Fig 1: Effect of siRNA knockdown of RAMP2 in CHO-K1-GCGR-RAMP2 cells on (a) human RAMP2 (hRAMP2) expression by qPCR, (b) cAMP accumulation, and (c) Ca2+ flux in response to glucagon. Each peptide concentration was tested in duplicate or triplicate in each experiment. Values calculated as a mean from a minimum of two separate experiments. **P < 0.01; ***P < 0.001. Values shown are ±standard error of the mean. ATP, adenosine triphosphate.
Fig 2: Human GCGR (hGCGR)–mediated β-arrestin recruitment in CHO-K1- βArr-GCGR cells with or without RAMP2 by endogenous ligands (a) glucagon, (b) GLP-1, and (c) oxyntomodulin and (d) analog G(X). Each peptide concentration was tested in duplicate or triplicate in each experiment. Results are expressed as a percentage of maximal glucagon-mediated β-arrestin recruitment. Values calculated as a mean from a minimum of four separate experiments. ****P < 0.0001 comparing Emax for CHO-K1-βArr-GCGR cells with or without RAMP2. Values represent the mean ± standard error of the mean. hRAMP2, human RAMP2.
Fig 3: Intracellular Ca2+ flux in response to varying doses of glucagon in real time in CHO-K1-GCGR cells (a) without RAMP2 and (b) with RAMP2 [measured in relative fluorescence unit (RFU) fold increase from baseline RFU]. Human GCGR (hGCGR)–mediated Ca2+ flux in CHO-K1-GCGR cells with or without RAMP2 by ligands (c) glucagon, (d) GLP-1, and (e) oxyntomodulin and (f) analog G(X). Each peptide concentration was tested in duplicate or triplicate in each experiment. Values calculated as a mean from a minimum of four separate experiments (unless stated otherwise). **P < 0.01 comparing Emax for CHO-K1-GCGR cells with or without RAMP2. Values represent the mean ± standard error of the mean. ATP, adenosine triphosphate; hRAMP2, human RAMP2.
Fig 4: Effect of RAMP2 on GCGR localization. (a) Representative image showing that GCGR-GFP (green) and RAMP2-CFP (red) can colocalize (yellow) within the cytosolic compartment following their overexpression (n = 15 cells) (zoomed images are inset to the right). (b) Representative image showing that GCGR-GFP expression is predominantly at the membrane/surface in HEK cells without RAMP2 overexpression (n = 8 cells). (c) A negative control indicating that the 405-nm laser does not excite GCGR-GFP (n = 3 cells). (d) Overexpression of nonnative protein (pcDNA3.1) does not interfere with the distribution of the GCGR-GFP, which remains at the membrane (n = 7 cells). (e) Overexpression of nontagged RAMP2 leads to redistribution of GCGR-GFP into the cell (n = 7 cells) (zoomed images are inset to the right). (f) Bar graph showing that overexpression of either RAMP2-CFP or nontagged RAMP2, but not pcDNA3.1, leads to a significant reduction in cell surface GCGR-GFP expression (**P < 0.01) (n = 8 to 14 cells from at least three independent experiments). Scale bar = 10 µm. Values represent the mean ± standard error of the mean. NS, not significant.
Fig 5: (a) Specific binding of I125-glucagon to the GCGR in CHO-K1-GCGR cells with or without RAMP2 (P < 0.0001). (b) The protein content was determined by Bradford assay (used here as a surrogate marker for the number of cells) for CHO-K1-GCGR cells with or without RAMP2. Whole cell binding of (c) glucagon, (d) GLP-1, (e) oxyntomodulin, and (f) analog G(X) to the human GCGR (hGCGR). Whole CHO-K1-GCGR cells with or without RAMP2 were used. I125-glucagon was used as the competing peptide in all assays and IC50 values were calculated as a mean of four separate experiments (except for GLP-1, for which n = 2), with each peptide concentration performed in duplicate or triplicate during an individual experiment. Values represent the mean ± standard error of the mean. hRAMP2, human RAMP2.
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