Insertion of a synthetic switch into insulin provides metabolite-dependent regulation of hormone–receptor activation

Insulin-signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to structural reorganization. To test the functional coupling between insulin’s “hinge opening” and receptor activation, we inserted an artificial ligand-dependent switch into the insulin molecule. Ligand-binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin-signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the hormone. Similarly, metabolite-regulated signaling was not observed in control studies of 1) an unmodified insulin analog or 2) an analog containing a diol sensor without internal tethering. Although secondary structure (as probed by circular dichroism) was unaffected by ligand-binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and allosteric holoreceptor signaling. In addition to this foundational finding, our results provide proof of principle for design of a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog to mitigate hypoglycemic risk in diabetes therapy.

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Insertion of a Synthetic Switch Into Insulin Provides Metabolite-Dependent Regulation of Hormone-Receptor Activation

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.899 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A440-A440

Author(s):

Yen-Shan Chen ◽

Jeremy Gleaton ◽

Yanwu Yang ◽

Balamurugan Dhayalan ◽

Nelson B Phillips ◽

...

Keyword(s):

Ligand Binding ◽

Insulin Signaling ◽

Large Scale ◽

Glucose Sensor ◽

Phenylboronic Acid ◽

Receptor Activation ◽

Insulin Analog ◽

Structural Reorganization ◽

Nmr Studies ◽

Downstream Signaling

Abstract Insulin signaling requires conformational change: whereas the free hormone and its receptor each adopt autoinhibited conformations, their binding leads to large-scale structural reorganization. To test the coupling between insulin’s “opening” and receptor activation, we inserted an artificial ligand-dependent switch into insulin. Ligand binding disrupts an internal tether designed to stabilize the hormone’s native closed and inactive conformation, thereby enabling productive receptor engagement. This scheme exploited a diol sensor (meta-fluoro-phenylboronic acid at GlyA1) and internal diol (3,4-dihydroxybenzoate at LysB28). The sensor recognizes monosaccharides (fructose > glucose). Studies of insulin signaling in human hepatoma-derived cells (HepG2) demonstrated fructose-dependent receptor autophosphorylation leading to appropriate downstream signaling events, including a specific kinase cascade and metabolic gene regulation (gluconeogenesis and lipogenesis). Addition of glucose (an isomeric ligand with negligible sensor affinity) did not activate the receptor. Similarly, metabolite-regulated signaling was not observed in control studies of (i) an unmodified insulin analog or (ii) an analog containing a diol sensor in the absence of internal tethering. Although as expected CD-detected secondary structure was unaffected by ligand binding, heteronuclear NMR studies revealed subtle local and nonlocal monosaccharide-dependent changes in structure. Insertion of a synthetic switch into insulin has thus demonstrated coupling between hinge-opening and holoreceptor signaling. In addition to this basic finding, our results provide proof of principle for a mechanism-based metabolite-responsive insulin. In particular, replacement of the present fructose sensor by an analogous glucose sensor may enable translational development of a “smart” insulin analog designed to mitigate risk of hypoglycemia in the treatment of diabetes mellitus.

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On the TRAIL of Better Therapies: Understanding TNFRSF Structure-Function

Cells ◽

10.3390/cells9030764 ◽

2020 ◽

Vol 9 (3) ◽

pp. 764 ◽

Cited By ~ 2

Author(s):

Éva S. Vanamee ◽

Denise L. Faustman

Keyword(s):

Cell Surface ◽

Ligand Binding ◽

Structure Function ◽

Apoptotic Cell ◽

Hexagonal Lattice ◽

Receptor Activation ◽

Tnf Receptor ◽

Downstream Signaling ◽

Site Cluster ◽

The Right

Tumor necrosis factor (TNF) superfamily ligands show diverse biological functions, such as the induction of apoptotic cell death or cell survival and proliferation, making them excellent therapeutic targets for cancer and autoimmunity. We review the latest literature on TNF receptor superfamily signaling with a focus on structure-function. Using combinatorics, we argue that receptors that cluster on the cell surface and are activated by membrane-bound ligands need to arrange in a highly ordered manner, as the probability of random ligand and receptor arrangements matching up for receptor activation is very low. A growing body of evidence indicates that antiparallel receptor dimers that sequester the ligand binding site cluster on the cell surface, forming a hexagonal lattice. Upon ligand binding, this arrangement puts the activated receptors at the right distance to accommodate the downstream signaling partners. The data also suggest that the same geometry is utilized regardless of receptor type. The unified model provides important clues about TNF receptor signaling and should aid the design of better therapies for cancer and various immune mediated diseases.

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Variants of Insulin-Signaling Inhibitor Genes in Type 2 Diabetes and Related Metabolic Abnormalities

International Journal of Genomics ◽

10.1155/2013/376454 ◽

2013 ◽

Vol 2013 ◽

pp. 1-13 ◽

Cited By ~ 9

Author(s):

Carlo de Lorenzo ◽

Annalisa Greco ◽

Teresa Vanessa Fiorentino ◽

Gaia Chiara Mannino ◽

Marta Letizia Hribal

Keyword(s):

Insulin Resistance ◽

Type 2 Diabetes ◽

Insulin Signaling ◽

Metabolic Diseases ◽

Receptor Activation ◽

Serine Threonine Kinase ◽

Downstream Signaling ◽

Genes Encoding ◽

Inhibitor Genes

Insulin resistance has a central role in the pathogenesis of several metabolic diseases, including type 2 diabetes, obesity, glucose intolerance, metabolic syndrome, atherosclerosis, and cardiovascular diseases. Insulin resistance and related traits are likely to be caused by abnormalities in the genes encoding for proteins involved in the composite network of insulin-signaling; in this review we have focused our attention on genetic variants of insulin-signaling inhibitor molecules. These proteins interfere with different steps in insulin-signaling: ENPP1/PC-1 and the phosphatases PTP1B and PTPRF/LAR inhibit the insulin receptor activation; INPPL1/SHIP-2 hydrolyzes PI3-kinase products, hampering the phosphoinositide-mediated downstream signaling; and TRIB3 binds the serine-threonine kinase Akt, reducing its phosphorylation levels. While several variants have been described over the years for all these genes, solid evidence of an association with type 2 diabetes and related diseases seems to exist only for rs1044498 of theENPP1gene and for rs2295490 of theTRIB3gene. However, overall the data recapitulated in this Review article may supply useful elements to interpret the results of novel, more technically advanced genetic studies; indeed it is becoming increasingly evident that genetic information on metabolic diseases should be interpreted taking into account the complex biological pathways underlying their pathogenesis.

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Residue 6.43 defines receptor function in class F GPCRs

Nature Communications ◽

10.1038/s41467-021-24004-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Ainoleena Turku ◽

Hannes Schihada ◽

Pawel Kozielewicz ◽

Carl-Fredrik Bowin ◽

Gunnar Schulte

Keyword(s):

Ligand Binding ◽

G Protein ◽

Conformational Changes ◽

Mutational Analysis ◽

Receptor Activation ◽

Receptor Function ◽

Protein Activation ◽

Downstream Signaling ◽

Activation Mechanisms ◽

Class F

AbstractThe class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO.

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Faculty Opinions recommendation of Quantitative NMR studies of high molecular weight proteins: application to domain orientation and ligand binding in the 723 residue enzyme malate synthase G.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1013349.190158 ◽

2003 ◽

Author(s):

H Jane Dyson

Keyword(s):

Molecular Weight ◽

Ligand Binding ◽

High Molecular Weight ◽

Malate Synthase ◽

Nmr Studies ◽

Quantitative Nmr ◽

Domain Orientation ◽

Malate Synthase G ◽

High Molecular Weight Proteins

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Functional role in ligand binding and receptor activation of an asparagine residue present in the sixth transmembrane domain of all muscarinic acetylcholine receptors.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)32248-2 ◽

1994 ◽

Vol 269 (29) ◽

pp. 18870-18876

Author(s):

K. Blüml ◽

E. Mutschler ◽

J. Wess

Keyword(s):

Ligand Binding ◽

Acetylcholine Receptors ◽

Functional Role ◽

Transmembrane Domain ◽

Receptor Activation ◽

Muscarinic Acetylcholine Receptors ◽

Muscarinic Acetylcholine ◽

Asparagine Residue

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Biosensors Monitor Ligand-Selective Effects at Kappa Opioid Receptors

10.1007/164_2020_427 ◽

2021 ◽

Author(s):

Lucie Oberhauser ◽

Miriam Stoeber

Keyword(s):

Subcellular Location ◽

Receptor Activation ◽

Cellular Level ◽

Two Dimensions ◽

Kappa Opioid Receptor ◽

Kappa Opioid ◽

Downstream Signaling ◽

Regulator Protein ◽

Cellular Context ◽

Selective Effects

AbstractThe kappa opioid receptor (KOR) has emerged as a promising therapeutic target for pain and itch treatment. There is growing interest in biased agonists that preferentially activate select signaling pathways downstream of KOR activation on the cellular level due to their therapeutic promise in retaining the analgesic and antipruritic effects and eliminating the sedative and dysphoric effects of KOR signaling on the physiological level. The concept of ligand-selective signaling includes that biased ligands promote KOR to selectively recruit one transducer or regulator protein over another, introducing bias into the signaling cascade at the very receptor-proximal level. Measuring agonist effects directly at the receptor has remained challenging and previous studies have focused on inferring agonist-selective KOR engagement with G protein relative to β-arrestin based on downstream signaling readouts. Here we discuss novel strategies to directly assess ligand-selective effects on receptor activation using KOR-interacting biosensors. The conformation-specific cytoplasmic biosensors are disconnected from the endogenous signaling machinery and provide a direct receptor-proxy readout of ligand effects in living cells. Receptor–biosensor interaction is ligand concentration dependent and can be used to determine relative ligand potency and efficacy. In addition, the biosensors reveal the existence of two dimensions of agonist bias in the cellular context: Firstly, agonists can selectively produce discrete protein-engaged KOR states and secondly, agonists can differ in the precise subcellular location at which they activate KOR. We discuss the value and the limitations of using orthogonal receptor-interacting biosensors in the quest to understand functional selectivity amongst KOR agonists in the cellular context.

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N‐glycosylation of the human neuropeptide QRFP receptor QRFPR is essential for ligand binding and receptor activation

Journal of Neurochemistry ◽

10.1111/jnc.15337 ◽

2021 ◽

Author(s):

Weiwei Wang ◽

Yanan Tian ◽

Xiaoliu Shi ◽

Qiang Ma ◽

Yue Xu ◽

...

Keyword(s):

Ligand Binding ◽

Receptor Activation

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Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

Molecular Endocrinology ◽

10.1210/me.2011-1160 ◽

2011 ◽

Vol 25 (10) ◽

pp. 1804-1818 ◽

Cited By ~ 37

Author(s):

K. Coopman ◽

R. Wallis ◽

G. Robb ◽

A. J. H. Brown ◽

G. F. Wilkinson ◽

...

Keyword(s):

Ligand Binding ◽

Structural Model ◽

Transmembrane Domain ◽

Transmembrane Helix ◽

Receptor Activation ◽

Agonist Affinity ◽

Camp Generation ◽

N Terminus ◽

Glucagon Like Peptide ◽

Glucagon Like Peptide 1

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.

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