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Ala(5)-galanin (2-11) is a GAL2R specific galanin analogue
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
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2016 (English)In: Neuropeptides, ISSN 0143-4179, E-ISSN 1532-2785, Vol. 60, 75-82 p.Article in journal (Refereed) Published
Abstract [en]

It is over 30years since the regulatory peptide galanin was discovered by Professor Mutt and co-workers. Galanin exerts its effects by binding to three galanin G-protein coupled receptors, namely GAL1R, GAL2R and GAL3R. Each galanin receptor has a different distribution in the central nervous system and the peripheral nervous system as well as distinctive signaling pathways, which implicates that the receptors are involved in different biological- and pathological effects. The delineation of the galaninergic system is however difficult due to a lack of stable, specific galanin receptor ligands. Herein, a new short GAL2R specific ligand, Ala(5)-galanin (2-11), is presented. The galanin (2-11) modified analogue Ala(5)-galanin (2-11) was tested in (125)I-galanin competitive binding studies for the three galanin receptors and the G-protein coupled receptor signaling properties was tested by the ability to influence second-messenger molecules like inositol phosphate and cyclic adenosine monophosphate. In addition, two different label-free real-time assays, namely EnSpire® based on an optical biosensor and xCELLigence® based on an electric biosensor, were used for evaluating the signaling properties using cell lines with different levels of receptor expression. Ala(5)-galanin (2-11) was subsequently found to be a full agonist for GAL2R with more than 375-fold preference for GAL2R compared to both GAL1R and GAL3R. The single amino acid substitution of serine to alanine at position 5 in the short ligand galanin (2-11) resulted in a ligand subsequently unable to bind neither GAL3R nor GAL1R, even at concentrations as high as 0.1mM.

Place, publisher, year, edition, pages
2016. Vol. 60, 75-82 p.
Keyword [en]
Agonist, Dynamic mass redistribution, Galanin, GAL2R specific ligand, GPCR, Impedance, Neuropeptide, Label-free real-time assay
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-136177DOI: 10.1016/j.npep.2016.08.008ISI: 000389398800011PubMedID: 27592409OAI: oai:DiVA.org:su-136177DiVA: diva2:1050820
Available from: 2016-11-30 Created: 2016-11-30 Last updated: 2017-02-20Bibliographically approved
In thesis
1. Design, Synthesis and Characterization of Galanin Receptor Selective Ligands
Open this publication in new window or tab >>Design, Synthesis and Characterization of Galanin Receptor Selective Ligands
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Galanin is a 29/30 amino acid long bioactive peptide discovered over 30 years ago when C-terminally amidated peptides were isolated from porcine intestines. The name galanin originates from a combination of the first and last amino acids - G from glycine and the rest from alanine. The first 15 amino acids are highly conserved throughout species, which indicates that the N-terminus is important for receptor recognition and binding. Galanin exerts its effects by binding to three different G protein-coupled receptors, which all differ according to regional distribution, the affinity for shortened galanin fragments, as well as the intracellular G-protein signaling cascade used. When first discovered, galanin was found to cause muscle contraction as well as hyperglycemia.  Over the years, galanin has been reported to be involved in a wide variety of biological functions, for example food intake and neurogenesis, and pathological functions, for example epilepsy and depression.

Determining the specific involvement of the three different galanin receptors in biological and pathological processes is limited by the small amount of galanin receptor selective/specific ligands available as research tools. Furthermore, the fast degradation of peptides limits the administration routes in animal studies.

This thesis aims at developing new galanin receptor-selective ligands to help delineate the involvement of the three different galanin receptors.

Paper 1 presents the shortest galanin fragment with a galanin receptor 2 specific binding preference where only a single amino acid substitution was made, Ala5Ser in galanin (2-11). In addition, G-protein coupled receptor signaling were evaluated through both a classical second messenger assay and a real-time label-free technique in cells overexpressing the receptor as well as low receptor expression.

Paper 2 demonstrates that the neuroprotective effects of galanin in a kainic acid-induced excitotoxic animal model were mediated through galanin receptor 1. Furthermore, a new robust protocol for evaluating G-protein signaling using a label-free real time impedance technique was presented and compared to two different classical second-messenger assays.

Paper 3 presents a series of systemically active galanin receptor 2 selective ligands subsequently evaluated in two different depression-like animal models.

Paper 4 investigates a mutated form of human galanin which was found in epilepsy patients and binding and signaling properties of the mutated associated ligand p.(A39E) was examined.

In conclusion, this thesis presents the discovery of eight new galanin ligands, which can be used to evaluate the galaninergic system as well as to help investigate the possible use of peptides as pharmaceuticals in different diseases.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2017. 96 p.
Keyword
galanin, GAL1R, GAL2R, depression, epilepsy
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-139880 (URN)978-91-7649-721-0 (ISBN)978-91-7649-722-7 (ISBN)
Public defence
2017-03-31, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2017-03-08 Created: 2017-02-20 Last updated: 2017-03-06Bibliographically approved

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