Dynorphin peptides differentially regulate the human kappa opioid receptor

Abstract

Dynorphins, endogenous peptides for the kappa opioid receptor, play important roles in many physiological and pathological functions. Here, we examined how prolonged treatment with three major prodynorphin peptides, dynorphin A (1-17) (Dyn A), dynorphin B (1-13) (Dyn B) and alpha-neoendorphin (alpha-Neo), regulated the human kappa opioid receptor (hKOR) stably expressed in Chinese hamster ovary (CHO) cells. Results from receptor binding and [(35)S]GTPgammaS binding assays showed that these peptides were potent full agonists of the hKOR with comparable receptor reserve and intrinsic efficacy to stimulate G proteins. A 4-h incubation with alpha-Neo at a concentration of approximately 600xEC(50) value (from [(35)S]GTPgammaS binding) resulted in receptor down-regulation to a much lower extent than the incubation with Dyn A and Dyn B at comparable concentrations ( approximately 10% vs. approximately 65%). Extending incubation period and increasing concentrations did not significantly affect the difference. The plateau level of alpha-Neo-mediated receptor internalization (30 min) was significantly less than those of Dyn A and Dyn B. Omission of the serum from the incubation medium or addition of peptidase inhibitors into the serum-containing medium enhanced alpha-Neo-, but not Dyn A- or Dyn B-, mediated receptor down-regulation and internalization; however, the degrees of alpha-Neo-induced adaptations were still significantly less than those of Dyn A and Dyn B. Thus, these endogenous peptides differentially regulate KOR after activating the receptor with similar receptor occupancy and intrinsic efficacy. Both stability in the presence of serum and intrinsic capacity to promote receptor adaptation play roles in the observed discrepancy among the dynorphin peptides.

Figure 1. Generation and amino acid sequences of naturally occurring dynorphins

(A). Schematic representation of human preprodynorphin shows that endogenous dynorphins are generated from the precursor through differential cleavages by proprotein convertases at paired and single basic amino acids followed by carboxypeptidases. (B). Amino acid sequences of prodynorphin gene products display that all peptides contain Leu⁵-Enk at the N-termini, but have different C-terminal extensions.

Figure 2. Dyn A, Dyn B and α-Neo differentially down-regulated the mature form of FLAG-hKOR

(A). Immunoblotting of the mature FLAG-hKOR. Cells were incubated with each peptide at 37°C for 4 h at a concentration ∼1000-fold of its EC₅₀ in stimulating [³⁵S]GTPγS binding. Subsequently 2×10⁵ cells were solubilized and subjected to SDS-PAGE. FLAG-hKOR was detected by immunoblotting with polyclonal anti-FLAG antibody. (B). Quantitation and comparison of peptide-induced down-regulation of mature receptor. Each value represents mean ± S.E. of four independent experiments performed with similar results. *** P < 0.001 compared to Dyn A- or Dyn B-treated cell group using one-way ANOVA followed by Tukey's post hoc test.

Figure 3. Time- and concentration-dependence of peptide-mediated down-regulation of mature FLAG-hKOR

Cells were treated with (A) Dyn A (0.2 μM), Dyn B (0.5 μM) or α-Neo (0.7 μM) for indicated time periods or (B) different concentrations of the peptides for 4 h. FLAG-hKOR was detected by immunoblotting and quantitated (mean ± S.E., n=3) by densitometry. *** P < 0.001 compared to Dyn A- or Dyn B-treated (16 h) cell group; ** P < 0.01 compared to Dyn A (20 μM)- and Dyn B (50 μM)-treated cell group using one-way ANOVA followed by Tukey's post hoc test.

Figure 4. Concentration-dependence of peptide-mediated internalization of surface FLAG-hKOR

Cells were treated with different concentrations of Dyn A, Dyn B and α-Neo for 30 min. Surface receptors were labeled by monoclonal M₁ anti-FLAG antibody and then Alexa Fluo 488-conjugated goat anti-mouse IgG antibody. Immunofluorescence intensity was determined using fluorescence activated cell sorter (FACS). Each value represents mean ± S.E. of three independent experiments. * P < 0.05 compared to Dyn A (20 μM)- and Dyn B (50 μM)-treated cell group using one-way ANOVA followed by Tukey's post hoc test.

Figure 5. Effects of serum and peptidase inhibitors on the peptide-promoted down-regulation and internalization of FLAG-hKOR

(A). Immunoblotting of mature FLAG-hKOR One hour prior to ligand treatment, complete medium was removed and Opti-MEM I reduced-serum medium with or without 10% FBS and/or peptidase inhibitor cocktail was added as indicated. Ligand treatment and immunoblotting assay were conducted as described in Fig. 2A. (B). Quantitation and comparison of peptide-induced down-regulation of mature receptor. (C). Quantitation and comparison of peptide-induced receptor internalization. Replacement of incubation medium and addition of 10% FBS and/or peptidase inhibitor cocktail followed the same protocol as described in (A), and the internalization assay for surface FLAG-hKOR was conducted as described in Fig. 4. Each value represents mean ± S.E. of three independent experiments performed with similar results. * P < 0.05 compared to corresponding Dyn A- or Dyn B-treated cell group; # P < 0.05 compared to α-Neo-treated cell group in the presence of 10% FBS by one-way ANOVA followed by Tukey's post hoc test.