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Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience. (Pharmacology)ORCID iD: 0000-0002-2913-514X
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The neuropeptide Y (NPY) system consists of 3-4 peptides and 4-7 receptors in vertebrates. It has powerful effects on appetite regulation and is involved in many other biological processes including blood pressure regulation, bone formation and anxiety. This thesis describes studies of the evolution of the NPY system by comparison of several vertebrate species and structural studies of the human Y2 receptor, which reduces appetite, to identify amino acid residues involved in peptide-receptor interactions.

The NPY system was studied in zebrafish (Danio rerio), western clawed frog (Xenopus tropicalis), and sea lamprey (Petromyzon marinus). The receptors were cloned and functionally expressed and their pharmacological profiles were determined using the native peptides in either binding studies or a signal transduction assay. Some peptide-receptor preferences were observed, indicating functional specialization.

A receptor family closely related to the NPY receptors, called the QRFP receptors, was investigated. A QRFP receptor was cloned from amphioxus, Branchistoma floridae, showing that the receptor arose before the origin of the vertebrates. Evolutionary studies demonstrated that the ancestral vertebrate had as many as four QRFP receptors, only one of which remains in mammals today. This correlates with the NPY receptor family, located in the same chromosomal regions, which had seven members in the ancestral vertebrate but only 4-5 in living mammals. Some vertebrates have considerably more complex NPY and QRFP receptor systems than humans and other mammals.

Two studies investigated interactions of NPY-family peptides with the human Y2 receptor. Candidate residues, selected based on structural modeling and docking, were mutated to disrupt possible interactions with peptide ligands. The modified receptors were expressed in cultured cells and investigated by measuring binding and functional responses. Several receptor residues were found to influence peptide-receptor interactions, some of which are involved in maintaining receptor structure. In a pilot study, the kinetics of peptide-receptor interaction were found to be very slow, of the order several hours.

In conclusion, this thesis clarifies evolutionary relationships for the complex NPY and QRFP peptide-receptor systems and improves the structural models of the human NPY-family receptors, especially Y2. These results will hopefully facilitate drug design for targeting of NPY-family receptors.

Place, publisher, year, edition, pages
Uppsala, Sweden: Acta Universitatis Upsaliensis, 2014. , 59 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1040
Keyword [en]
Neuropeptide Y, genome duplication, Evolution, vertebrate, Pharmacology, Modelling, Kinetics
National Category
Evolutionary Biology Pharmacology and Toxicology Cell and Molecular Biology Neurosciences Biochemistry and Molecular Biology Cell Biology Structural Biology
Research subject
Bioinformatics; Biology with specialization in Evolutionary Genetics; Biology with specialization in Evolutionary Functional Genomics; Pharmaceutical Pharmacology
Identifiers
URN: urn:nbn:se:uu:diva-233461ISBN: 978-91-554-9059-1 (print)OAI: oai:DiVA.org:uu-233461DiVA: diva2:752819
Public defence
2014-11-21, C2, 305, Husargatan 3, BMC, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-10-31 Created: 2014-10-06 Last updated: 2015-02-02
List of papers
1. Cloning and pharmacological characterization of the neuropeptide Y receptor Y5 in the sea lamprey, Petromyzon marinus
Open this publication in new window or tab >>Cloning and pharmacological characterization of the neuropeptide Y receptor Y5 in the sea lamprey, Petromyzon marinus
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2013 (English)In: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 39, 64-70 p.Article in journal (Refereed) Published
Abstract [en]

The neuropeptide Y system is known to have expanded in early vertebrate evolution. Three neuropeptide Y receptors have been proposed to have existed before the two basal vertebrate tetraploidizations, namely a VI-like, a Y2-like, and a Y5-like receptor, with their genes in the same chromosomal region. Previously we have described a VI-subfamily and a Y2-subfamily receptor in the river lamprey, Lampetra fluviatilis. Here we report the identification of a Y5 receptor in the genome of the sea lamprey, Petromyzon marinus. In phylogenetic analyses, the Y5 receptor clusters together with gnathostome Y5 receptors with high bootstrap value and shares the long intracellular loop 3. This lamprey receptor has an even longer loop 3 than the gnathostome Y5 receptors described so far, with the expansion of amino acid repeats. Functional expression in a human cell line, co-transfected with a modified human G-protein, resulted in inositol phosphate turnover in response to the three lamprey NPY-family peptides NPY, PYY and PMY at nanomolar concentrations. Our results confirm that the Y1-Y2-Y5 receptor gene triplet arose before the cyclostome-gnathostome divergence. However, it is not clear from the NPY receptors whether cyclostomes diverged from the gnathostome lineage after the first or the second tetraploidization. Duplicates resulting from the tetraploidizations exist for both Y1 and Y2 in gnathostomes, but only a single copy of Y5 has survived in all vertebrates characterized to date, making the physiological roles of Y5 interesting to explore.

Keyword
Lamprey, Neuropeptide Y, Peptide YY, Y5 receptor
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-198074 (URN)10.1016/j.peptides.2012.11.007 (DOI)000315839300011 ()
Available from: 2013-04-09 Created: 2013-04-08 Last updated: 2017-12-06Bibliographically approved
2. Neuropeptide Y family receptors Y1 and Y2 from sea lamprey, Petromyzon marinus: cloning and pharmacological characterization
Open this publication in new window or tab >>Neuropeptide Y family receptors Y1 and Y2 from sea lamprey, Petromyzon marinus: cloning and pharmacological characterization
2015 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 222, 106-115 p.Article in journal (Other academic) Published
Abstract [en]

The vertebrate gene family for neuropeptide Y (NPY) receptors expanded by duplication of the chromosome carrying the ancestral Y1–Y2–Y5 gene triplet. After loss of some duplicates, the ancestral jawed vertebrate had seven receptor subtypes forming the Y1 (including Y1, Y4, Y6, Y8), Y2 (including Y2, Y7) and Y5 (only Y5) subfamilies. Lampreys are considered to have experienced the same chromosome duplications as gnathostomes and should also be expected to have multiple receptor genes. However, previously only a Y4-like and a Y5 receptor have been cloned and characterized. Here we report the cloning and characterization of two additional receptors from the sea lamprey Petromyzon marinus. Sequence phylogeny alone could not with certainty assign their identity, but based on synteny comparisons of P. marinus and the Arctic lamprey, Lethenteron camtschaticum, with jawed vertebrates, the two receptors most likely are Y1 and Y2. Both receptors were expressed in human HEK293 cells and inositol phosphate assays were performed to determine the response to the three native lamprey peptides NPY, PYY and PMY. The three peptides have similar potencies in the nanomolar range for Y1. No obvious response to the three peptides was detected for Y2. Synteny analysis supports identification of the previously cloned receptor as Y4. No additional NPY receptor genes could be identified in the presently available lamprey genome assemblies. Thus, four NPY-family receptors have been identified in lampreys, orthologs of the same subtypes as in humans (Y1, Y2, Y4 and Y5), whereas many other vertebrate lineages have retained additional ancestral subtypes.

National Category
Evolutionary Biology Endocrinology and Diabetes
Identifiers
urn:nbn:se:uu:diva-233437 (URN)10.1016/j.ygcen.2015.08.005 (DOI)000366438000012 ()26255155 (PubMedID)
Funder
Swedish Research Council
Available from: 2014-10-06 Created: 2014-10-05 Last updated: 2017-12-05Bibliographically approved
3. Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes
Open this publication in new window or tab >>Characterization of the neuropeptide Y system in the frog Silurana tropicalis (Pipidae): three peptides and six receptor subtypes
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2012 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 177, no 3, 322-331 p.Article in journal (Refereed) Published
Abstract [en]

Neuropeptide Y and its related peptides PYY and PP (pancreatic polypeptide) are involved in feeding behavior, regulation of the pituitary and the gastrointestinal tract, and numerous other functions. The peptides act on a family of G-protein coupled receptors with 4-7 members in jawed vertebrates. We describe here the NPY system of the Western clawed frog Silurana (Xenopus) tropicalis. Three peptides, NPY, PYY and PP, were identified together with six receptors, namely subtypes Y1, Y2, Y4, Y5, Y7 and Y8. Thus, this frog has all but one of the ancestral seven gnathostome NPY-family receptors, in contrast to mammals which have lost 2-3 of the receptors. Expression levels of mRNA for the peptide and receptor genes were analyzed in a panel of 19 frog tissues using reverse transcriptase quantitative PCR. The peptide mRNAs had broad distribution with highest expression in skin, blood and small intestine. NPY mRNA was present in the three brain regions investigated, but PYY and PP mRNAs were not detectable in any of these. All receptor mRNAs had similar expression profiles with high expression in skin, blood, muscle and heart. Three of the receptors, Y5, Y7 and Y8, could be functionally expressed in HEK-293 cells and characterized with binding studies using the three frog peptides. PYY had the highest affinity for all three receptors (K(i) 0.042-0.34 nM). Also NPY and PP bound to the Y8 receptor with high affinity (0.14 and 0.50 nM). The low affinity of NPY for the Y5 receptor (100-fold lower than PYY) differs from mammals and chicken. This may suggest a less important role of NPY on Y5 in appetite stimulation in the frog compared with amniotes. In conclusion, our characterization of the NPY system in S. tropicalis with its six receptors demonstrates not only greater complexity than in mammals but also some interesting differences in ligand-receptor preferences.

Keyword
NPY, PYY, G-protein-coupled receptor, Silurana tropicalis, evolution
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-129517 (URN)10.1016/j.ygcen.2012.04.027 (DOI)000306390100005 ()22565163 (PubMedID)
Note

Erratum in General and Comparative Endocrinology 2015:215, doi:10.1016/j.ygcen.2014.11.014.

Available from: 2010-08-18 Created: 2010-08-18 Last updated: 2017-12-12Bibliographically approved
4. Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a, and Y8b
Open this publication in new window or tab >>Interactions of zebrafish peptide YYb with the neuropeptide Y-family receptors Y4, Y7, Y8a, and Y8b
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2013 (English)In: Frontiers in Neuroscience, ISSN 1662-4548, E-ISSN 1662-453X, Vol. 7, 29Article in journal (Refereed) Published
Abstract [en]

The neuropeptide Y (NPY) system influences numerous physiological functions including feeding behavior, endocrine regulation, and cardiovascular regulation. In jawed vertebrates it consists of 3-4 peptides and 4-7 receptors. Teleost fishes have unique duplicates of NPY and PYY as well as the Y8 receptor. In the zebrafish, the NPY system consists of the peptides NPYa, PYYa, and PYYb (NPYb appears to have been lost) and at least seven NPY receptors: Y1, Y2, Y2-2, Y4, Y7, Y8a, and Y8b. Previously PYYb binding has been reported for Y2 and Y2-2. To search for peptide-receptor preferences, we have investigated PYYb binding to four of the remaining receptors and compared with NPYa and PYYa. Taken together, the most striking observations are that PYYa displays reduced affinity for Y2 (3 nM) compared to the other peptides and receptors and that all three peptides have higher affinity for Y4 (0.028-0.034 nM) than for the other five receptors. The strongest peptide preference by any receptor selectivity is the one previously reported for PYYb by the Y2 receptor, as compared to NPY and PYYa. These affinity differences may be helpful to elucidate specific details of peptide-receptor interactions. Also, we have investigated the level of mRNA expression in different organs using qPCR. All peptides and receptors have higher expression in heart, kidney, and brain. These quantitative aspects on receptor affinities and mRNA distribution help provide a more complete picture of the NPY system.

Keyword
Evolution, genome duplication, NPY, elephant shark
National Category
Natural Sciences Neurology
Research subject
Neuroscience
Identifiers
urn:nbn:se:uu:diva-205599 (URN)10.3389/fnins.2013.00029 (DOI)000346567300029 ()23508731 (PubMedID)
Funder
Swedish Research Council
Available from: 2013-08-20 Created: 2013-08-20 Last updated: 2017-12-06Bibliographically approved
5. Characterization of peptide QRFP (26RFa) and its receptor from amphioxus, Branchiostoma floridae
Open this publication in new window or tab >>Characterization of peptide QRFP (26RFa) and its receptor from amphioxus, Branchiostoma floridae
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2015 (English)In: General and Comparative Endocrinology, ISSN 0016-6480, E-ISSN 1095-6840, Vol. 210, 107-113 p.Article in journal (Refereed) Published
Abstract [en]

A peptide ending with RFamide (Arg-Phe-amide) was discovered independently by three different laboratories in 2003 and named 26RFa or QRFP. In mammals, a longer version of the peptide, 43 amino acids, was identified and found to bind to the orphan G protein-coupled receptor GPR103. We searched the genome database of Branchiostoma floridae (Bfl) for receptor sequences related to those that bind peptides ending with RFa or RYa (including receptors for NPFF, PRLH, GnIH, and NPY). One receptor clustered in phylogenetic analyses with mammalian QRFP receptors. The gene has 3 introns in Bfl and 5 in human, but all intron positions differ, implying that the introns were inserted independently. A QRFP-like peptide consisting of 25 amino acids and ending with RFa was identified in the amphioxus genome. Eight of the ten last amino acids are identical between Bfl and human. The prepro-QRFP gene in Bfl has one intron in the propeptide whereas the human gene lacks introns. The Bfl QRFP peptide was synthesized and the receptor was functionally expressed in human cells. The response was measured as inositol phosphate (IP) turnover. The Bfl QRFP peptide was found to potently stimulate the receptor's ability to induce IP turnover with an EC50 of 0.28nM. Also the human QRFP peptides with 26 and 43 amino acids were found to stimulate the receptor (1.9 and 5.1nM, respectively). Human QRFP with 26 amino acids without the carboxyterminal amide had dramatically lower potency at 1.3μM. Thus, we have identified an amphioxus QRFP-related peptide and a corresponding receptor and shown that they interact to give a functional response.

Keyword
Neuropeptide, receptor, evolution
National Category
Neurosciences
Research subject
Evolutionary Genetics; Neuroscience
Identifiers
urn:nbn:se:uu:diva-240039 (URN)10.1016/j.ygcen.2014.10.010 (DOI)000346886400012 ()25449662 (PubMedID)
Available from: 2015-01-05 Created: 2015-01-05 Last updated: 2017-12-05Bibliographically approved
6. Unexpected multiplicity of QRFP receptors in early vertebrate evolution
Open this publication in new window or tab >>Unexpected multiplicity of QRFP receptors in early vertebrate evolution
2014 (English)In: Frontiers in Neuroscience, ISSN 1662-4548, E-ISSN 1662-453X, Vol. 8, 337- p.Article in journal (Refereed) Published
Abstract [en]

The neuropeptide QRFP, also called 26RFa, and its G protein-coupled receptor GPR103 have been identified in all vertebrates investigated. In mammals, this peptide-receptor pair has been found to have several effects including stimulation of appetite. Recently, we reported that a QRFP peptide is present in amphioxus, Branchiostoma floridae, and we also identified a QRFP receptor (QRFPR) that mediates a functional response to sub-nanomolar concentrations of the amphioxus peptide as well as short and long human QRFP (Xu et al., submitted). Because the ancestral vertebrate underwent two tetraploidizations, it might be expected that duplicates of the QRFP gene and its receptor gene may exist. Indeed, we report here the identification of multiple vertebrate QRFPR genes. Three QRFPR genes are present in the coelacanth Latimeria chalumnae, representing an early diverging sarcopterygian lineage. Three QRFPR genes are present in the basal actinopterygian fish, the spotted gar. Phylogenetic and chromosomal analyses show that only two of these receptor genes are orthologous between the two species, thus demonstrating a total of four distinct vertebrate genes. Three of the QRFPR genes resulted from the early vertebrate tetraploidizations and were copied along with syntenic neuropeptide Y receptor genes. The fourth QRFPR gene may be an even older and distinct lineage. Because mammals and birds have only a single QRFPR gene, this means that three genes have been lost in these lineages, and at least one of these was lost independently in mammals and birds because it is still present in a turtle. In conclusion, these results show that the QRFP system gained considerable complexity in the early stages of vertebrate evolution and still maintains much of this in some lineages, and that it has been secondarily reduced in mammals.

Keyword
Neuropeptide, receptor, evolution
National Category
Natural Sciences
Research subject
Neuroscience; Evolutionary Genetics
Identifiers
urn:nbn:se:uu:diva-240037 (URN)10.3389/fnins.2014.00337 (DOI)000346532900001 ()25386115 (PubMedID)
Available from: 2015-01-05 Created: 2015-01-05 Last updated: 2017-12-05Bibliographically approved
7. 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
8. Detecting ligand interactions with G protein-coupled receptors in real-time on living cells
Open this publication in new window or tab >>Detecting ligand interactions with G protein-coupled receptors in real-time on living cells
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2013 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 441, no 4, 820-824 p.Article in journal (Refereed) Published
Abstract [en]

G protein-coupled receptors (GPCRs) are a large group of receptors of great biological and clinical relevance. Despite this, the tools for a detailed analysis of ligand-GPCR interactions are limited. The aim of this paper was to demonstrate how ligand binding to GPCRs can be followed in real-time on living cells. This was conducted using two model systems, the radiolabeled porcine peptide YY (pPYY) interacting with transfected human Y2 receptor (hY2R) and the bombesin antagonist RM26 binding to the naturally expressed gastrin-releasing peptide receptor (GRPR). By following the interaction over time, the affinity and kinetic properties such as association and dissociation rate were obtained. Additionally, data were analyzed using the Interaction Map method, which can evaluate a real-time binding curve and present the number of parallel interactions contributing to the curve. It was found that pPYY binds very slowly with an estimated time to equilibrium of approximately 12 h. This may be problematic in standard end-point assays where equilibrium is required. The RM26 binding showed signs of heterogeneity, observed as two parallel interactions with unique kinetic properties. In conclusion, measuring binding in real-time using living cells opens up for a better understanding of ligand interactions with GPCRs.

Keyword
GPCR, Real-time, LigandTracer, Interaction Map, Kinetics, Heterogeneity
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-215943 (URN)10.1016/j.bbrc.2013.10.149 (DOI)000328434800022 ()
Note

De två första författarna delar första författarskapet.

Available from: 2014-01-17 Created: 2014-01-17 Last updated: 2017-12-06Bibliographically approved

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