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

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.