Peptides Identify the Critical Hotspots Involved in the Biological Activation of the Insulin Receptor

Pillutla, Renuka; Hsiao, Ku-chuan; Beasley, James R.; Brandt, Jakob; Østergaard, Søren; Hansen, Per; Spetzler, Jane C.; Danielsen, Gillian M.; Andersen, Asser S.; Brissette, Renée; Lennick, Michael; Fletcher, Paul W.; Blume, Arthur J.; Schäffer, Lauge; Goldstein, Neil I.

doi:10.1074/jbc.m202119200

Cited by 75 publications

(87 citation statements)

References 21 publications

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“…3,4 Furthermore we have earlier published on other peptides that bind to the insulin receptor with regard to affinity for the receptor and ability to stimulate lipogenesis in mouse adipocytes (8,9), but intracellular signaling was not investigated.…”

Section: Discussionmentioning

confidence: 99%

“…The other major signaling pathway activated by IRS proteins is the phosphatidylinositol 3-kinase (PI 3-kinase) pathway that includes activation of PKB and leads to activation of glycogen synthase and other enzymes/proteins necessary for the acute metabolic effects of insulin (3). Synthetic peptides that bind to the insulin receptor have been identified by investigation of phage display libraries (ranging in size from 20 -40 amino acids) (8). The identified peptides bound to three spots on the insulin receptor (sites 1, 2, and 3).…”

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confidence: 99%

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Activation of the Insulin Receptor by Insulin and a Synthetic Peptide Leads to Divergent Metabolic and Mitogenic Signaling and Responses

Jensen¹,

Hansen

Meyts³

et al. 2007

Journal of Biological Chemistry

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Recently, single chain peptides have been designed that target the insulin receptor and mimic insulin action. The aim of this study is to explore if activation of the insulin receptor with such an optimized peptide (S597) leads to the same activation of signaling pathways and biological endpoints i.e. stimulation of glycogen synthesis and cell proliferation as stimulation with insulin. We find that surface activation of the insulin receptor A-isoform with S597 leads to activation of protein kinase B (PKB) and glycogen synthesis comparable to activation by insulin, even though the level of insulin receptor phosphorylation is lower. In contrast, both Src homology 2/␣ collagen-related (Shc) and extracellular signal-regulated kinase (ERK) 2 activation are virtually absent upon stimulation with S597. Cell proliferation is only stimulated slightly by S597, suggesting that it depends on signals from Shc and ERK. The differences in signaling response could explain both the earlier reported differences in gene expression, and the reported differences in cell proliferation and glycogen synthesis induced by insulin and S597. In conclusion, despite binding equipotency, insulin, and S597 initiate different signaling and biological responses through the same insulin receptor isoform. We show for the first time that it is possible to design insulin receptor ligand mimetics with metabolic equipotency but low mitogenicity.The insulin receptor is a disulfide linked heterodimeric protein consisting of two 135-kDa extracellular ␣-subunits and two 95-kDa transmembrane spanning ␤-subunits, containing the intracellular tyrosine kinase domain that is activated upon ligand binding (1, 2). Binding of insulin to the receptor results in a wide range of metabolic and mitogenic responses initially mediated by phosphorylation of tyrosines on several intracellular protein substrates, including insulin receptor substrates (IRS) 2 1 and 2 and Shc (3). IRS1 and IRS2 are the major insulin receptor substrates leading to glucose homeostasis and have distinct and overlapping roles in diverse organs. Furthermore alterations in both IRS1 and IRS2 have been shown to be strongly involved in the development of diabetes mellitus (4, 5). The finding of specific functions of IRS1 and IRS2 in the murine pre-muscle cell line (L6 cells) are summarized by Thirone et al.(6) but the relative contributions of IRS1 and IRS2 to insulin signaling and to the development of insulin resistance is not yet conclusive. The majority of the published literature in this field suggests that IRS1 is the major substrate leading to stimulation of glucose transport in muscle and adipose tissues, whereas in liver IRS1 and IRS2 have complementary roles in insulin signaling and metabolism. In contrast Shc does not appear to be directly involved in metabolic signaling of insulin but plays a critical role in insulin-induced mitogenesis (6, 7). Both IRS and Shc initiate the mitogenactivated pathway kinase/extracellular-regulated kinase (MAPK/ERK) pathway, including activation of ERK1/2 i...

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Activation of the Insulin Receptor by Insulin and a Synthetic Peptide Leads to Divergent Metabolic and Mitogenic Signaling and Responses

Jensen¹,

Hansen

Meyts³

et al. 2007

Journal of Biological Chemistry

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“…In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extracellular region and that had affinities in the high nanomolar to low micromolar range (1). Based on competition studies, the putative epitopes of these peptides were grouped into three non-overlapping sites, termed Sites 1, 2, and 3.…”

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confidence: 99%

“…The highest affinity Site 1 peptides contained the motif FYXWF, whereas Site 2 peptides were characterized by either a short or long disulfide loop (1).…”

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Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Lawrence

Margetts

Menting

et al. 2016

Journal of Biological Chemistry

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Sets of synthetic peptides that interact with the insulin receptor ectodomain have been discovered by phage display and reported in the literature. These peptides were grouped into three classes termed Site 1, Site 2, and Site 3 based on their mutual competition of binding to the receptor. Further refinement has yielded, in particular, a 36-residue Site 2-Site 1 fusion peptide, S519, that binds the insulin receptor with subnanomolar affinity and exhibits agonist activity in both lipogenesis and glucose uptake assays. Here, we report three-dimensional crystallographic detail of the interaction of the C-terminal, 16-residue Site 1 component (S519C16) of S519 with the first leucinerich repeat domain (L1) of the insulin receptor. Our structure shows that S519C16 binds to the same site on the L1 surface as that occupied by a critical component of the primary binding site, namely the helical C-terminal segment of the insulin receptor ␣-chain (termed ␣CT). In particular, the two phenylalanine residues within the FYXWF motif of S519C16 are seen to engage the insulin receptor L1 domain surface in a fashion almost identical to the respective ␣CT residues Phe 701 and Phe 705 . The structure provides a platform for the further development of peptidic and/or small molecule agents directed toward the insulin receptor and/or the type 1 insulin-like growth factor receptor.In 2002, sets of synthetic peptides were identified that could compete for insulin binding to the human insulin receptor extracellular region and that had affinities in the high nanomolar to low micromolar range (1). Based on competition studies, the putative epitopes of these peptides were grouped into three non-overlapping sites, termed Sites 1, 2, and 3.3 Some Site 1 peptides were able to activate the receptor tyrosine kinase and act as agonists in an insulin-dependent fat cell assay, whereas Site 2 and Site 3 peptides were found to act as antagonists both in phosphorylation and fat cell assays. The highest affinity Site 1 peptides contained the motif FYXWF, whereas Site 2 peptides were characterized by either a short or long disulfide loop (1).Optimized versions of the Site 1 and Site 2 peptides were subsequently described (2), in particular those that were linked in tandem in various ways. Several of these linked peptides were found to function as either potent antagonists (e.g. peptide S661, a Site 1-Site 2 combination) or as potent agonists (e.g. peptide S597, a Site 2-Site 1 combination), depending on the order in which the individual Site 1 and Site 2 peptides were linked (2, 3). In earlier studies (4, 5), we provided isothermal titration calorimetry (ITC) 4 data that suggested how Site 1 peptides might bind to the insulin receptor. The insulin receptor (IR) itself is a disulfide-linked (␣␤) 2 homodimer; each ␣␤ monomer comprises, from its N terminus, two homologous leucine-rich repeat domains (L1 and L2) separated by a cysteine-rich region (CR) comprising eight disulfide-linked modules (6). These domains are followed by three fibronectin type III d...

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“…Considerable opportunity exists for new chemistry in this area (14). Synthetic peptides that target "hot spots" on protein surfaces are a logical entry to drug-discovery programs (15)(16)(17)(18)(19). However, native peptides generally have poor pharmacological properties (20).…”

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Template-constrained macrocyclic peptides prepared from native, unprotected precursors

Lawson

Rose

Harran

2013

Proc. Natl. Acad. Sci. U.S.A.

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Peptide-protein interactions are important mediators of cellularsignaling events. Consensus binding motifs (also known as short linear motifs) within these contacts underpin molecular recognition, yet have poor pharmacological properties as discrete species. Here, we present methods to transform intact peptides into stable, templated macrocycles. Two simple steps install the template. The key reaction is a palladium-catalyzed macrocyclization. The catalysis has broad scope and efficiently forms large rings by engaging native peptide functionality including phenols, imidazoles, amines, and carboxylic acids without the necessity of protecting groups. The tunable reactivity of the template gives the process special utility. Defined changes in reaction conditions markedly alter chemoselectivity. In all cases examined, cyclization occurs rapidly and in high yield at room temperature, regardless of peptide composition or chain length. We show that conformational restraints imparted by the template stabilize secondary structure and enhance proteolytic stability in vitro. Palladium-catalyzed internal cinnamylation is a strong complement to existing methods for peptide modification. S ynthetic peptides and peptidomimetics play wide-ranging roles in pharmacology and drug discovery. Interest in these substances continues to grow, particularly as medicinal chemistry pushes further into control of cellular-signaling events mediated by protein-protein interactions (PPIs) (1-3). The subset of socalled "druggable" PPIs includes those mediated by consensus peptides, where binding determinants are localized within defined motifs (4-8). Experimental data as well as computational/ bioinformatic efforts (9-11) to identify "short linear motifs" within signaling proteins suggest that the number of druggable PPIs has been underestimated (12, 13). Considerable opportunity exists for new chemistry in this area (14). Synthetic peptides that target "hot spots" on protein surfaces are a logical entry to drug-discovery programs (15-19). However, native peptides generally have poor pharmacological properties (20). Modifications that offset those limitations while stably recapitulating proteinbinding conformations are of considerable interest (14,(21)(22)(23).Ring-forming reactions are prominent among alterations found to improve the stability and performance of peptides (24-27). They find broad utility in synthesis and, increasingly, in combination with phage, ribosome, and mRNA display technologies (28-32). Relative to their acyclic counterparts, cyclic peptides have more defined conformations and are less prone to aggregate (33). Head-to-tail lactamization is the most common method to synthesize cyclic peptides (34-36). Internal disulfide bonding is also used (37, 38), as are newer techniques such as ring-closing olefin metathesis (39) and catalyzed cycloaddition of azides to alkynes (40-42). These procedures rely on judicious use of protecting groups and/or tailored amino acid residues (Fig. 1A) (43, 44). Careful attention must be paid to...

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Peptides Identify the Critical Hotspots Involved in the Biological Activation of the Insulin Receptor

Cited by 75 publications

References 21 publications

Activation of the Insulin Receptor by Insulin and a Synthetic Peptide Leads to Divergent Metabolic and Mitogenic Signaling and Responses

Activation of the Insulin Receptor by Insulin and a Synthetic Peptide Leads to Divergent Metabolic and Mitogenic Signaling and Responses

Insulin Mimetic Peptide Disrupts the Primary Binding Site of the Insulin Receptor

Template-constrained macrocyclic peptides prepared from native, unprotected precursors

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