Receptor-induced internalization of selective peptidic μ and δ opioid ligands

Abstract

The binding and internalization of radioiodinated and fluorescent μ and δ opioid peptides in mammalian cells were quantitatively studied by biochemical techniques and directly visualized by confocal microscopy. The labeled peptides were prepared by inserting either a 125I-Bolton-Hunter group or a fluorescent probe into the C-terminal part of 5-aminopentylamide derivatives of deltorphin-I and [Lys7]dermorphin. The purified derivatives kept most of their specificity and selectivity toward δ and μ opioid receptors, respectively. Biochemical and confocal microscopy data showed that both and δ opioid peptides were internalized in mammalian cells transfected with the corresponding opioid receptor according to a receptor-mediated mechanism. The internalization process was time- and temperature-dependent and was completely blocked by the endocytosis inhibitor phenylarsine oxyde. Internalization of both δ and μ ligands occurred from a single large cap at one pole of the cell, indicating that polymerization of ligand-receptor complexes preceeded internalization. Finally, green and red fluorescent analogues of deltorphin-I and [Lys7]dermorphin, respectively, were found to internalize through partly distinct endocytic pathways in cells co- transfected with μ and δ receptors, suggesting that each of these receptors interacts with distinct proteins mediating intracellular sorting and trafficking.