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

doi:10.1074/jbc.272.5.2880

Receptor-induced Internalization of Selective Peptidic μ and δ Opioid Ligands*

From the Institut de Pharmacologie Moleculaire et Cellulaire, Centre National de la Recherche Scientifique-UPR 411, 660, Route des lucioles, 06560 Valbonne, France and the
^‡ Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada H3A 2B4

^§ To whom all correspondence should be addressed. Tel.: 33-493-95-77-65; Fax: 33-493-95-77-08; E-mail: Jean-Pierre.VINCENT{at}unice.fr

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 μ 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.

Footnotes

↵* This work was supported by the Center National de la Recherche Scientifique and by the Association Claude Bernard. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

DLT-I 5APA

[D-Ala²]deltorphin-I 5-aminopentylamide (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH-(CH₂)₅-NH₂)

[K7]DRM 5APA

[Lys⁷]dermorphin 5-aminopentylamide (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Lys-NH-(CH₂)₅-NH₂)

BH*

3-(4-hydroxy-5-[¹²⁵I]iodophenyl)propionyl group

ω-BH*DLT-I 5APA

DLT-I 5APA amidated on the ω-amine function of its C-terminal aliphatic chain with BH*

ϵ-BH* [K7]DRM 5APA

[K7]DRM 5APA amidated on the ϵ-amine function of Lys⁷ with BH*

PTH

phenylthiohydantoin

PAO

phenylarsine oxide

PBS

phosphate-buffered saline.
- Received August 1, 1996.
- Revision received November 4, 1996.