IDENTIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE HUMAN BRAIN GALANIN RECEPTOR

Human galanin (hGal) is an important neuromodulator present in the bra in, gastrointestinal system and the hypothalamo-pituitary axis. A spec ific receptor for hGal has been identified in various areas in human b rain. A single class of high affinity binding sites was found on plasm a membranes of the amygdala (K-d 0.23 nM, B-max 44 fmol/mg), the hypot halamus (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, thalamu s or pens, expressed binding sites of identical high affinity in lower quantities (B-max <3 fmol/mg). Specific binding of I-125-labelled hGa l was found to be reversible, time- and temperature-dependent and inhi bited by Ca2+, Na+ and K+ ions at a concentration of 5 mM. Non-hydroly sable 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 f or the hGal receptor. Using yl)dimethylammonio]-2-hydroxy-1-propanesul phonate, hGal receptors were successfully solubilized from human corti cal membranes, exhibiting no significant loss of binding affinity. Aff inity 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 tr eatment with endoglycosidase F. Receptors bound to I-125-labelled hGal could be specifically adsorbed to wheat germ agglutinin and ricinus c ommunis agglutinin, suggesting that receptor glycosylation involves N- acetyl glucosamine and galactose respectively.