1. Academic Validation
  2. Ala5-galanin (2-11) is a GAL2R specific galanin analogue

Ala5-galanin (2-11) is a GAL2R specific galanin analogue

  • Neuropeptides. 2016 Dec:60:75-82. doi: 10.1016/j.npep.2016.08.008.
Kristin Webling 1 Johan Runesson 2 Andreas Lang 3 Indrek Saar 4 Barbara Kofler 3 Ülo Langel 5
Affiliations

Affiliations

  • 1 Department of Neurochemistry, Stockholm University, Svante Arrheniusv. 16B, SE-10691 Stockholm, Sweden. Electronic address: kristin.webling@neurochem.su.se.
  • 2 Department of Neurochemistry, Stockholm University, Svante Arrheniusv. 16B, SE-10691 Stockholm, Sweden.
  • 3 Research Program for Receptorbiochemistry and Tumormetabolism, Laura Bassi Centre of Expertise THERAPEP, Department of Pediatrics, University Hospital Salzburg, Paracelsus Medical University, Müllner Hauptstr. 48, A-5020 Salzburg, Austria.
  • 4 Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
  • 5 Department of Neurochemistry, Stockholm University, Svante Arrheniusv. 16B, SE-10691 Stockholm, Sweden; Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
Abstract

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, Ala5-galanin (2-11), is presented. The Galanin (2-11) modified analogue Ala5-galanin (2-11) was tested in 125I-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. Ala5-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.

Keywords

Agonist; Dynamic mass redistribution; GAL(2)R specific ligand; GPCR; Galanin; Impedance; Label-free real-time assay; Neuropeptide.

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