Receptor-induced Internalization of Selective Peptidic μ and Δ Opioid Ligands

Georges Gaudriault,Dominique Nouel,Claude Dal Farra,Alain Beaudet,Jean-Pierre Vincent

doi:10.1074/jbc.272.5.2880

Abstract

The binding and internalization of radioiodinated and fluorescent mu and delta 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 delta and mu opioid receptors, respectively. Biochemical and confocal microscopy data showed that both mu and delta 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 delta and mu 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 mu and delta receptors, suggesting that each of these receptors interacts with distinct proteins mediating intracellular sorting and trafficking.

Highlights

The pharmacological, behavioral, and binding properties of ␮, ␦, and ␬ opioid receptors have been extensively studied
The binding and internalization of ␮ and ␦ opioid peptides in mammalian cells were quantitatively studied by means of biochemical techniques and directly visualized by confocal microscopy
The radiolabeled and fluorescent analogues developed for this purpose were synthesized by inserting either an 125I-labeled Bolton-Hunter group or a fluorescent probe into the C-terminal part of 5-aminopentylamide derivatives of deltorphin-I and dermorphin

Summary

Introduction

The pharmacological, behavioral, and binding properties of ␮, ␦, and ␬ opioid receptors have been extensively studied. Confocal microscopic studies carried out on transfected cells have shown a rapid endocytosis of ␮ [13, 14] and ␦ [13] antigenic epitope-tagged receptors following exposure to enkephalins, but not to morphine Whether this endocytosis occurs in conjunction with that of the bound ligand, remains unclear. Dermorphin, isolated from the skin of the frog Phylomedusa sauvagei [15], was the first natural peptide described as having high affinity and selectivity for the ␮ opioid receptor Another unusual property of dermorphin is the D configuration of the alanine residue in position 2, which is responsible for its strong resistance to enzymatic degradation and for its good opioid binding site recognition. Radioactive compounds were used to quantitatively assess the binding and internalization of the opioid derivatives, while fluorescent compounds were used to study their distribution in the confocal microscope, an approach that has been successfully resorted to for a variety of other neuropeptides, including fluorescent analogues of cholecystokinin [18], gastrin-releasing peptide [19], neurotensin [20], thyrotropin-releasing hormone [21, 22], substance P [23, 24], and somatostatin [25]

Methods

Results

Conclusion