complexes during membrane preparation.

IGF-1-dependent subunit communication of the IGF-1 holoreceptor: interactions between alpha beta heterodimeric receptor halves.

Examination of 125I-IGF-1 affinity cross-linking and beta-subunit autophosphorylation has indicated that IGF-1 induces a covalent association of isolated alpha beta heterodimeric IGF-1 receptors into an alpha 2 beta 2 heterotetrameric state, in a similar manner to that observed for the insulin receptor [Morrison, B.D., Swanson, M.L., Sweet, L.J., & Pessin, J.E. (1988) J. Biol. Chem. 263, 7806-7813]. The formation of the alpha 2 beta 2 heterotetrameric IGF-1 receptor complex from the partially purified alpha beta heterodimers was time dependent with half-maximal formation in approximately 30 min at saturating IGF-1 concentrations. The IGF-1-dependent association of the partially purified alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state was specific for the IGF-1 receptors since IGF-1 was unable to stimulate the protein kinase activity of the purified alpha beta heterodimeric insulin receptor complex. Incubation of the alpha 2 beta 2 heterotetrameric IGF-1 holoreceptor with the specific sulfhydryl agent iodoacetamide (IAN) did not alter 125I-IGF-1 binding of IGF-1 stimulation of protein kinase activity. In addition, IAN did not affect the Mn/MgATP-dependent noncovalent association of IGF-1 receptor alpha beta heterodimers into an alpha 2 beta 2 heterotetrameric state. However, IAN treatment of the alpha beta heterodimeric IGF-1 receptors inhibited the IGF-1-dependent covalent formation of the disulfide-linked alpha 2 beta 2 heterotetrameric complex. These data indicate that IGF-1 induces the covalent association of isolated alpha beta heterodimeric IGF-1 receptor complexes into a disulfide-linked alpha 2 beta 2 heterotetrameric state whereas Mn/MgATP induces a noncovalent association.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of insulin/insulin-like growth factor-1 hybrid receptors in vitro

Insulin and Mn/MgATP treatment of immunoaffinity-purified alpha beta heterodimeric insulin receptors induced the formation of an alpha 2 beta 2 heterotetrameric insulin receptor complex. In contrast, insulin-like growth factor-1 (IGF-1) treatment was completely ineffective in inducing the association of alpha beta heterodimeric insulin receptors. Similarly, IGF-1 or Mn/MgATP, but not insulin, treatment of immunoaffinity-purified alpha beta heterodimeric IGF-1 receptors induced the formation of an alpha 2 beta 2 heterotetrameric IGF-1 receptor complex. A monoclonal antibody specific for the insulin receptor (MA5) completely immunoprecipitated all the insulin binding activity from both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes but did not immunoprecipitate IGF-1 receptors. Conversely, the IGF-1 receptor-specific monoclonal antibody (alpha IR-3) immunoprecipitated all the IGF-1 binding activity, but not insulin receptors. The simultaneous treatment of pooled equal amounts of alpha beta heterodimeric insulin and IGF-1 receptors with a combination of insulin and IGF-1 resulted in the formation of alpha 2 beta 2 heterotetrameric insulin and IGF-1 receptor complexes. However, in the mixed alpha 2 beta 2 heterotetrameric receptor fraction MA5 immunoprecipitated 94% of the insulin binding in addition to 27% of the IGF-1 binding activity whereas alpha IR-3 immunoprecipitated 97% of the IGF-1 binding in addition to 38% of the insulin binding activity. Treatment of the mixed alpha beta heterodimeric insulin and IGF-1 receptors with Mn/MgATP also resulted in the formation of cross-immunoreactive (42-46%) alpha 2 beta 2 heterotetrameric receptors. These data directly demonstrate the formation of insulin/IGF-1 hybrid receptors by both a combination of insulin plus IGF-1 or Mn/MgATP treatment of purified human placenta alpha beta heterodimeric insulin and IGF-1 half-receptors in vitro.

High affinity insulin binding in the human placenta insulin receptor requires alpha beta heterodimeric subunit interactions.

Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0 mM DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot. 125I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an alpha beta heterodimeric insulin receptor complex from the alpha 2 beta 2 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional alpha beta heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional alpha beta heterodimeric insulin receptor complex. (ii) the alpha beta heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the alpha 2 beta 2 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the alpha beta heterodimers.

Wheat Germ Agglutinin Stimulation ofαβHeterodimeric Insulin Receptor β-Subunit Autophosphorylation by Noncovalent Association into an α2/β2Heterotetrameric State*

The purified human placental alpha 2 beta 2 heterotetrameric insulin-receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Wheat germ agglutinin (WGA) was observed to stimulate the beta-subunit autophosphorylation of both the alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric complexes in the absence of insulin. However, WGA was without effect on the insulin stimulation of beta-subunit autophosphorylation in these insulin-receptor complexes. In contrast, monomeric WGA was unable to stimulate the basal exogenous substrate protein kinase activity in either the alpha 2 beta 2 heterotetrameric or alpha beta heterodimeric insulin receptor complexes. The stimulatory effect of WGA was biphasic, increasing the basal insulin receptor beta-subunit autophosphorylation at low concentrations (250 ng/ml to 2.5 micrograms/ml) and inhibiting at high concentrations (greater than 25 micrograms/ml). Similarly, equilibrium tracer insulin binding was not significantly altered by low concentrations of WGA (less than 1 microgram/ml) but was dramatically reduced at high WGA concentrations (greater than 2.5 micrograms/ml). In contrast to the insulin-induced covalent association of the alpha beta heterodimeric insulin receptors to form a disulfide-linked alpha 2 beta 2 heterotetrameric complex, nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the WGA stimulation of beta-subunit autophosphorylation in the alpha beta heterodimer preparation occurred in the absence of covalent association. Nondenaturing Bio-Gel A-1.5m gel filtration chromatography and [125I]insulin affinity cross-linking demonstrated that WGA treatment of the alpha beta heterodimeric insulin receptors induced a noncovalent association into an alpha 2 beta 2 heterotetrameric state. These data support the hypothesis that the insulin receptor protein kinase activity, although dependent upon alpha beta heterodimeric subunit interactions, does not necessarily require covalent disulfide bond formation between the individual alpha beta heterodimeric species.

Relationship between insulin receptor subunit association and protein kinase activation: insulin-dependent covalent and manganese/magnesium ATP dependent noncovalent association of .alpha..beta. heterodimeric insulin receptors into an .alpha.2.beta.2 heterotetrameric state

The purified human placenta alpha 2 beta 2 heterotetrameric insulin receptor was reduced and dissociated into a functional alpha beta heterodimeric complex by a combination of alkaline pH and dithiothreitol treatment. In the presence of Mn/MgATP, insulin binding to the isolated alpha beta heterodimeric insulin receptor was found to induce the formation of a covalent disulfide-linked alpha 2 beta 2 heterotetrameric complex. In the absence of insulin, a noncovalent association of the alpha beta heterodimeric insulin receptor complex into an alpha 2 beta 2 heterotetrameric state required the continuous presence of both a divalent metal ion (Mn or Mg) and an adenine nucleotide (ATP, ADP, or AMPPCP). Thus, Mn/MgATP binding and not insulin receptor autophosphorylation was responsible for the noncovalent association into the alpha 2 beta 2 heterotetrameric state. However, the divalent metal ions or NaATP separately was ineffective in inducing the noncovalent association between the alpha beta heterodimers. The specific sulfhydryl agent iodoacetamide (IAN) was observed to inhibit the insulin-dependent covalent association of the alpha beta heterodimers without affecting the Mn/MgATP-induced noncovalent association into the alpha 2 beta 2 heterotetrameric state. Insulin treatment of the isolated alpha beta heterodimeric complex in the presence of IAN demonstrated that the Mn/MgATP-induce noncovalent association into the alpha 2 beta 2 heterotetrameric state was sufficient for insulin stimulation of beta-subunit autophosphorylation and exogenous substrate protein kinase activity. These data indicate that although interaction between the individual insulin receptor alpha beta heterodimers is necessary for insulin stimulation of protein kinase activity it does not require covalent disulfide bond formation.

Insulin-dependent covalent reassociation of isolated alpha beta heterodimeric insulin receptors into an alpha 2 beta 2 heterotetrameric disulfide-linked complex.

The purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex was reduced and dissociated into functional alpha beta heterodimers by a combination of alkaline pH and dithiothreitol treatment. Insulin treatment of the isolated alpha beta heterodimeric complex was observed to induce the complete reassociation to an alpha 2 beta 2 heterotetrameric state when analyzed by nondenaturing Bio-Gel A-1.5m gel filtration chromatography. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-insulin affinity cross-linked and 32P-autophosphorylated alpha beta heterodimers demonstrated that the insulin-dependent reassociation to the alpha 2 beta 2 heterotetrameric state occurred both covalently and noncovalently under these conditions. Comparison by reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the insulin-dependent covalent reassociation to an alpha 2 beta 2 heterotetrameric complex was due to the formation of a disulfide linkage(s) between the alpha beta heterodimers. beta subunit autophosphorylation of the control alpha 2 beta 2 heterotetrameric insulin receptor preparation was maximally stimulated within 5 min of insulin preincubation and occurred exclusively in the Mr = 400,000 alpha 2 beta 2 complex. Similarly, maximal insulin-stimulated beta subunit autophosphorylation of the alpha beta heterodimeric preparation occurred within 5 min of insulin pretreatment in the Mr = 210,000 alpha beta complex. However, 4 h of insulin pretreatment of the alpha beta heterodimer preparation induced the formation (6-fold) of a covalent 32P-labeled alpha 2 beta 2 heterotetrameric complex. Maximal stimulation of substrate phosphorylation for the alpha 2 beta 2 heterotetrameric complex was also observed to occur within 5 min of insulin treatment, whereas maximal insulin-stimulated substrate phosphorylation of the alpha beta heterodimeric complex required greater than 4 h. These data demonstrate that (i) insulin treatment can induce the reassociation of the alpha beta heterodimeric complex into a covalent alpha 2 beta 2 heterotetrameric state, and (ii) insulin-dependent protein kinase activation of the alpha beta heterodimeric insulin receptor correlates with the covalent reassociation into a disulfide-linked alpha 2 beta 2 heterotetrameric complex.

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

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

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

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

Isolation of functional alpha beta heterodimers from the purified human placental alpha 2 beta 2 heterotetrameric insulin receptor complex. A structural basis for insulin binding heterogeneity.

To investigate the role of subunit communication in the insulin binding and tyrosine-specific protein kinase activities of the purified human placental insulin receptor, we have developed the methodology to isolate a functional alpha beta heterodimeric insulin receptor complex from the native alpha 2 beta 2 heterotetrameric disulfide-linked state. The dissociation of the alpha 2 beta 2 heterotetrameric insulin receptor into an alpha beta heterodimer was found to be approximately 50% efficient by treatment with alkaline pH (8.75) and dithiothreitol (2 mM). Removal of the dithiothreitol and pH neutralization (pH 7.60) by rapid Sephadex G-50 gel filtration resulted in the preservation of tracer insulin binding activity. The nondissociated alpha 2 beta 2 heterotetrameric and alpha beta heterodimeric insulin receptor complexes could then be effectively separated by Bio-Gel A-1.5m gel filtration. Scatchard analyses of insulin binding to the alpha 2 beta 2 heterotetrameric control or dithiothreitol-treated but nondissociated alpha 2 beta 2 heterotetrameric insulin receptor complexes demonstrated a curvilinear binding isotherm with a maximum of 1 mol of insulin bound/mol of alpha 2 beta 2 heterotetrameric complex. However, binding analyses performed on the isolated alpha beta heterodimeric complex yielded a nearly linear binding curve also, with 1 mol of insulin bound/mol of alpha beta heterodimeric complex at saturation. These data demonstrate that the insulin half-site binding reactivity observed in the alpha 2 beta 2 heterotetrameric insulin receptor complex results from either an asymmetric assembly of identical alpha beta heterodimers or from absolute negative cooperativity.