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.

In-frame exon 2 deletion in insulin receptor RNA in a family with extreme insulin resistance in association with defective insulin binding: a case report

1996 ◽  
Vol 135 (3) ◽  
pp. 357-363 ◽  
Author(s):  
Wolfgang Moritz ◽  
Marianne Böni-Schnetzler ◽  
Wayne Stevens ◽  
E Rudolf Froesch ◽  
James R Levy
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Receptor Binding ◽  
Type A ◽  
Insulin Binding ◽  
Exon 2 ◽  
Receptor Mrna ◽  
Mrna Transcripts ◽  
Extreme Insulin Resistance ◽  
Type A Insulin Resistance

Moritz W, Böni-Schnetzler M, Stevens W, Froesch ER, Levy JR. In-frame exon 2 deletion in insulin receptor RNA in a family with extreme insulin resistance in association with defective insulin binding. Eur J Endocrinol 1996;135:357–63. ISSN 0804–4643 The phenotype and allelic expression of the insulin receptor gene is presented in a family with a patient with type A insulin resistance. Compared to controls, insulin receptor binding in transformed lymphocytes was 100%, 33% and 13% in the father, mother and proband, respectively. Reduced insulin receptor binding co-segregated with altered insulin receptor mRNA expression; the mother and daughter expressed eight insulin receptor mRNA species, including a set of four normal sized and a set of four shorter mRNA transcripts. In the proband the levels of the normal sized mRNA transcripts were suppressed relative to the shorter transcripts. Reverse polymerase chain reaction (PCR) revealed that the shorter transcripts contained an in-frame deletion of exon 2. Sequencing of the entire insulin receptor coding region revealed a paternally inherited A to T substitution in nucleotide 3205, converting isoleucine 996 to phenylalanine. which does not co-segregate with reduced binding. Therefore, we hypothesize that two findings are necessary for the presentation of type A insulin resistance in this patient: an in-frame deletion of the insulin receptor exon 2 that codes for amino acids crucial for insulin binding; and an inhibition of expression of the paternal insulin receptor allele. Marianne Böni-Schnetzler, Division of Endocrinology and Metabolism, Department of Internal Medicine, University Hospital, 8091 Zurich, Switzerland

scholarly journals Capturing Peptide–GPCR Interactions and Their Dynamics

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4724
Author(s):  
Anette Kaiser ◽  
Irene Coin
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Structural Changes ◽  
Structural Heterogeneity ◽  
Receptor Interaction ◽  
Spectroscopic Techniques ◽  
Peptide Receptors ◽  
Physiological Processes ◽  
High Dynamics ◽  
G Protein Coupled

Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.

scholarly journals Insulin causes insulin-receptor internalization in human erythrocyte ghosts

1987 ◽  
Vol 241 (1) ◽  
pp. 93-97 ◽  
Author(s):  
R S Kelleher ◽  
E F Murray ◽  
S W Peterson
Keyword(s):  
Insulin Receptor ◽  
Receptor Binding ◽  
Human Erythrocyte ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Erythrocyte Ghost ◽  
Receptor Internalization ◽  
Erythrocyte Ghosts ◽  
And Control ◽  
Vesicular Compartment

The effect of incubation with insulin on insulin-receptor internalization by erythrocyte ghosts was investigated. The number of surface insulin receptors decreased by 30-40% after incubation of ghosts with insulin. Total insulin-receptor binding to solubilized ghosts was the same in insulin-incubated and control ghosts, whereas insulin binding to an internal vesicular fraction was substantially increased in insulin-incubated ghosts. Our findings suggest that erythrocyte-ghost insulin receptors are internalized to a vesicular compartment in response to incubation with insulin.

scholarly journals The Science Behind G Protein-Coupled Receptors (GPCRs) and Their Accurate Visual Representation in Scientific Research

2017 ◽  
Vol 41 (1) ◽  
Author(s):  
Amy Sojka ◽  
Kevin Brennan ◽  
Evelyn Maizels ◽  
Christine Young
Keyword(s):  
Ligand Binding ◽  
G Protein ◽  
Conformational Changes ◽  
Intracellular Signaling ◽  
Three Dimensional ◽  
G Protein Coupled Receptors ◽  
Membrane Topology ◽  
Extracellular Domains ◽  
Gpcr Structure ◽  
G Protein Coupled

G Protein-Coupled Receptors (GPCRs) are transmembrane (TM) proteins that span the cell membrane seven times, and contain intracellular and extracellular domains, comprised of connecting loops, as well as terminal extension sequences. GPCRs bind ligands within their transmembrane and/or extracellular domains. Ligand binding elicits conformational changes that initiate downstream intracellular signaling events through arrestins and G proteins. GPCRs play central roles in many physiological processes, from sensory to neurological, cardiovascular, endocrine, and reproductive functions. This paper strives to provide an entry point to current GPCR science, and to identify visual approaches to communicate select aspects of GPCR structure and function with clarity and accuracy. The overall GPCR structure, primary sequence and the implications of sequence for membrane topology, ligand binding and helical rearrangements accompanying activation are considered and discussed in the context of visualization strategies, including two-dimensional topological diagrams, three-dimensional representations, and common errors that arise from these representation.

A Small-Molecule Antagonist to Integrin LFA-1 Reveals a Crucial Inter-Domain Communication as a Novel Therapeutic Target.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 650-650
Author(s):  
Motomu Shimaoka ◽  
Azucena Salas ◽  
Wei Yang ◽  
Gabriele Weitz-Schmidt ◽  
Timothy A. Springer
Keyword(s):  
Ligand Binding ◽  
Small Molecule ◽  
Conformational Changes ◽  
Therapeutic Target ◽  
Metal Ion ◽  
Cell Mediated Immunity ◽  
Β Subunit ◽  
Surface Plasmon Resonance Analysis ◽  
Α Subunit ◽  
High Affinity

Abstract The integrin LFA-1 (αLβ2) is an αβ heterodimeric adhesion molecule critical in the effective trafficking of leukocytes and in facilitating subsequent antigen-specific inter-action. Participation of αLβ2 in multiple steps critical for T-cell-mediated immunity in vivo makes αLβ2 a valid therapeutic target for anti-inflammation therapy. Many small-molecule antagonists to αLβ2 have been developed as anti-inflammatory agents, out of which polysubstituted (S)-2-benzoylamino propionic acids, represented by XVA143 (XVA), have emerged as the most potent antagonists. αLβ2 is a large glycoprotein with a complex multi-domain organization, where a conserved von Willebrand factor-type A domain is contained in each subunit, the inserted (I) domain in the α-subunit and the I-like domain in the β subunit. The α-subunit I domain directly binds ligand, whereas the β-subunit I-like domain is thought to play a regulatory role by interacting with a part of the I domain. Thus far, it remains to be elucidated which domain the antagonists bind to and how they inhibit αLβ2 function. Here we investigate a mechanistic basis of XVA activity. XVA blocked the αLβ2-ICAM-1 interaction with EC50 of < 1 nM and suppressed mixed lymphocyte reaction as potently as cyclosporin A. XVA did not block ligand binding by αLβ2 directly, as it did not block αLβ2 containing a mutant I domain that is stabilized in the high-affinity conformation. Rather, XVA interfered with conformational changes that convert the I domain to the high-affinity state. Surface plasmon resonance analysis using an isolated I domain showed that XVA did not target the I domain. Interestingly, XVA stabilized non-covalent αβ association sufficiently to make it resistant to denaturation with SDS. Stabilization of mutant αβ complexes was utilized to test compound binding to αLβ2 mutants and locate the inhibitor-binding site. As binding of XVA was found to be metal-dependent, alanine-scanning of the metal binding sites indicated that this compound binds to the metal ion-dependent adhesion site in the I-like domain, where it disrupts the interaction of the I-like domain with the I domain. XVA inhibits αLβ2 allosterically by perturbing the inter-domain communication that is critical to relay conformational signals which induce the active I domain conformation. Furthermore, XVA stabilized a global conformation of αLβ2 in the active extended form, whereas the ligand binding I domain was left in the inactive conformation, as demonstrated by exposure of activation-dependent epitopes in αLβ2 on the cell surface and electron microscopic images of the soluble recombinant αLβ2. The results strongly suggest that XVA would serve as a mimetic for the intrinsic ligand that is involved in receptor-ligand like interaction between the I domain and I-like domain. This inhibitor revealed a crucial intersection for relaying conformational signals within the integrin αLβ2. While blocking signals in one direction (to the I domain), the antagonists induce the active conformation of the I-like domain as well as the rest of domains, and thus transmit conformational signals in the opposite direction toward the transmembrane domains.

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.

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 Nectin-Like Interactions between Poliovirus and Its Receptor Trigger Conformational Changes Associated with Cell Entry

2015 ◽  
Vol 89 (8) ◽  
pp. 4143-4157 ◽  
Author(s):  
Mike Strauss ◽  
David J. Filman ◽  
David M. Belnap ◽  
Naiqian Cheng ◽  
Roane T. Noel ◽  
...  
Keyword(s):  
Cell Surface ◽  
Capsid Protein ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Three Dimensional ◽  
Cell Surface Receptor ◽  
Membrane Structures ◽  
Lipid Molecule ◽  
Surface Receptor ◽  
Capsid Protein Vp1

ABSTRACTPoliovirus infection is initiated by attachment to a receptor on the cell surface called Pvr or CD155. At physiological temperatures, the receptor catalyzes an irreversible expansion of the virus to form an expanded form of the capsid called the 135S particle. This expansion results in the externalization of the myristoylated capsid protein VP4 and the N-terminal extension of the capsid protein VP1, both of which become inserted into the cell membrane. Structures of the expanded forms of poliovirus and of several related viruses have recently been reported. However, until now, it has been unclear how receptor binding triggers viral expansion at physiological temperature. Here, we report poliovirus in complex with an enzymatically partially deglycosylated form of the 3-domain ectodomain of Pvr at a 4-Å resolution, as determined by cryo-electron microscopy. The interaction of the receptor with the virus in this structure is reminiscent of the interactions of Pvr with its natural ligands. At a low temperature, the receptor induces very few changes in the structure of the virus, with the largest changes occurring within the footprint of the receptor, and in a loop of the internal protein VP4. Changes in the vicinity of the receptor include the displacement of a natural lipid ligand (called “pocket factor”), demonstrating that the loss of this ligand, alone, is not sufficient to induce particle expansion. Finally, analogies with naturally occurring ligand binding in the nectin family suggest which specific structural rearrangements in the virus-receptor complex could help to trigger the irreversible expansion of the capsid.IMPORTANCEThe cell-surface receptor (Pvr) catalyzes a large structural change in the virus that exposes membrane-binding protein chains. We fitted known atomic models of the virus and Pvr into three-dimensional experimental maps of the receptor-virus complex. The molecular interactions we see between poliovirus and its receptor are reminiscent of the nectin family, by involving the burying of otherwise-exposed hydrophobic groups. Importantly, poliovirus expansion is regulated by the binding of a lipid molecule within the viral capsid. We show that receptor binding either causes this molecule to be expelled or requires it, but that its loss is not sufficient to trigger irreversible expansion. Based on our model, we propose testable hypotheses to explain how the viral shell becomes destabilized, leading to RNA uncoating. These findings give us a better understanding of how poliovirus has evolved to exploit a natural process of its host to penetrate the membrane barrier.

Suppression of insulin receptor binding by prolonged enteral or parenteral nutrient infusion in the rat: role of gastric inhibitory polypeptide

1989 ◽  
Vol 67 (9) ◽  
pp. 1105-1109 ◽  
Author(s):  
A. R. Baer ◽  
J. Dupré
Keyword(s):  
Insulin Resistance ◽  
Steady State ◽  
Insulin Receptor ◽  
Receptor Binding ◽  
Plasma Insulin ◽  
Gastric Inhibitory Polypeptide ◽  
Insulin Binding ◽  
Down Regulation ◽  
Insulin Receptor Binding ◽  
Nutrient Infusion

In the rat, prolonged enteral or parenteral alimentation with a high-carbohydrate diet results in hyperinsulinemia, which is substantially greater with the parenteral route. Supplementing the parenteral infusate with porcine gastric inhibitory polypeptide (GIP) to approximate plasma immunoreactive GIP levels achieved with enteral feeding further increases steady-state plasma insulin and glucose concentrations, suggesting insulin resistance. We examined the effects of sustained hyperinsulinemia elicited by continuous nutrient infusion on insulin binding to isolated rat adipocytes and the modification of this response by GIP. Compared with a baseline group, both enterally and parenterally alimented groups showed decreased insulin receptor binding affinity. However, despite substantially different steady-state plasma insulin levels, insulin binding was similar with either infusion route. Factors other than plasma insulin concentration alone therefore contribute to insulin receptor down-regulation during prolonged enteral alimentation. Supplementing the parenteral infusate with exogenous GIP resulted in a further reduction in insulin receptor affinity. Thus, adaptation to continuous nutrient infusion is characterized by insulin receptor down-regulation regardless of the route of nutrient delivery. An additional suppression of insulin receptor binding may in part be responsible for the insulin resistance elicited by prolonged exogenous GIP administration.Key words: gastric inhibitory polypeptide, insulin receptor binding, hyperinsulinemia.

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