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Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
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2010 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 163, no 1-3, 120-129 p.Article in journal (Refereed) Published
Abstract [en]

Neuropeptide Y (NPY) and peptide YY (PYY) share similar to 70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share similar to 30% sequence identity Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i e Tyr(2 64), Val(6 58) and Tyr(7 31) Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3-36) and NPY(13-36) as well as the non-peptide antagonist BIIE0246 Results from mutants where Tyr(2 64) has been substituted by Ala suggest that Tyr(2 64) is involved in the interaction with all investigated ligands whereas position Tyr(7 31) seems to be more important for interaction with the truncated peptide PYY(3-36) than with intact NPY Surprisingly, substitution of Tyr(7 31) with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY The third position. Val(6 58), did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr(2 64) and Tyr(7 31) in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1 This suggests that the receptor-ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.

Place, publisher, year, edition, pages
2010. Vol. 163, no 1-3, 120-129 p.
Keyword [en]
Site-directed mutagenesis, G-protein coupled receptor, Three dimensional model, Evolution
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-135756DOI: 10.1016/j.regpep.2010.04.011ISI: 000280050000018OAI: oai:DiVA.org:uu-135756DiVA: diva2:375574
Note

Manuscript title: Investigation of receptor-ligand interactions of the human neuropeptide Y receptor Y2 by site-directed mutagenesis: comparison with the structurally divergent Y1 subtype

Available from: 2010-12-08 Created: 2010-12-08 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Functional Studies of the Neuropeptide Y System: Receptor-Ligand Interaction and Regulation of Food Intake
Open this publication in new window or tab >>Functional Studies of the Neuropeptide Y System: Receptor-Ligand Interaction and Regulation of Food Intake
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

Place, publisher, year, edition, pages
Uppsala: Universitetsbiblioteket, 2009. 67 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 415
Keyword
neuropeptide Y, G-protein coupled receptor (GPCR), site-directed mutagenesis, pancreatic polypeptide, food intake, dog
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-9533 (URN)978-91-554-7393-8 (ISBN)
Public defence
2009-02-20, B21, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2009-01-30 Created: 2009-01-30 Last updated: 2013-01-07Bibliographically approved
2. Studies of the Neuropeptide Y Receptor Y2 in Human and Zebrafish
Open this publication in new window or tab >>Studies of the Neuropeptide Y Receptor Y2 in Human and Zebrafish
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs.

The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes.

This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity.

A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246.

In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 55 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 687
Keyword
Y2-receptor, NPY, PYY, G-protein-coupled receptor, mutagenesis, receptor binding, zebrafish, evolution
National Category
Pharmacology and Toxicology
Research subject
Pharmacology
Identifiers
urn:nbn:se:uu:diva-156635 (URN)978-91-554-8117-9 (ISBN)
Public defence
2011-09-21, B:41, Uppsala Biomedical Center (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2011-08-31 Created: 2011-08-04 Last updated: 2011-09-08Bibliographically approved
3. Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes
Open this publication in new window or tab >>Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods.

We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target.  In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance.

Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 61 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1105
Keyword
computer simulations, molecular dynamics, ligand binding, free energy perturbation, linear interaction energy, binding free energy, homology modelling, structure prediction, alanine scanning, site-directed mutagenesis, hERG, GPCR, neuropeptide Y, HIV-1 reverse transcriptase, integron integrase
National Category
Theoretical Chemistry Structural Biology Biochemistry and Molecular Biology
Research subject
Molecular Biotechnology
Identifiers
urn:nbn:se:uu:diva-212103 (URN)978-91-554-8823-9 (ISBN)
Public defence
2014-01-31, B42, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-01-10 Created: 2013-12-05 Last updated: 2014-01-24

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Gutierrez-de-Teran, Hugo

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