Constitutive and ligand-induced internalization of EGFP-tagged galanin R2 and Rl receptors in PC12 cells

In the present experiments trafficking of the galanin R1-(GALR1) and, in particular, the galanin R2 receptor (GALR2) was studied after fusion with enhanced green fluorescent protein (GALR1-EGFP and GALR2-EGFP) and transfection into PC12 cells. Both fusion proteins were predominantly localized on the plasma membrane and internalized in a dose dependent manner after incubation with galanin. Preincubation with M35 or M40 did not prevent galanin-induced internalization of GALR1-EGFP or GALR2-EGFP. However, AR-M1896, a selective GALR2 agonist, caused GALR2, but not GALR1 internalization. Hyperosmotic sucrose inhibited internalization of GALR2-EGFP. After co-incubation with galanin, GALR2-EGFP was co-localized with internalized Texas Red transferrin, a marker of the clathrin endocytic pathway. Experiments with protein synthesis inhibition and Texas Red transferrin suggest that GALR2 is constitutively internalized. Studies in progress will show if this is the case also for GALR1.