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 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.
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CURE: VA/UCLA Gastroenteric Biology Center, University of California 90095, USA. Islice@med1.medsch.ucla.edu
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D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001667:Binding, Competitive; D001839:Bombesin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D004847:Epithelial Cells; D005456:Fluorescent Dyes; D019886:Gastrin-Releasing Peptide / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D018942:Macrolides; D008856:Microscopy, Fluorescence; D051381:Rats; D018004:Receptors, Bombesin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection
D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D007457:Iodine Radioisotopes; D008164:Luminescent Proteins; D018942:Macrolides; D018004:Receptors, Bombesin; D011994:Recombinant Proteins; C057620:bafilomycin A; D049452:Green Fluorescent Proteins; D013395:Sucrose; D019886:Gastrin-Releasing Peptide; D001839:Bombesin; D002118:Calcium