Publications

The mechanisms by which naive helper T cells differentiate into potent cytokine-expressing effectors remain critical to understanding both successful and aberrant immune responses. Studies using Leishmania major infection of mice have revealed genetic contributions to factors that influence this differentiative process. Further, antigen recognition at the level of the T cell repertoire can also profoundly affect the outcome of disease and the appearance of discrete T cell subsets. It is likely that such mechanisms also underpin genetic susceptibility to diverse other infectious and autoimmune diseases.

Gastrin releasing peptide (GRP) regulates critical gastrointestinal functions via the GRP receptor (GRPR). GRPR internalization and recycling have been 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 living cells. A chimeric protein, consisting of GRPR fused to green fluorescent protein (GFP), was expressed in epithelial cells. Ligand and receptor interactions were examined with radiolabeled agonist and fluorescent imaging. In comparison 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 trafficking was directly visualized. Upon ligand binding, the receptor-ligand complex coalesced into vesicles prior to internalization and migration to the perinuclear space. Whereas a portion of the receptors were observed to return to the plasma membrane, the ligand remained in the perinuclear space. Hyperosmolar solution prevented ligand and receptor internalization, and bafilomycin inhibited receptor recycling. We demonstrate that GRPR-GFP is physiologically similar to wild-type GRPR, and permits direct visualization of intracellular trafficking processes in individual living cells with minimal toxicity over several hours.