Does GEC1 Enhance Expression and Forward Trafficking of the Kappa Opioid Receptor (KOR) via Its Ability to Interact with NSF Directly?

Abstract

We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.

Keywords: GABARAP; GATE16; GEC1; Kappa opioid receptor; NSF; Tubulin.

Fig 1.. GEC1 interacts with NSF depending on the form of nucleotide bound to NSF.

Pull-down experiments were carried out as described in Experimental Procedures. The purified His₆-NSF (~100 nM) was incubated with glutathione-Sepharose 4B beads pre-loaded with GST and GST-GEC1 for 1 h at RT, in buffer BB with the presence or absence of ATP (5 mM), AMP-PNP (5 mM) or ADP (5 mM), EDTA (5 mM) and/or MgCl₂ (10 mM). For upper panel, the His₆-NSF was detected by blotting with HisProbe-HRP (Pierce) followed by enhanced chemiluminescence reagents. For middle panel, the signals from 4–8 times of independent experiments, similar to the representative one shown in the upper panel, were captured via the Fujifilm LAS1000 plus imaging system, quantified by the affiliated Image Gauge software, and normalized by the signals under the condition of lane 9 (with ATP and Mg⁺⁺). For lower panel, the same membrane of upper panel was stained with 0.5% Ponceau S in 5% acetic acid, which shows the molecular weights and amounts of the GST and GST-GEC1.

Fig 2.. GEC1 interacts with D1 and/or D2 domain(s) of NSF, but not N domain.

Pull-down experiments were carried out as described in Experimental Procedures. Each of the purified His₆-NSF full-length or domains (~200 nM) was incubated with glutathione-Sepharose 4B beads pre-loaded with GST and GST-GEC1 for 1 h at RT, in buffer BB with ATP (5 mM) and MgCl₂ (10 mM). His₆-NSF was detected by blotting with HisProbe-HRP (Pierce) followed by enhanced chemiluminescence reagents. The figures represent one of three experiments performed with similar results.

Fig. 3.. NSF does not interact with the kappa opioid receptor c-tail (KCT) directly (A), but it forms a protein complex in vitro with KCT via GEC1 (B).

(A) The purified His₆-NSF (~100 nM) was incubated with glutathione-Sepharose 4B beads pre-loaded with GST, GST-GEC1 or GST-KCT for 1 h at RT, in buffer BB with ATP (5 mM) and MgCl₂ (10 mM). For upper panel, the bound proteins were washed, eluted from the beads by 2 × SDS-PAGE sample buffer, resolved by 8% SDS-PAGE, transferred onto a PVDF membrane, and detected as described in Fig. 1. For lower panel, the same membrane of upper panel was stained with 0.5% ponceau S in 5% acetic acid, which shows the molecular weights and amounts of the GST and GST fusion proteins; (B) The purified His₆-S.tag-GEC1 (1–6 μg / μl, ~100–600 nM) was incubated with glutathione-Sepharose 4B beads pre-loaded with GST or GST-KCT for 1 h at RT, in the presence or absence of His₆-NSF (~100 nM) (The molar ratio is 1 NSF hexamer: 6–36 GEC1), in buffer BB with ATP (5 mM) and MgCl₂ (10 mM). The figures represent one of 2–4 experiments performed with similar results.

Fig. 4.. NSF and/or α-SNAP do not disrupt KCT-GEC1 interaction.

The purified His₆-S.tag-GEC1 (~100–600 nM) was incubated with glutathione-Sepharose 4B beads pre-loaded with GST or GST-KCT for 1 h at RT, in the presence or absence of His₆-NSF (~100 nM) and/or His₆-α-SNAP (1 μg / 300 μl, ~50 nM) (The molar ratio is 1 NSF hexamer : 3 α-SNAP : 6–36 GEC1), in buffer BB with ATP (5 mM) and MgCl₂ (10 mM). The bound proteins were washed, eluted from the beads by 2 × SDS-PAGE sample buffer, resolved by 15% SDS-PAGE, transferred and detected as described in Fig. 1. The figures represent one of four experiments performed with similar results.