PMC

The C terminus of the wild-type GABARAP, but not the G116A mutant (GABARAP-AL), is cleaved, similar to GEC1, in CHO cells (A) and in Neuro 2A cells (B). The experiment was performed similarly as described in the legend for . The blots represent one of the three experiments. IB, immunoblot.

A, amino acid sequence comparison among GEC1 and its three analogues GABARAP, GATE16, and Atg8. The residue(s) C-terminal to the conserved glycine (equivalent to Gly¹¹⁶ of GEC1) of these analogues are cleaved, and the conserved glycine becomes the C terminus. Under starvation conditions, the glycine is conjugated to phosphatidylethanolamine. B, cDNA constructs used in this study. cDNA constructs of the mutants and wild types of GEC1 and GABARAP shown are inserted into the pcDNA3.1 or pCMV-HA mammalian expression vector.

Contrary to GEC1, the C-terminal cleavage is essential for GABARAP to enhance KOPR total and cell surface expression. Experiments were carried out as described in the legend for . A, each value is mean ± S.E. of four experiments. [³H]DIP, [³H]diprenorphine. B, the immunoblot (IB) represents one of the three experiments. *, p < 0.05, ***, p < 0.0005 when compared with the vector control by one-way ANOVA followed by Dunnett's post hoc test.

A, GEC1 undergoes C-terminal cleavage, but GEC1AK and GEC1A do not. Each construct was transfected into CHO cells with Lipofectamine. Forty hours later, cells were collected, dissolved in SDS loading buffer, and loaded onto 12% SDS-PAGE (4 × 10⁵ cells/lane), and immunoblotting (IB) was performed with the antibodies indicated. B, HA-GEC1-Myc and the HA-GEC1AK-Myc were transfected into Neuro 2A cells and immunoblotted in a similar manner as in A. Note that C-terminal cleavage of HA-GEC1-Myc was incomplete, suggesting less robust Atg4B activity in Neuro 2A cells. The figures shown are the results of one of three independent experiments.

GEC1 and GEC1AK affect KOPR expression with similar time courses. Transfection and intact cell binding were performed as described in the legend for . KOPR binding was performed with ∼1 nm [³H]diprenorphine ([³H]DIP) on intact cells using 10 μm Naloxone to define nonspecific binding. Each value is mean ± S.E. of three or four experiments. ***, p < 0.0005, **, p < 0.005, *, p < 0.05 when compared with the vector control group at same time point by one-way ANOVA followed by Dunnett's post hoc test.

Co-localization of KOPR with GEC1, GEC1-A, and GABARAP, but not with GABARAP-A and GEC1F60A. HEK293 cells were cultured on coverslips for 24 h and then co-transfected with KOPR-EGFP (hκ-EGFP) and HA-tagged constructs as indicated with Lipofectamine 2000. Twenty-four hours after transfection, cells were fixed in 4% paraformaldehyde. Immunofluorescence was performed with mouse anti-HA antibodies followed by Texas Red-conjugated anti-mouse IgG for wild-type and mutant GEC1/GABARAP (red) and rabbit anti-giantin antibody and then Alexa Fluor-350-conjugated anti-rabbit IgG for the Golgi marker giantin (blue). Images shown are KOPR-EGFP (green, first column) co-expressed with HA-GEC1 (A), HA-GEC1-A (B), HA-GABARAP (C), HA-GABARAP-A (D), and HA-GEC1F60A (E) (red, second column). The Golgi marker giantin is shown in the third column (blue). Merged images are shown in the fourth column. These images are representatives of at least 50 images per row from four independent experiments.

C-terminal cleavage is not required for GEC1 to enhance total and cell surface KOPR expression. CHO cells stably expressing FLAG-hKOPR were transfected as described in the legend for , and cells were harvested 40 h later. A, KOPR binding was performed with 1 nm [³H]diprenorphine ([³H]DIP) on intact cells using 10 μm Naloxone and 1 μm dynorphin to define nonspecific binding for total and cell surface receptors, respectively. The results are presented as mean ± S.E. of five experiments. ***, p < 0.0005 when compared with the vector group by one-way ANOVA followed by Dunnett's post hoc test. B, aliquots of each transfected cells from A were immunoblotted (IB) with the antibodies indicated. Each lane was loaded with 20 μg of proteins. The blot represents one of the four experiments performed.

Knockdown of endogenous GEC1 or GABARAP reduces both total and cell surface KOPR expression in Neuro 2A cells. siRNAs targeting mouse genes gec1 (siGEC1) or gabarap (siGABARAP) were transfected into Neuro 2A cells stably expressing 3HA-hKOPR. siControl, a non-targeting siRNA, was used as the negative control. Thirty hours after transfection, cells were collected for receptor binding and immunoblotting assays. A, total and cell surface receptor binding was conducted with 1 nm [³H]diprenorphine ([³H]DIP) using 10 μm Naloxone and 1 μm dynorphin to define nonspecific binding for total and cell surface receptors, respectively. The results are presented as mean ± S.E. of four independent experiments. ***, p < 0.0005, *, p < 0.05 when compared with siControl group by one-way ANOVA followed by Dunnett's post hoc test. B, aliquots of transfected cells from A were immunoblotted (IB) with the antibodies indicated. Each lane was loaded with 20 μg of proteins. The blot represents one of the three experiments performed.

GEC1 exhibits higher affinity for KOPR-C-tail than GABARAP. Protein pulldown assays were performed to compare the interaction strength of the KOPR-C-tail with GEC1 and GABARAP. Purified GST-KOPR-C-tail (GST-KCT) or GST-DOPR-C-tail (GST-DCT) proteins were coupled to glutathione-Sepharose 4B beads. HA-GEC1 and HA-GABARAP were obtained by cleavage with thrombin of GST-HA-GEC1 and GST-HA-GABARAP coupled to glutathione-Sepharose 4B beads. Ten μl each of GST-KCT or GST-DCT beads was incubated overnight at 4 °C with different concentrations of HA-GEC1 or HA-GABARAP followed by immunoblotting analysis. A, one of the four immunoblotting (IB) results obtained. An aliquot of HA-GEC1 or HA-GABARAP from each concentration (10, 20, 40, and 80 μg/ml) was loaded as loading control (upper right) for every experiment and was used to generate standard curves as shown in B. DLU, digital line unit. C, the bound HA-GEC1 or HA-GABARAP was quantified using the standard curves in each data set and plotted against the incubation concentration. The result was presented as mean ± S.E. of four experiments. *, p < 0.05, **, p < 0.01 by one-way ANOVA followed by Dunnett's post hoc test.