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Characterization of peptide QRFP (26RFa) and its receptor from amphioxus, Branchiostoma floridae
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Pharmacology.
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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.

Place, publisher, year, edition, pages
2015. Vol. 210, 107-113 p.
Keyword [en]
Neuropeptide, receptor, evolution
National Category
Neurosciences
Research subject
Evolutionary Genetics; Neuroscience
Identifiers
URN: urn:nbn:se:uu:diva-240039DOI: 10.1016/j.ygcen.2014.10.010ISI: 000346886400012PubMedID: 25449662OAI: oai:DiVA.org:uu-240039DiVA: diva2:775798
Available from: 2015-01-05 Created: 2015-01-05 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
Open this publication in new window or tab >>Evolutionary and Pharmacological Studies of NPY and QRFP Receptors
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
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:nbn:se:uu:diva-233461 (URN)978-91-554-9059-1 (ISBN)
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

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Xu, BoBergqvist, Christina ASundström, GörelLarhammar, Dan

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