Leucine-2-alanine enkephalin induced delta opioid receptors internalization expressed stably in CHO cells
Abstract
AIM:
To characterize the internalization of delta opioid receptors (DOR) stably expressed in Chinese hamster ovary (CHO) cells and the role of the C-terminal in this process.
METHODS:
Receptor membrane anchoring was shown by immunofluorescence microscopy. Receptor internalization was assessed by measuring the radioligand binding resistant to the acid-buffer wash.
RESULTS:
Originally, all the wild-type (CHO-W) and C-truncated (CHO-T) DOR expressed were localized to the membrane. Agonist [3H] leucine-2-alanine enkephalin (LAE) but not the antagonist [3H]diprenorphine (Dip) induced rapid receptor internalization. The internalization of C-truncated DOR in CHO-T was similar to that of the wild-type in maximal level, but climbed up more slowly. DOR internalization was extracellular osmolarity- and temperature-sensitive. Pertussis toxin and universal protein kinase inhibitor staurosporine had no effect on it.
CONCLUSION:
DOR internalization is an agonist and clathrin-coated pits dependent, but post-receptor cellular signal transduction independent process; moreover, the C-terminal of DOR, not engaged in membrane anchoring, affects the initialization of DOR internalization.
Keywords:
To characterize the internalization of delta opioid receptors (DOR) stably expressed in Chinese hamster ovary (CHO) cells and the role of the C-terminal in this process.
METHODS:
Receptor membrane anchoring was shown by immunofluorescence microscopy. Receptor internalization was assessed by measuring the radioligand binding resistant to the acid-buffer wash.
RESULTS:
Originally, all the wild-type (CHO-W) and C-truncated (CHO-T) DOR expressed were localized to the membrane. Agonist [3H] leucine-2-alanine enkephalin (LAE) but not the antagonist [3H]diprenorphine (Dip) induced rapid receptor internalization. The internalization of C-truncated DOR in CHO-T was similar to that of the wild-type in maximal level, but climbed up more slowly. DOR internalization was extracellular osmolarity- and temperature-sensitive. Pertussis toxin and universal protein kinase inhibitor staurosporine had no effect on it.
CONCLUSION:
DOR internalization is an agonist and clathrin-coated pits dependent, but post-receptor cellular signal transduction independent process; moreover, the C-terminal of DOR, not engaged in membrane anchoring, affects the initialization of DOR internalization.