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Studies of the Neuropeptide Y Receptor Y2 in Human and Zebrafish
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
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 [en]
Y2-receptor, NPY, PYY, G-protein-coupled receptor, mutagenesis, receptor binding, zebrafish, evolution
National Category
Pharmacology and Toxicology
Research subject
Pharmacology
Identifiers
URN: urn:nbn:se:uu:diva-156635ISBN: 978-91-554-8117-9 (print)OAI: oai:DiVA.org:uu-156635DiVA: diva2:432641
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
List of papers
1. Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions
Open this publication in new window or tab >>Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions
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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.

Keyword
Site-directed mutagenesis, G-protein coupled receptor, Three dimensional model, Evolution
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-135756 (URN)10.1016/j.regpep.2010.04.011 (DOI)000280050000018 ()
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
2. Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes
Open this publication in new window or tab >>Identification of positions in the human neuropeptide Y/peptide YY receptor Y2 that contribute to pharmacological differences between receptor subtypes
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2011 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 45, no 4, 293-300 p.Article in journal (Refereed) Published
Abstract [en]

The members of the neuropeptide Y (NPY) family are key players in food-intake regulation. In humans this family consists of NPY, peptide YY (PYY) and pancreatic polypeptide (PP) which interact with distinct preference for the four receptors showing very low sequence identity, i.e. Y1, Y2, Y4 and Y5. The binding of similar peptides to these divergent receptors makes them highly interesting for mutagenesis studies. We present here a site-directed mutagenesis study of four amino acid positions in the human Y2 receptor. T(3.40) was selected based on sequence alignments both between subtypes and between species and G(2.68), L(4.60) and Q(6.55) also on previous binding studies of the corresponding positions in the Y1 receptor. The mutated receptors were characterized pharmacologically with the peptide agonists NPY, PYY, PYY(3-36), NPY(13-36) and the non-peptide antagonist BIIE0246. Interestingly, the affinity of NPY and PYY(3-36) increased for the mutants T(3.40)I and Q(6.55)A. Increased affinity was also observed for PYY to Q(6.55)A. PYY(3-36) displayed decreased affinity for G(2.68)N and L(4.60)A whereas binding of NPY(13-36) was unaffected by all mutations. The antagonist BIIE0246 showed decreased affinity for T(3.40)I, L(4.60)A and Q(6.55)A. Although all positions investigated were found important for interaction with at least one of the tested ligands the corresponding positions in hY1 seem to be of greater importance for ligand binding. Furthermore these data indicate that binding of the agonists and the antagonist differs in their points of interaction. The increase in the binding affinity observed may reflect an indirect effect caused by a conformational change of the receptor. These findings will help to improve the structural models of the human NPY receptors.

Keyword
Y2 receptor, Y1 receptor, Binding experiment, Neuropeptide Y, Peptide YY, BIIE0246, Appetite regulation
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-156633 (URN)10.1016/j.npep.2011.05.006 (DOI)000295349600006 ()21696823 (PubMedID)
Available from: 2011-08-04 Created: 2011-08-04 Last updated: 2017-12-08Bibliographically approved
3. Mutagenesis and Computational Modeling of Human G‑Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY
Open this publication in new window or tab >>Mutagenesis and Computational Modeling of Human G‑Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY
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2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, 7987-7998 p.Article in journal (Refereed) Published
Abstract [en]

Neuropeptide Y and peptide YY receptor type 2 (Y2) is involved in appetite regulation and several other physiological processes. We have investigated the structure of the human Y2 receptor. Computational modeling of receptor–agonist interactions was used as a guide to design a series of receptor mutants, followed by binding assays using full-length and truncated peptide agonists and the Y2-specific antagonist BIIE0246. Our model suggested a hydrogen bond network among highly conserved residues Thr2.61, Gln3.32, and His7.39, which could play roles in ligand binding and/or receptor structure. In addition, the C-terminus of the peptide could make contact with residues Tyr5.38 and Leu6.51. Mutagenesis of all these positions, followed by binding assays, provides experimental support for our computational model: most of the mutants for the residues forming the proposed hydrogen bond network displayed reduced peptide agonist affinities as well as reduced hPYY3-36 potency in a functional assay. The Ala and Leu mutants of Gln3.32 and His7.39 disrupted membrane expression of the receptor. Combined with the modeling, the experimental results support roles for these hydrogen bond network residues in peptide binding as well as receptor architecture. The reduced agonist affinity for mutants of Tyr5.38 and Leu6.51 supports their role in a binding pocket surrounding the invariant tyrosine at position 36 of the peptide ligands. The results for antagonist BIIE0246 suggest several differences in interactions compared to those of the peptides. Our results lead to a new structural model for NPY family receptors and peptide binding.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013
National Category
Natural Sciences Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-154994 (URN)10.1021/bi400830c (DOI)000330017700012 ()
Available from: 2011-08-04 Created: 2011-06-14 Last updated: 2017-12-08Bibliographically approved
4. Neuropeptide Y/peptide YY receptor Y2 duplicate in zebrafish with unique introns displays distinct peptide binding properties
Open this publication in new window or tab >>Neuropeptide Y/peptide YY receptor Y2 duplicate in zebrafish with unique introns displays distinct peptide binding properties
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2011 (English)In: Comparative Biochemistry and Physiology - Part B: Biochemistry & Molecular Biology, ISSN 1096-4959, E-ISSN 1879-1107, Vol. 160, no 4, 166-173 p.Article in journal (Other academic) Published
Abstract [en]

The neuropeptide Y-family peptides and receptors are involved in a broad range of functions including appetite regulation. Both the peptide genes and the receptor genes are known to have duplicated in early vertebrate evolution. The ancestral jawed vertebrate had 7 NPY receptors but the number varies between 4 and 7 in extant vertebrates. Herein we describe the identification of an additional NPY receptor in two fish species, zebrafish and medaka. They cluster together with the Y2 receptors in phylogenetic analyses and seem to be orthologous to each other that is why we have named them Y2-2. Their genes differ from Y2 in having introns in the coding region. Binding studies with zebrafish Y2-2 receptors show that the three endogenous peptides NPY, PYYa and PYYb have similar affinities, 0.15-0.66nM. This is in contrast to the Y2 receptor where they differed considerably from one another. N-terminally truncated NPY binds poorly and the Y2 antagonist BIIE0246 binds well to Y2-2, results that are reversed in comparison to Y2. Zebrafish Y2-2 mRNA was detected by PCR in the intestine and the eye, but not in the brain. In conclusion, we have found a novel Y2-like NPY/PYY receptor that probably arose in early teleost fish evolution.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-154990 (URN)10.1016/j.cbpb.2011.08.001 (DOI)000296679800004 ()21855645 (PubMedID)
Available from: 2011-08-04 Created: 2011-06-14 Last updated: 2017-12-08Bibliographically approved

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