Similarly, dissociated primary mouse ��-cells were transfected with Epac-camps and mCherry-SUMO-1 or mCherry alone as control, driven by rat insulin promoter-2 to ensure ��-cell-specific expression. Addition of 100 nM exendin-4 caused a fivefold increase in the FRET ratio in MIN6 cells further info expressing mCherry compared with cells expressing mCherry-tagged SUMO-1 (Fig. 2, A and C). Mouse primary ��-cells expressing mCherry showed a 3.7-fold increase in FRET ratio in response to exendin-4 compared with cells expressing mCherry-SUMO-1 (Fig. 2, B and C). This result shows that both, in insulinoma cells and primary ��-cells overexpressing SUMO-1, stimulation of GLP-1 receptor by exendin-4 did not cause an increase in cAMP. Fig. 2. Overexpression of SUMO-1 decreases cAMP response following agonist stimulation of glucagon-like peptide-1 receptor (GLPP-1R).

MIN6 cells and mouse primary islet cells were transfected with Epac-camps and mCherry-SUMO or mCherry vector. Dynamic changes … Next, we assessed total cAMP produced by exendin-4 treatment in MIN6 cells overexpressing SUMO-1 and compared it with control cells using a cAMP-specific ELISA. MIN-6 cells were transfected with GFP or GFP-tagged SUMO-1. The cells were sorted by FACS 48 h posttransfection and treated with 100 nM exendin-4. Control cells expressing GFP alone showed a 1.9-fold increase in total cAMP concentration, whereas cells expressing GFP-tagged SUMO showed only a 1.3-fold increase following exendin-4 treatment (Fig. 2D).

A similar response was seen when exendin-4 treatment was done in the presence of phoshodiesterase inhibitor IBMX despite slight elevation in basal cAMP compared with cells not treated with IBMX. This assay confirmed that enhanced expression of SUMO-1 resulted in significantly reduced cAMP response following agonist treatment and that SUMO-mediated downregulation of GLP-1R signaling is independent of phoshodiesterase activity (Fig. 2D). We next investigated whether SUMO-1 directly modified the GLP-1 receptor. SUMO-1 Binds Covalently and Noncovalently to the GLP-1 Receptor Similar to ubiquitin, SUMO covalently binds to its target proteins through a lysine residue. However, SUMO-1 is also known to interact noncovalently with some proteins, and noncovalent interaction was shown to be required for efficient E3 SUMO ligase activity (15, 23).

To detect noncovalent interaction, COOH-terminally tagged GLP-1R-HA was expressed in MIN6 cells and coimmunoprecipitated with an anti-HA antibody. A double band corresponding to GFP-SUMO-1 coimmunoprecipitated with the GLP-1R-HA protein, demonstrating the presence of a noncovalent interaction between the two proteins (Fig. 3A). To investigate whether SUMO-1 covalently interacts with GLP-1R, we transfected MIN6 cells with 1d4 epitope-tagged GLP-1R and GFP-SUMO-1 or with a conjugation-deficient GFP-SUMO��GG construct where the Drug_discovery last four amino acids containing the diglycine motif were deleted.