The ratio of membrane vs. cytosol-associated GLP-1R was drastically reduced by fivefold in three independent experiments when the receptor was coexpressed with SUMO-1 (Fig. 5, A and B). Together, these results demonstrate that sumoylation interferes with the selleck chemicals Pazopanib cell surface trafficking of the receptor, causing decreased receptor density at the membrane. Fig. 4. Intracellular retention of GLP-1R when coexpressed with SUMO-1. A: GLP-1R-GFP expressed with mCherry vector shows a predominant plasma membrane fluorescence of the GLP-1R-GFP. B: GLP-1R-GFP expressed with mCherry-SUMO-1 shows decreased plasma membrane … Fig. 5. Impaired cell surface trafficking of GLP-1R coexpressed with SUMO-1. A: top, MIN6 cells transfected with GLP-1R-GFP and empty vector or untagged SUMO-1 and cell surface biotinylation was carried out to isolate plasma membrane-bound proteins, purified .

.. Next, we tested whether partial knock down of Ubc-9 is able to rescue SUMO-mediated intracellular retention of GLP-1R. MIN6 cells transfected with GLP-1R-GFP and mCherry SUMO-1 were transduced with retroviral particles expressing shRNA against Ubc-9. Reduced expression of Ubc-9 resulted in diminished nuclear mCherry-SUMO, and GLP-1R-GFP was predominantly localized at the plasma membrane (Fig. 6). Fig. 6. Partial knock down of Ubc-9 rescues SUMO-mediated impaired trafficking of GLP-1R. MIN6 cells transfected with GLP-1R-GFP and mCherry SUMO-1 were transduced with retroviral particles expressing scrambled or short-hairpin RNA (shRNA) against Ubc-9. A: MIN6 …

Overexpression of SUMO-1 Results in Reduced Insulin Content and Agonist-Stimulated Insulin Secretion Transcription factors that are involved in insulin gene expression such as MafA and cleaved COOH-termini of ICA512 are targets of sumoylation (32) . SUMO-1 was not found to affect insulin content when overexpressed by transient transfection (6). However, MIN6 cells stably expressing GFP-SUMO-1 showed a 6.3-fold reduction in total insulin content compared with control cells that express empty vector. Similarly, GFP-SUMO-1 stable cells also showed a 2.3-fold reduction in secreted insulin when stimulated by exendin-4 compared with control cells (Fig. 7, A and B). These results indicate that prolonged expression of SUMO-1 reduces insulin content and GLP-1R agonist-stimulated insulin secretion. Fig. 7.

Overexpression of SUMO-1 results in reduced insulin content and secretion. MIN6 cells, stably expressing GFP-SUMO-1 cells, and control cells were stimulated with 100 nM exendin-4 for 3 h. Entinostat Insulin content in the supernatant and lysate was quantified by … We tested the presence of an endoplasmic reticulum (ER) stress-induced gene ��Chop�� in GFP-SUMO-overexpressing and control GFP-expressing cells by RT-PCR. cDNA was prepared from MIN6 cells overexpressing GFP-SUMO-1 and untransfected cells.