Hormone-triggered conformational changes within the insulin-receptor ectodomain: requirement for transmembrane anchors

2001 ◽  
Vol 360 (1) ◽  
pp. 189-198 ◽  
Author(s):  
Ralf-Rudiger FLÖRKE ◽  
Kerstin SCHNAITH ◽  
Waltraud PASSLACK ◽  
Marc WICHERT ◽  
Lothar KUEHN ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Insulin Receptor ◽  
Conformational Changes ◽  
Sedimentation Coefficient ◽  
Insulin Binding ◽  
Underlying Structure ◽  
Structural Constraints ◽  
Wild Type ◽  
Recombinant Human Insulin ◽  
High Affinity

Interaction between two αβ half-receptors within the (αβ)2 holoreceptor complex is required for insulin binding with high affinity and for insulin-triggered changes of size and shape. To understand the underlying structure–function relationship, two truncated receptor constructs have been characterized. Reduction in the Stokes radius and increase in the sedimentation coefficient, which are characteristic for wild-type receptors, were entirely lacking for the recombinant human insulin receptor (HIR) ectodomain (HIR-ED). Stokes radii of about 5.8nm and sedimentation coefficients of 10.2S were found for both insulin-bound and free HIR-EDs. However, attaching the membrane anchors to the ectodomain, as with the recombinant membrane-anchored ectodomain (HIR-MAED) construct, was sufficient to restore not only high-affinity hormone binding but also the marked insulin-inducible alterations in hydrodynamic properties. The Stokes radii of HIR-MAED complexes, as assessed by non-denaturing PAGE, decreased upon insulin binding from 9.5nm to 7.9nm. In parallel, the sedimentation coefficient was increased from 9.0S to 9.8S. CD and fluorescence spectroscopy of HIR-MAED revealed only minor insulin-induced changes in the secondary structure. Similarity with wild-type receptors has also been demonstrated by the differential insertion of insulin-bound and free HIR-MAED complexes into artificial bilayer membranes of Triton X-114. The results are consistent with a model of receptor function that ensures a global insulin-triggered reorientation of subdomains within the ectodomain moieties while the secondary structure is essentially retained. For the rearrangement of such subdomains, the transmembrane anchors confer essential structural constraints on the receptor ectodomain.

scholarly journals The β-tail domain (βTD) regulates physiologic ligand binding to integrin CD11b/CD18

Blood ◽  
2006 ◽  
Vol 109 (8) ◽  
pp. 3513-3520 ◽  
Author(s):  
Vineet Gupta ◽  
Annette Gylling ◽  
José Luis Alonso ◽  
Takashi Sugimori ◽  
Petre Ianakiev ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Conformational Changes ◽  
Divalent Cations ◽  
Contact Region ◽  
Allosteric Modulator ◽  
Tail Domain ◽  
Wild Type ◽  
High Affinity

Abstract Crystallographic and electron microscopy studies revealed genuflexed (bent) integrins in both unliganded (inactive) and physiologic ligandbound (active) states, suggesting that local conformational changes are sufficient for activation. Herein we have explored the role of local changes in the contact region between the membrane-proximal β-tail domain (βTD) and the ligand-binding βA domain of the bent conformation in regulating interaction of integrin CD11b/CD18 (αMβ2) with its physiologic ligand iC3b. We replaced the βTD CD loop residues D658GMD of the CD18 (β2) subunit with the equivalent D672SSG of the β3 subunit, with AGAA or with NGTD, expressed the respective heterodimeric receptors either transiently in epithelial HEK293T cells or stably in leukocytes (K562), and measured their ability to bind iC3b and to conformation-sensitive mAbs. In the presence of the physiologic divalent cations Ca2+ plus Mg2+ (at 1 mM each), the modified integrins showed increased (in HEK293) or constitutive (in K562) binding to iC3b compared with wild-type receptors. K562 expressing the βTD-modified integrins bound in Ca2+Mg2+ to the βA-directed high-affinity reporter mAb 24 but not to mAb KIM127, a reporter of the genu-straightened state. These data identify a role for the membrane proximal βTD as an allosteric modulator of integrin activation.

scholarly journals Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

2000 ◽  
Vol 346 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Richard GRIEßLER ◽  
Sabato D'AURIA ◽  
Reinhard SCHINZEL ◽  
Fabio TANFANI ◽  
Bernd NIDETZKY
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Active Site ◽  
Thermal Denaturation ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Reversible Inactivation ◽  
The Stability ◽  
Maltodextrin Phosphorylase

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each ≈ 90-kDa subunit contains active-site pyridoxal 5ʹ-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 → Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at ≈ 45 °C. They are manifested by slight increases in unordered structure and 1H/2H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at ≈ 45 °C without prior release into solution of pyridoxal 5ʹ-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 °C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 °C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a ≈ 15 °C higher value on the temperature scale. Apart from greater stability, the Asn-133 → Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.

scholarly journals Spectroscopic characterization of insulin and small molecule ligand binding to the insulin receptor

2002 ◽  
Vol 16 (3-4) ◽  
pp. 147-159 ◽  
Author(s):  
Morten Schlein ◽  
Svend Ludvigsen ◽  
Helle B. Olsen ◽  
Michael F. Dunn ◽  
Niels C. Kaarsholm
Keyword(s):  
Ligand Binding ◽  
Insulin Receptor ◽  
Small Molecule ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Insulin Analogue ◽  
Three Dimensional ◽  
Insulin Binding ◽  
Spectroscopic Characterization ◽  
Spectroscopic Techniques

We have applied spectroscopic techniques to study two kinds of ligand binding to the insulin receptor. First, a fluorescently labelled insulin analogue is used to characterize the mechanism of reversible 1 :1 complex formation with a fragment of the insulin receptor ectodomain. The receptor induced fluorescence enhancement of the labelled insulin analogue provides the basis for stopped flow kinetic experiments. The kinetic data are consistent with a bimolecular binding event followed by a conformational change. This emphasizes the importance of insulin induced conformational changes in the activation of the insulin receptor. Second, the binding of fluorescein derivatives to the insulin receptor is studied. These small molecule ligands displace insulin from its receptor with micromolar affinity. The binding is verified by transferred NOESY NMR experiments. Their chromophoric properties are used to measure the affinity by UV-vis and fluorescence difference spectroscopies and the resulting Kdvalues are similar to those observed in the displacement receptor binding assay. However, these experiments and a stoichiometry determination indicate multiple binding sites, of which one overlaps with the insulin binding site. These two examples illustrate how spectroscopy complements biochemical receptor binding assays and provides information on ligand–insulin receptor interactions in the absence of three dimensional structures.

scholarly journals The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals.

1992 ◽  
Vol 118 (4) ◽  
pp. 831-839 ◽  
Author(s):  
J M Backer ◽  
S E Shoelson ◽  
M A Weiss ◽  
Q X Hua ◽  
R B Cheatham ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Insulin Receptor ◽  
Insulin Binding ◽  
Beta Turns ◽  
Cho Cell ◽  
Beta Turn ◽  
Tyrosine Residues ◽  
Juxtamembrane Region ◽  
Mutant Receptors

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.

High affinity insulin binding and insulin receptor-effector coupling: Modulation by Ca2+

Cell Calcium ◽  
1990 ◽  
Vol 11 (8) ◽  
pp. 547-556 ◽  
Author(s):  
P.F. Williams ◽  
I.D. Caterson ◽  
G.J. Cooney ◽  
R.R. Zilkens ◽  
J.R. Turtle
Keyword(s):  
Insulin Receptor ◽  
Insulin Binding ◽  
High Affinity

scholarly journals Characterization of insulin/IGF hybrid receptors: contributions of the insulin receptor L2 and Fn1 domains and the alternatively spliced exon 11 sequence to ligand binding and receptor activation

2007 ◽  
Vol 403 (3) ◽  
pp. 603-613 ◽  
Author(s):  
Samira Benyoucef ◽  
Katharina H. Surinya ◽  
Dirk Hadaschik ◽  
Kenneth Siddle
Keyword(s):  
Ligand Binding ◽  
Insulin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Binding ◽  
Receptor Activation ◽  
Type I ◽  
High Affinity ◽  
Igf I ◽  
Exon 11

The IR (insulin receptor) and IGFR (type I insulin-like growth factor receptor) are found as homodimers, but the respective pro-receptors can also heterodimerize to form insulin–IGF hybrid receptors. There are conflicting data on the ligand affinity of hybrids, and especially on the influence of different IR isoforms. To investigate further the contribution of individual ligand binding epitopes to affinity and specificity in the IR/IGFR family, we generated hybrids incorporating both IR isoforms (A and B) and IR/IGFR domain-swap chimaeras, by ectopic co-expression of receptor constructs in Chinese hamster ovary cells, and studied ligand binding using both radioligand competition and bioluminescence resonance energy transfer assays. We found that IR-A–IGFR and IR-B–IGFR hybrids bound insulin with similar relatively low affinity, which was intermediate between that of homodimeric IR and homodimeric IGFR. However, both IR-A–IGFR and IR-B–IGFR hybrids bound IGF-I and IGF-II with high affinity, at a level comparable with homodimeric IGFR. Incorporation of a significant fraction of either IR-A or IR-B into hybrids resulted in abrogation of insulin- but not IGF-I-stimulated autophosphorylation. We conclude that the sequence of 12 amino acids encoded by exon 11 of the IR gene has little or no effect on ligand binding and activation of IR–IGFR hybrids, and that hybrid receptors bind IGFs but not insulin at physiological concentrations regardless of the IR isoform they contained. To reconstitute high affinity insulin binding within a hybrid receptor, chimaeras in which the IGFR L1 or L2 domains had been replaced by equivalent IR domains were co-expressed with full-length IR-A or IR-B. In the context of an IR-A–IGFR hybrid, replacement of IR residues 325–524 (containing the L2 domain and part of the first fibronectin domain) with the corresponding IGFR sequence increased the affinity for insulin by 20-fold. We conclude that the L2 and/or first fibronectin domains of IR contribute in trans with the L1 domain to create a high affinity insulin-binding site within a dimeric receptor.

scholarly journals Intrasteric Inhibition of ATP Binding Is Not Required To Prevent Unregulated Autophosphorylation or Signaling by the Insulin Receptor

2001 ◽  
Vol 21 (13) ◽  
pp. 4197-4207 ◽  
Author(s):  
Mark Frankel ◽  
Ararat J. Ablooglu ◽  
Joseph W. Leone ◽  
Elena Rusinova ◽  
J. B. Alexander Ross ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Conformational Changes ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Kinase Domain ◽  
Atp Binding ◽  
Activation Loop ◽  
Wild Type ◽  
Solution Studies ◽  
Activated State

ABSTRACT Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase ink cat and a 15-fold-lowerK m ATP althoughK m peptide was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.

scholarly journals High affinity insulin binding by soluble insulin receptor extracellular domain fused to a leucine zipper

FEBS Letters ◽  
2000 ◽  
Vol 479 (1-2) ◽  
pp. 15-18 ◽  
Author(s):  
Peter A. Hoyne ◽  
Leah J. Cosgrove ◽  
Neil M. McKern ◽  
John D. Bentley ◽  
Neva Ivancic ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Leucine Zipper ◽  
Insulin Binding ◽  
Extracellular Domain ◽  
High Affinity ◽  
Soluble Insulin ◽  
Soluble Insulin Receptor

scholarly journals A conformational switch controlling the toxicity of the prion protein

2021 ◽  
Author(s):  
Karl Frontzek ◽  
Marco Bardelli ◽  
Assunta Senatore ◽  
Anna Henzi ◽  
Regina R. Reimann ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Neurological Impairment ◽  
Wild Type ◽  
Specific Expression ◽  
Conformational Switch ◽  
Affinity Ligands ◽  
Prion Infection ◽  
High Affinity ◽  
Prion Propagation ◽  
Cerebellar Cultures

SummaryPrion infections cause conformational changes of PrPC and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond (“H-latch”) altering the flexibility of the α2-α3 and β2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation, prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is causally linked to prion neurotoxicity.

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