This thesis is devoted to the deeper understanding of the molecular details of the interaction between the neuropeptide galanin and its target protein, the G-protein coupled 7TM galanin receptor. The enzymatic control of galanin degradation and second messenger systems triggered by galanin receptor activation are also studied.
Galanin analogs with substitutions of amino acids by units of L-Ala residues have been developed as selective ligands to galanin receptor subtypes in the rat hypothalamus and in the rat jejunum. It is shown that galanin receptors in the jejunum require also the C-terminal segment KHGLT25-29for receptor recognition in contrast to receptors in the hypothalamus which only interact with the N-terminal portion (1-13) of galanin.
It is confirmed that the N-terminal portion (1-13) of galanin is essential for receptor recognition in the rat hypothalamus and that the C-terminus of the ligand can be extended with sequences of amino acids or other organic structures to increase the affinity of the N-terminal (1-13) fragment of galanin. It is also concluded that a minimum motif required for galanin receptor recognition should contain the indol structure of a Trp residue and the aromatic side chain of a Tyr residue plus one additional bulky highly hydrophobic structure as well as some positively charged structures.
The galanin binding domains of the receptor, GalR1 have been studied by site directed mutagenesis and a structural model for the interaction of the major pharmacophores of galanin is proposed. The charged N-terminus of galanin is suggested to interact with Phe115 in TM III by cation-p interaction, the Trp2 residue of galanin is proposed to interact with a pair of His residues located at the top of TM VI and the Tyr9 of galanin is interacting with another phenylalanine, Phe282 in the EC III of the receptor. One of the two histidine residues (264,267); His267is believed not only to be important for ligand binding but also is stabilising the active conformation of the GalR1 receptor, important for the G-protein coupling of the agonist occupied receptor.
It is shown that galanin binding sites are present at significantly higher numbers in the ventral part of the rat hippocampus compared to the dorsal area. Furthermore it is determined that the efficiency of the inhibitory coupling to basal and forskolin stimulated cAMP production by galanin receptors is 250 fold less potent in the dorsal region of the rat hippocampus. The differences in regulation of adenylate cyclase suggests an unequal receptor subtype distribution in the dorsal and ventral hippocampus with higher levels of Gicoupled GalR1 receptors in the ventral segments of the hippocampal formation.
An internally quenched fluorescent substrate for galanin degrading enzymes has been developed and used for purification of a galanin inactivating 70 kDa membrane associated metallo-endopeptidase from bovine spinal cord. The enzyme that has a inhibitor profile different from NEP-24.11 primarily cleaves galanin between Trp2 and Thr3 and recognises both galanin and the galanin mimicking fluorescent artificial substrate with high affinity. This cleavage generates products that are inactive at both GalR1 and GalR2 receptors.
Stockholm: Stockholm University , 1998. , 60 p.