Ala5-galanin (2–11) is a GAL2R specific galanin analogue. (December 2016)

• Record Type: Journal Article
• Authors: Webling, Kristin; Runesson, Johan; Lang, Andreas; Saar, Indrek; Kofler, Barbara; Langel, Ülo
• Is Part Of: Neuropeptides. Volume 60(2016)
• Page Start: 75
• Page End: 82
• Publication Date: 2016-12
• Subjects: BBB blood-brain barrier -- CHO cells Chinese hamster ovary cells -- CNS central nervous system -- EDTA ethylenediaminetetraacetic acid -- FBS fetal bovine serum -- Fmoc 9-fluorenylmethyloxycarbonyl -- FSK forskolin -- GAL1R Galanin receptor 1 -- GAL2R Galanin receptor 2 -- GAL3R Galanin receptor 3 -- GIP GPCR interacting proteins -- GPCRs G-protein coupled receptors -- HKR HEPES buffered Krebs-Ringer solution -- HPLC high performance liquid chromatography -- IP inositol phosphate -- MBHA p-methylbenzhydrylamine -- PBS phosphate-buffered saline -- PNS peripheral nervous system -- Sar sarcosine; Agonist -- Dynamic mass redistribution -- Galanin -- GAL2R specific ligand -- GPCR -- Impedance -- Neuropeptide -- Label-free real-time assay; Neuropeptides -- Periodicals; Neuropeptides; Neuropeptides -- Périodiques; Neuropeptides; Electronic journals; Periodicals; 572.65
• DOI: 10.1016/j.npep.2016.08.008
• Languages: English
• ISSNs: 0143-4179

Abstract

Abstract: It is over 30 years 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 GAL1 R, GAL2 R and GAL3 R. 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 GAL2 R 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 GAL2 R with more than 375-fold preference for GAL2 R compared to both GAL1 R and GAL3 R. 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 GAL3 R nor GAL1 R, even at concentrations as high as 0.1 mM. Highlights: Ala 5 -galanin (2–11) is the shortest galanin receptor 2 specific agonist reported. Classical second messenger assays were compared to real-time label-free techniques for GPCR signaling. The importance of Ser 5 for GAL3 R binding from an in silico study were experimentally verified.