IDENTIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE HUMAN BRAIN GALANIN RECEPTOR

Human galanin (hGal) is an important neuromodulator present in the brain, gastrointestinal system and the hypothalamo-pituitary axis. A specific receptor for hGal has been identified in various areas in human brain. A single class of high affinity binding sites was found on plasma membranes of the amygdala (K-d 0.23 nM, B-max 44 fmol/mg), the hypothalamus (K-d 0.20 nM, B-max 25 fmol/mg) and the cortex cerebri (K-d 0.11 nM, B-max 8.2 fmol/mg). Other brain areas, i.e. cerebellum, thalamus or pens, expressed binding sites of identical high affinity in lower quantities (B-max <3 fmol/mg). Specific binding of I-125-labelled hGal was found to be reversible, time- and temperature-dependent and inhibited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydrolysable guanosine nucleotides potently reduced specific binding of I-125-labelled hGal by more than 80%. Synthetic hGal analogues substituted in the N-terminal region exhibited strongly reduced binding affinity for the hGal receptor. Using 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulphonate, hGal receptors were successfully solubilized from human cortical membranes, exhibiting no significant loss of binding affinity. Affinity cross-linking to I-125-labelled hGal revealed a labelled band of approximately 60 kDa sensitive to unlabelled Gal. This putative hGal receptor is glycosylated since its molecular size was reduced after treatment with endoglycosidase F. Receptors bound to I-125-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus communis agglutinin, suggesting that receptor glycosylation involves N-acetyl glucosamine and galactose respectively.