Visualization of internalization and recycling of the gastrin releasing peptide receptor green fluorescent protein chimera expressed in epithelial cells

Gastrin releasing peptide (GRP) regulates critical grastrointestinal functi ons via the GRP receptor (GRPR). GRPR internalization and recycling have be en proposed to play an important role in the cellular response to GRP, Our aim was to develop a direct method for investigating GRPR trafficking in li ving cells. A chimeric protein, consisting of GRPR fused to green fluoresce nt protein (GFP), was expressed in epithelial cells. Ligand and receptor in teractions were examined with radiolabeled agonist and fluorescent imaging. In comparisen with epithelial cells expressing wild-type GRPR, the GRPR - GFP expressing cells showed similar ligand binding affinity, GRP-stimulated Ca2+ signaling, and GRP-initiated internalization. In GRPR-GFP expressing cells treated with fluorescently labeled ligand, receptor and ligand traffi cking was directly visualized. Upon ligand binding, the receptor-ligand com plex coalesced into vesicles prior to internalization and migration to the perinuclear space. Whereas a portion of the receptors were observed to retu rn to the plasma membrane, the ligand remained in the perinuclear space. Hy perosmolar solution prevented ligand and receptor internalization, and bafi lomycin inhibited receptor recycling. We demonstrate that GRPR-GFP is physi ologically similar to wild-type GRPR, and permits direct visualization of i ntracellular trafficking processes in individual living cells with minimal toxicity over several hours.