Panoply™ Human GCGR Over-expressing Stable Cell Line

Endogenous satiety hormones offer an attractive target for anti-obesity drugs. Glucagon promotes weight loss by reducing food intake and increasing energy expenditure. To further understand the cellular mechanisms by which glucagon and its related ligands activate the glucagon receptor (GCGR), researchers investigated the interaction between GCGR and receptor activity-modifying protein (RAMP) 2 (a member of the receptor activity-modifying protein family). They investigated the effects of RAMP2 on GCGR using a combination of competition binding experiments, cell-surface enzyme-linked immunosorbent assays, functional assays assessing Gαs and Gαq pathways and β-arrestin recruitment, and small interfering RNA knockdown. This study demonstrated that co-expression of RAMP2 with GCGR reduced GCGR cell surface expression. Confocal microscopy confirmed this, demonstrating that RAMP2 colocalized with GCGR and led to a significant intracellular redistribution of GCGR. Furthermore, the presence of RAMP2 impaired signaling in the Gαs and Gαq pathways, as well as β-arrestin recruitment. This study suggests that RAMP2 may alter the agonist activity and trafficking of GCGR, which may be relevant for the generation of novel peptide analogs with selective agonist activity.

To investigate whether the RAMP2-induced changes in GCGR cAMP accumulation apply to other cell types, the researchers measured cAMP accumulation after glucagon stimulation in human hepatoma 7 cells overexpressing the human GCGR (Huh7-GCGR) with or without RAMP2 knockdown. Huh7-GCGR cells have low levels of endogenous RAMP2 expression, and RAMP2 silencing resulted in approximately 70% RAMP2 knockdown. In Huh7-GCGR cells with RAMP2 knockdown, there was no statistically significant change in glucagon titer, and a trend toward decreased Emax was observed, but this was not statistically significant (Figure 1).

Figure 1. In Huh7-GCGR cells, the knockdown of RAMP2 resulted in no change in potency and a trend toward decreased efficacy. (Cegla J, et al., 2017)