Mapping of an NH--terminal Ligand Binding Site of the Insulin Receptor by Alanine Scanning Mutagenesis

Williams, Paul F.; Mynarcik, Dennis C.; Yu, Gui Qin; Whittaker, Jonathan

doi:10.1074/jbc.270.7.3012

Cited by 93 publications

(114 citation statements)

References 27 publications

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“…These results suggest that the Ala-37-Thr change may affect ligand binding but to a lesser extent than the mutations that are within the IGF binding pocket. This region also coincides with residues 34-44 of the IR that are shown to be important for insulin binding (25). Alignment of IGFIR with the IR shows that Ala-37 is just one residue away from IR Phe-39, a residue important for insulin ligand specificity (26).…”

Section: Discussionmentioning

confidence: 99%

Functionally significant insulin-like growth factor I receptor mutations in centenarians

Suh

Atzmon

Cho

et al. 2008

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

636

474

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Rather than being a passive, haphazard process of wear and tear, lifespan can be modulated actively by components of the insulin/ insulin-like growth factor I (IGFI) pathway in laboratory animals. Complete or partial loss-of-function mutations in genes encoding components of the insulin/IGFI pathway result in extension of life span in yeasts, worms, flies, and mice. This remarkable conservation throughout evolution suggests that altered signaling in this pathway may also influence human lifespan. On the other hand, evolutionary tradeoffs predict that the laboratory findings may not be relevant to human populations, because of the high fitness cost during early life. Here, we studied the biochemical, phenotypic, and genetic variations in a cohort of Ashkenazi Jewish centenarians, their offspring, and offspring-matched controls and demonstrated a gender-specific increase in serum IGFI associated with a smaller stature in female offspring of centenarians. Sequence analysis of the IGF1 and IGF1 receptor (IGF1R) genes of female centenarians showed overrepresentation of heterozygous mutations in the IGF1R gene among centenarians relative to controls that are associated with high serum IGFI levels and reduced activity of the IGFIR as measured in transformed lymphocytes. Thus, genetic alterations in the human IGF1R that result in altered IGF signaling pathway confer an increase in susceptibility to human longevity, suggesting a role of this pathway in modulation of human lifespan.IGF1 receptor ͉ human longevity ͉ genetic variation

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Section: Discussionmentioning

confidence: 99%

Functionally significant insulin-like growth factor I receptor mutations in centenarians

Suh

Atzmon

Cho

et al. 2008

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

636

474

View full text Add to dashboard Cite

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“…Insulin Binding-We have previously characterized the functional epitopes of the secreted recombinant insulin receptor extracellular domain by alanine-scanning mutagenesis (23)(24)(25). To determine significance of these epitopes for insulin binding in a physiological context, we subcloned the alanine mutations used to characterize them into the full-length B isoform receptor cDNA-tagged at its 3Ј end with the coding sequence for a triple repeat of the AU5 epitope tag.…”

Section: Resultsmentioning

confidence: 99%

“…Construction of Receptor Mutants-Alanine mutations of the cDNA encoding the recombinant secreted receptor extracellular domain have been described previously (23)(24)(25). These were used to generate fulllength epitope-tagged mutants of the full-length receptor by standard subcloning techniques.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Whittaker

Whittaker²

2005

Journal of Biological Chemistry

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Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the ␣ subunit (amino acids [705][706][707][708][709][710][711][712][713][714][715] , and Phe 89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC 50 commensurate with their effect on the affinity of the receptor for insulin.The initiating event in the insulin-signaling cascade is the binding of insulin to a specific plasma membrane receptor. This interaction has been studied extensively and was found to be extremely complex (see Ref. 1 for review). Scatchard plots (2) of equilibrium binding data are concave and curvilinear, suggesting heterogeneity of ligand binding sites, negatively cooperative site-site interactions, or a combination of both (1). These properties and high affinity interactions with insulin are dependent on the dimeric structure of the receptor; insulin binds non-cooperatively to a single population of binding sites in the monomeric receptor (3-5). The stoichiometry of binding to the native receptor appears to be one insulin molecule to one receptor dimer (6). The secreted recombinant extracellular domain of the receptor exhibits properties similar to those of the receptor monomer and has a stoichiometry of two insulin molecules to one receptor dimer (6, 7).A number of hypothetical models of insulin-receptor interactions have been proposed to explain these findings (7-9). The model that best explains the experimental findings is that of De Meyts (1). This proposes that insulin has two topographically distinct receptor binding sites and that the receptor has two topographically distinct insulin binding sites/monomer. In this model, insulin binds asymmetrically to the insulin dimer, with each of its binding sites contacting its cognate receptor site on a separate monomer. Although the detailed structural basis of this model has yet to be elucidated, the structure-function relationships of the insulin molecule and the receptor have been studied extensively and provide support for the essentials of the model.Insulin (1, 10). However, hagfish insulin, in which these residues and the tertiary structure of the molecule are conserved (11), displays anomalous receptor binding behavior (11,12), suggesting that other residues outside this region might also be involved in receptor interactions. This is supported by the finding that two recombinant insulin analogues with substitutions in residues located on the opposite side of the molecule, Leu A13 to Ser and Leu B17 to Gln, exhibited similar receptor binding behavior to hagfish insulin (7,8).The structure and structure-function relationships of the insulin receptor are not as well documented. It is a dimeric transmembrane protein (see Ref. 1 for review). Each monomer consists of disulfide-linked ␣ and ␤ subunits. The ␣ subunits are wholly extracellular and c...

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“…Conservation of both Phe B24 and its cognate binding pocket among vertebrate sequences is striking. Ala scanning mutagenesis of the pocket-lining residues Asn15, Leu37, Phe39, and Phe714 impairs high-affinity hormone binding to the holoreceptor (33) or ectodomain (34).…”

Section: Discussionmentioning

confidence: 99%

Protective hinge in insulin opens to enable its receptor engagement

Menting

Yang

Chan

et al. 2014

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

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147

257

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Insulin provides a classical model of a globular protein, yet how the hormone changes conformation to engage its receptor has long been enigmatic. Interest has focused on the C-terminal Bchain segment, critical for protective self-assembly in β cells and receptor binding at target tissues. Insight may be obtained from truncated "microreceptors" that reconstitute the primary hormone-binding site (α-subunit domains L1 and αCT). We demonstrate that, on microreceptor binding, this segment undergoes concerted hinge-like rotation at its B20-B23 β-turn, coupling reorientation of Phe B24 to a 60°rotation of the B25-B28 β-strand away from the hormone core to lie antiparallel to the receptor's L1-β 2 sheet. Opening of this hinge enables conserved nonpolar side chains (Ile A2 , Val A3 , Val B12 , Phe B24 , and Phe B25 ) to engage the receptor. Restraining the hinge by nonstandard mutagenesis preserves native folding but blocks receptor binding, whereas its engineered opening maintains activity at the price of protein instability and nonnative aggregation. Our findings rationalize properties of clinical mutations in the insulin family and provide a previously unidentified foundation for designing therapeutic analogs. We envisage that a switch between free and receptorbound conformations of insulin evolved as a solution to conflicting structural determinants of biosynthesis and function.diabetes mellitus | signal transduction | receptor tyrosine kinase | metabolism | protein structure H ow insulin engages the insulin receptor has inspired speculation ever since the structure of the free hormone was determined by Hodgkin and colleagues in 1969 (1, 2). Over the ensuing decades, anomalies encountered in studies of analogs have suggested that the hormone undergoes a conformational change on receptor binding: in particular, that the C-terminal β-strand of the B chain (residues B24-B30) releases from the helical core to expose otherwise-buried nonpolar surfaces (the detachment model) (3-6). Interest in the B-chain β-strand was further motivated by the discovery of clinical mutations within it associated with diabetes mellitus (DM) (7). Analysis of residuespecific photo-cross-linking provided evidence that both the detached strand and underlying nonpolar surfaces engage the receptor (8).The relevant structural biology is as follows. The insulin receptor is a disulfide-linked (αβ) 2 receptor tyrosine kinase (Fig. 1A), the extracellular α-subunits together binding a single insulin molecule with high affinity (9). Involvement of the two α-subunits is asymmetric: the primary insulin-binding site (site 1*) comprises the central β-sheet (L1-β 2 ) of the first leucine-rich repeat domain (L1) of one α-subunit and the partially helical Cterminal segment (αCT) of the other α-subunit (Fig. 1A) (10). Such binding initiates conformational changes leading to transphosphorylation of the β-subunits' intracellular tyrosine kinase (TK) domains. Structures of wild-type (WT) insulin (or analogs) bound to extracellular receptor fragments were recently...

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Mapping of an NH--terminal Ligand Binding Site of the Insulin Receptor by Alanine Scanning Mutagenesis

Cited by 93 publications

References 27 publications

Functionally significant insulin-like growth factor I receptor mutations in centenarians

Functionally significant insulin-like growth factor I receptor mutations in centenarians

Characterization of the Functional Insulin Binding Epitopes of the Full-length Insulin Receptor

Protective hinge in insulin opens to enable its receptor engagement

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