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Evidence for hydrophobic interaction between galanin and the Ga1R1 galanin receptor and Ga1R1-mediated ligand internalization: fluorescent probing with a fluorescein-galanin

Wang, S., Clemmons, A., Strader, C., Bayne, M.
Biochemistry 1998 v.37 no.26 pp. 9528-9535
Chinese hamsters, G-protein coupled receptors, animal ovaries, binding sites, flow cytometry, fluorescein, fluorescence, hydrophilicity, hydrophobic bonding, hydrophobicity, ligands, neuropeptides, plasma membrane, rats, sucrose
Galanin is a neuropeptide that activates specific receptors to modulate several physiological functions including food intake, nociception, and learning and memory. The molecular nature of the interaction between galanin and its receptors and the fate of the galanin/receptor complex after the binding event are not understood. A fluorescein-N-galanin (F-Gal) was generated to measure the interaction between galanin and rat GalR1 galanin receptor (rGalR1) and rGalR1-mediated ligand internalization using flow cytometry in transfected Chinese hamster ovary (CHO) cells. Like, galanin, bound rGalR1 with high affinity and stimulated intracellular signaling events. Fluorescence quenching by soluble KI of rGalR1-bound F-Gal revealed a highly protected environment around the fluorescein, suggesting that the N-terminal portion of galanin, which constitutes the binding site of galanin for the receptor, binds to a protected hydrophobic binding pocket within the receptor. Exposure to F-Gal stimulated rapid (t1,2 approximately 10 min) and extensive (78%) internalization of surface F-Gal into rGalR1/CHO cells at 37 degrees C but not at 0 degrees C. In addition, the internalization did not occur in parental CHO cells at either 0 or 37 degrees C and was inhibited by addition of 0.25 M sucrose in the medium, indicating a GalR1-mediated energy-requiring endocytic process. These results revealed a hydrophobic interaction between galanin and the GalR1 receptor, which is in contrast to those of other G protein-coupled receptors that mainly require hydrophilic interaction with their peptide ligands near or outside the plasma membrane surface, and illustrated that the initial binding interaction is followed by rapid cellular internalization of the agonist/GalR1 complex.