Structure−Function Analysis of a Series of Novel GIP Analogues Containing Different Helical Length Linkers

Manhart, Susanne; Hinke, Simon A.; McIntosh, Christopher H.S.; Pederson, Raymond A.; Demuth, Hans‐Ulrich

doi:10.1021/bi026868e

Cited by 30 publications

(18 citation statements)

References 44 publications

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“…We have also found that GIP is 20% helical in 20 mM phosphate buffer, pH 7.0, increasing up to 26% when in 20 mM acetate buffer, pH 4.0. These results are in contrast with studies of Manhart et al (38), in which they reported a helical content of 11% for GIP in 20 mM phosphate buffer, pH 7. Studies in the near-UV (results not shown), carried out for 0.95 mM GIP samples in phosphate buffer (20 mM, pH 7.0) and unbuffered (pH 3.0, uncorrected), show spectra similar to those found for folded proteins (37).…”

Section: Discussioncontrasting

confidence: 99%

NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

Alana

Parker

Gault³

et al. 2006

Journal of Biological Chemistry

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Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that stimulates the secretion of insulin after ingestion of food. GIP also promotes the synthesis of fatty acids in adipose tissue. Therefore, it is not surprising that numerous literature reports have shown that GIP is linked to diabetes and obesity-related diseases. In this study, we present the solution structure of GIP in water determined by NMR spectroscopy. The calculated structure is characterized by the presence of an ␣-helical motif between residues Ser 11 and Gln 29 . The helical conformation of GIP is further supported by CD spectroscopic studies. Six GIP-(1-42)Ala 1-7 analogues were synthesized by replacing individual N-terminal residues with alanine. Alanine scan studies of these N-terminal residues showed that the GIP-(1-42)Ala 6 was the only analogue to show insulin-secreting activity similar to that of the native GIP. However, when compared with glucose, its insulinotropic ability was reduced. For the first time, these NMR and modeling results contribute to the understanding of the structural requirements for the biological activity of GIP. GIP3 (YAEGTFISDY SIAMDKIHQQ DFVNWLLAQK GKKND-WKHNI TQ) is an incretin hormone synthesized and secreted in the gut (1). In addition to its prominent glucose-lowering action, GIP promotes pro-insulin gene expression, proliferation of ␤-cells, and ␤-cell survival (2, 3). GIP also plays a role in fat metabolism stimulating the synthesis of fatty acids (4). Furthermore, a direct relationship between GIP suppression and obesity has also been found (5, 6). However, GIP is rapidly degraded by the enzyme dipeptidyl peptidase IV immediately after secretion (7-9). The resulting fragment, GIP-(3-42), has been shown to be a GIP antagonist in vivo (10). These results have encouraged the development of GIP analogues (11, 12) and dipeptidyl peptidase IV inhibitors (13, 14) as potential new targets for the treatment of type 2 diabetes and obesity-related disorders. We are interested in assessing the importance of the structure of GIP and its analogues in determining their biological activity. In this study, we present the three-dimensional solution structure of native GIP in water, as determined by NMR spectroscopy and molecular modeling. The structure of GIP consists of an ␣-helical motif between residues Ser 11 and Gln 29 , unlike the other major incretin hormone, GLP-1, which mainly adopts a random coil conformation, under physiological conditions (15). Previous solution structural studies carried out in our research laboratory showed that the GIP-(1-30)amide also adopts a random coil conformation when dissolved in water, whereas in a TFE-d 3 /water-mixed solvent, the peptide showed a well defined full-length ␣-helix (16). The results presented here could help in the design of new drugs for diabetes and obesityrelated disorders. , and GIP-(1-42)Ala 7 were sequentially synthesized on an Applied Biosystems 432A automated peptide synthesizer using standard solid-phase Fmoc procedure, starting from an Fmo...

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Section: Discussioncontrasting

confidence: 99%

NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

Alana

Parker

Gault³

et al. 2006

Journal of Biological Chemistry

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“…4, Table 2). In agreement with previous work using other cell jpet.aspetjournals.org lines (Manhart et al, 2003), equilibrium binding of the radioligand to HEK293 cells expressing the recombinant GIP-R was reached within a 7-h incubation period at 4°C (data not shown). Consistent with the marked reduction in agonist potency at the C46S isoform (Fig.…”

Section: Resultssupporting

confidence: 92%

Pharmacological Characterization of Human Incretin Receptor Missense Variants

Fortin¹,

Schroeder²,

Zhu³

et al. 2009

J Pharmacol Exp Ther

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Glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are gut-derived incretin hormones that regulate blood glucose levels. In addition to their widely accepted insulinotropic role, there is evidence that GLP-1 modulates feeding behavior and GIP regulates lipid metabolism, thereby promoting postprandial fat deposition. In this study, we investigated whether naturally occurring polymorphisms in the GLP-1 receptor (GLP-1R) and the GIP receptor (GIP-R) affect the pharmacological properties of these proteins. After transient expression of the receptors in human embryonic kidney 293 cells, basal and ligand-induced cAMP production were assessed by use of luciferase reporter gene assays. Our data reveal that the wild-type GIP-R displays a considerable degree of ligand-independent activity. In comparison, the GIP-R variants C46S, G198C, R316L, and E354Q show a marked decrease in basal signaling that may, at least in part, be explained by reduced cell surface expression. When stimulated with GIP, the C46S and R316L mutants display significantly reduced potency (Ͼ1000 and 25-fold, respectively) compared with wild type. Complementary competition binding assays further demonstrate that the C46S variant fails to bind radio-iodinated GIP, whereas all other GIP-R mutants maintain normal ligand affinity. In contrast to the GIP-R, the wild-type GLP-1R lacks constitutive activity. Furthermore, none of the 10 GLP-1R missense mutations showed an alteration in pharmacological properties versus wild type. The extent to which abnormalities in GIP-R function may lead to physiological changes or affect drug sensitivity in selected populations (e.g., obese, diabetic individuals) remains to be further investigated.The incretin hormones glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are homologous peptides released from intestinal enteroendocrine cells in response to food intake. Both hormones are important modulators of metabolic function. In the pancreas, GLP-1 and GIP potentiate nutrient-stimulated insulin secretion and promote the expansion of pancreatic islet mass via induction of ␤-cell proliferation and survival (Kim et al., 2005;Kim and Egan, 2008).

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“…Several studies have shown that N-terminal GIP residues confer significant affinity toward the full-length receptor and are essential for activation of the GIPR (37)(38)(39)(40). It has also been reported that the biological activity can be influenced by modifying the degree of ␣-helicity of the ligand (41). In the present study, thermodynamic and spectroscopic analysis of the GIPR ECD-GIP 1-42 complex formation provide evidence for structural reorganization during binding (SI Text).…”

Section: Discussionsupporting

confidence: 54%

Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor

Parthier¹,

Kleinschmidt

Neumann³

et al. 2007

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

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Incretins, endogenous polypeptide hormones released in response to food intake, potentiate insulin secretion from pancreatic ␤ cells after oral glucose ingestion (the incretin effect). This response is signaled by the two peptide hormones glucose-dependent insulinotropic polypeptide (GIP) (also known as gastric inhibitory polypeptide) and glucagon-like peptide 1 through binding and activation of their cognate class 2 G protein-coupled receptors (GPCRs). Because the incretin effect is lost or significantly reduced in patients with type 2 diabetes mellitus, glucagon-like peptide 1 and GIP have attracted considerable attention for their potential in antidiabetic therapy. A paucity of structural information precludes a detailed understanding of the processes of hormone binding and receptor activation, hampering efforts to develop novel pharmaceuticals. Here we report the crystal structure of the complex of human GIP receptor extracellular domain (ECD) with its agonist, the incretin GIP 1-42. The hormone binds in an ␣-helical conformation in a surface groove of the ECD largely through hydrophobic interactions. The N-terminal ligand residues would remain free to interact with other parts of the receptor. Thermodynamic data suggest that binding is concomitant with structural organization of the hormone, resulting in a complex mode of receptor-ligand recognition. The presentation of a well structured, ␣-helical ligand by the ECD is expected to be conserved among other hormone receptors of this class.glucose-dependent insulinotropic polypeptide ͉ hormone binding ͉ diabetes mellitus ͉ x-ray

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Structure−Function Analysis of a Series of Novel GIP Analogues Containing Different Helical Length Linkers

Cited by 30 publications

References 44 publications

NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

NMR and Alanine Scan Studies of Glucose-dependent Insulinotropic Polypeptide in Water

Pharmacological Characterization of Human Incretin Receptor Missense Variants

Crystal structure of the incretin-bound extracellular domain of a G protein-coupled receptor

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