EVIDENCE FOR HYDROPHOBIC INTERACTION BETWEEN GALANIN AND THE GA1R1 GALANIN RECEPTOR AND GA1R1-MEDIATED LIGAND INTERNALIZATION - FLUORESCENTPROBING WITH A FLUORESCEIN-GALANIN

Galanin is a neuropeptide that activates specific receptors to modulat e several physiological functions including food intake, nociception, and learning and memory. The molecular nature of the interaction betwe en galanin and its receptors and the fate of the galanin/receptor comp lex after the binding event are not understood. A fluorescein-N-galani n (F-Gal) was generated to measure the interaction between galanin and rat GalR1 galanin receptor (rGalR1) and rGalR1-mediated ligand intern alization using flow cytometry in transfected Chinese hamster ovary (C HO) cells. Like galanin, F-Gal bound rGalR1 with high affinity and sti mulated intracellular signaling events. Fluorescence quenching by solu ble KI of rGalR1-bound F-Gal revealed a highly protected environment a round the fluorescein, suggesting that the N-terminal portion of galan in, which constitutes the binding site of galanin for the receptor, bi nds to a protected hydrophobic binding pocket within the receptor. Exp osure to F-Gal stimulated rapid (t(1/2) similar to 10 min) and extensi ve (78%) internalization of surface F-Cal 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 i nhibited by addition of 0.25 M sucrose in the medium, indicating a Gal R1-mediated energy-requiring endocytic process. These results revealed a hydrophobic interaction between galanin and the GalR1 receptor, whi ch is in contrast to those of other G protein-coupled receptors that m ainly require hydrophilic interaction with their peptide ligands near or outside the plasma membrane surface, and illustrated that the initi al binding interaction is followed by rapid cellular internalization o f the agonist/GalR1 complex.