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A Constitutively Internalizing and Recycling Mutant of the μ‐Opioid Receptor
Author(s) -
Segredo Veronica,
Burford Neil T.,
Lameh Jelveh,
Sadée Wolfgang
Publication year - 1997
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1997.68062395.x
Subject(s) - mutant , opioid , receptor , opioid receptor , chemistry , neuroscience , microbiology and biotechnology , biology , biochemistry , gene
Internalization and recycling of G protein‐coupled receptors (GPCRs), such as the μ‐opioid receptor, largely depend on agonist stimulation, whereas certain other receptor types recycle constitutively, e.g., the transferrin receptor. To investigate structural domains involved in μ‐opioid receptor internalization, we constructed two truncation mutants bracketing a Ser/Thr‐rich domain ( 354 ThrSerSerThrIleGluGlnGlnAsn 362 ) unique to the C‐terminus of the μ‐opioid receptor (mutants Trunc354 and Trunc363). Ligand binding did not differ substantially, and G protein coupling was slightly lower for these μ‐receptor constructs, in particular for Trunc363. To permit localization of the receptor by immunocytochemistry, an epitope tag was added to the N‐terminus of the wildtype and mutant receptors. Both the wild‐type μ‐opioid receptor and Trunc363 resided largely at the plasma membrane and internalized into vesicles upon stimulation with the agonist [ d ‐Ala 2 , N ‐Me‐Phe 4 ,Gly‐ol 5 ]‐enkephalin. Internalization occurred into vesicles that contain transferrin receptors, as shown previously, as well as clathrin, but not caveolin. In contrast, even without any agonist present, Trunc354 colocalized in intracellular vesicles with clathrin and transferrin receptors, but not caveolin. On blocking internalization by hyperosmolar sucrose or acid treatment, Trunc354 translocated to the plasma membrane, indicating that the mutant internalized into clathrin‐coated vesicles and recycled constitutively. Despite agonist‐independent internalization of Trunc354, basal G protein coupling was not elevated, suggesting distinct mechanisms for coupling and internalization. Furthermore, a portion of the C‐terminus, particularly the Ser/Thr domain, appears to suppress μ‐receptor internalization, which can be overcome by agonist stimulation. These results demonstrate that a mutant GPCR can be constructed such that internalization, normally an agonist‐dependent process, can occur spontaneously without concomitant G protein activation.