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. 1996 Nov 15;271(46):29279-85.
doi: 10.1074/jbc.271.46.29279.

Sequestration of the delta opioid receptor. Role of the C terminus in agonist-mediated internalization

N Trapaidze  1 , D E Keith, S Cvejic, C J Evans, L A Devi
Affiliations

Sequestration of the delta opioid receptor. Role of the C terminus in agonist-mediated internalization

N Trapaidze et al. J Biol Chem. .

Abstract

The primary structure of the opioid receptors have revealed that many of the structural features that are conserved in other G protein-coupled receptors are also conserved in the opioid receptors. Upon exposure to agonists, some G protein-coupled receptors internalize rapidly, whereas other structurally homologous G protein-coupled receptors do not. It is not known whether opioid receptors are regulated by rapid endocytosis. In transfected Chinese hamster ovary cells expressing the epitope-tagged wild type delta opioid receptor, exposure to 100 nM [D-Ala2,D-Leu5]enkephalin causes internalization of the receptor within 30 min as determined by confocal microscopy. The rate of internalization of the wild type receptor is rapid with a half-maximal reduction by about 10 min, as determined by the reduction in mean surface receptor fluorescence intensity measured using flow cytometry. In contrast, the cells expressing receptors lacking the C-terminal 15 or 37 amino acids exhibit a substantially slower rate of internalization. Furthermore, the cells expressing receptors with point mutations of any of the Ser/Thr between Ser344 and Ser363 in the C-terminal tail exhibit a significant reduction in the rate of receptor internalization. These results suggest that a portion of the C-terminal tail is involved in receptor internalization. Agents that block the formation of clathrin-coated pits considerably reduce the extent of agonist-mediated internalization of the wild type receptor. Taken together, these results suggest that the wild type opioid receptor undergoes rapid agonist-mediated internalization via a classic endocytic pathway and that a portion of the C-terminal tail plays an important role in this internalization process.

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Figures

Fig. 1
Fig. 1. Schematic representation of the C-terminal tail of wild type and mutant δ opioid receptors
The C-terminal tail residues 333–372 of the wild type receptor is in a single-letter amino acid code. The asterisks point to the residues selected for generating mutants, and the numbers indicate the amino acid positions; the numbering is according to Evans et al. (4). The amino acid sequence of the mutants identical to the wild type are represented by a line and the changes are as indicated.
Fig. 2
Fig. 2. Confocal immunofluorescence microscopy of epitope-tagged wild type and mutant δ opioid receptors expressed in CHO cells
CHO cells expressing wild type (A and B) or ΔC15 receptor (C and D) were incubated in the absence (A and C) or presence of 100 nM DADLE (B and D) for 30 min. Fixation, permeabilization, and immunofluorescence staining of the receptors with the monoclonal antibody against the epitope tag are as described under “Experimental Procedures.” Cells were imaged by confocal fluorescence microscopy, using a plane of focus adjusted 3– 6 mm above the surface of the coverslip. This produces a cross-section through the center of the cell. Bright staining of the plasma membrane is apparent in A, C, and D, while prominent intracellular staining that appears as punctate staining within the cytoplasm is seen in C.
Fig. 3
Fig. 3. Kinetics of receptor internalization
The cells expressing wild type (□), ΔC7 (●), ΔC15 (■), or ΔC37 (○) were treated with 100 nM DADLE at 37 °C for the times indicated, stained, and analyzed by flow cytometry as described under “Experimental Procedures.” Internalization of the receptors is indicated by a reduction in the fluorescence measured in the cell population. The mean fluorescence after subtracting autofluorescence of cells (stained with second antibody alone) without DADLE treatment is taken as 100%. Percent internalization is defined as the percent reduction in mean fluorescence following various times of treatment with DADLE. The data represent the mean ± S.E. of three to four experiments. The data for cells expressing 1–2 × 105 receptors/cell are presented; similar dose-response curves were observed with additional transfected cultures expressing different numbers of receptors.
Fig. 4
Fig. 4. Dose response for the DADLE-induced internalization
The cells expressing wild type (□), ΔC7 (●), ΔC15 (■), or ΔC37 (○) were treated with various concentrations of DADLE for 30 min at 37 °C for the times indicated, stained, and analyzed by flow cytometry as described under “Experimental Procedures.” Internalization of the receptors is indicated by a reduction in the fluorescence measured in a cell population. The mean fluorescence after subtracting autofluorescence of cells is taken as 100%. Percent internalization is defined as the percent reduction in mean fluorescence following various doses of treatment with DADLE. The data represent the mean ± S.E. of three to four experiments. The data for cells expressing 1–2 × 105 receptors/cell are presented; similar dose-response curves were observed with additional transfected cultures expressing different numbers of receptors.
Fig. 5
Fig. 5. Kinetics of the mutant receptor internalization
The cells expressing wild type (□——□), Ser344 →Gly (●·····●), Thr352 →Ala (○- - - - -○), Thr353 → Ala (■– – –■), Thr358 → Ala (×–·–·–×), Thr361 → Ala (▲– – –▲), or Ser363 →Ala (•·····•) were treated with 100 nM DADLE at 37 °C for the times indicated, stained, and analyzed by flow cytometry as described under “Experimental Procedures.” Internalization of the receptors is indicated by a reduction in the fluorescence measured in the cell population. Percent internalization is defined as the percent reduction in mean fluorescence following various times of treatment with DADLE; mean fluorescence of cells without DADLE treatment is taken as (control) 100%. The data for cells expressing 1–2 × 105 receptors/cell are presented; similar dose-response curves were observed with additional transfected cultures expressing different numbers of receptors.
Fig. 6
Fig. 6. Time course of down-regulation in cell lines expressing mutant receptors
The cells expressing Ser344 → Gly (●·····●), Thr352 → Ala (○- - - - -○), Thr353 → Ala (■– – –■), Thr358-Ala (×–·–·–×), Thr361 → Ala (▲– – –▲), and Ser363 → Ala (•·····•) were treated with 100 nM DADLE for various periods of time, and the diprenorphine binding was carried out as described under “Experimental Procedures.” The receptor number in cells treated for 1–3 min with DADLE is taken as “control” (100%). The data represent mean ± S.E. of three to four experiments. The data for cells expressing 1–2 × 105 receptors/cell are presented; similar dose-response curves were observed with additional transfected cultures expressing different numbers of receptors.
Fig. 7
Fig. 7. Effect of various agents on the DADLE-induced rapid internalization
The cells expressing the wild type receptors were treated with various agents at indicated concentrations 15 or 30 min prior to the agonist treatment. The wells were treated with 100 nM DADLE for 30 min at 37 °C, chilled, washed, stained, and analyzed by flow cytometry as described under “Experimental Procedures.” The mean fluorescence of the cells not treated with DADLE is taken as control (100%). The data represent mean ± S.E. of three to four experiments. DA, DADLE; TPA, 12-O-tetradecanoylphorbol-13-acetate; Cal-phos; calphostin C; Stauro, staurosporin; OAG, 1-oleoyl-2-acetyl-sn-glycerol; H89, N-[2((3-(4-bromophenyl)-2-propenyl)-amino)ethyl]-5-isoquinoline sulfonamide; Okadaic, okadaic acid.
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