| Glucagon levels are elevated in some liver diseases and in diabetes. In recent decades, diabetes has become a global epidemic and a serious threat to public health. Diabetes is characterized by high blood glucose levels due to impaired glucagon-insulin balance. Increased glucagon secretion leads to abnormal stimulation of glucagon receptors and consequently elevated glucose production in the liver. Glucagon receptor (GR), a member of the G protein-coupled receptor (GPCR) superfamily, has emerged as an attractive target for treatment of diabetes. The scope of this study was to elucidate the key pathways of GR desensitization, internalization and downregulation. Glucagon treatment recruited PKCalpha to the plasma membrane where it colocalized with GR. The interaction between GR and PKCalpha was confirmed by immunoprecipitation. While PMA alone had no effect on internalization, 100 nM glucagon induced GR internalization with a maximum of 40--70% of total receptors internalized at 30 min of treatment. Stimulation of PKC with PMA increased GR internalization by 25--40% over control. We observed that GRK2, GRK3 and GRK5 enhanced GR internalization by 25--35%. The effect of PMA was increased in the presence of GRK2 overexpression and decreased when dominant negative PKCalpha was overexpressed. These results suggest that PKCalpha enhances ligand-induced GR internalization through stimulation of GRK2.; In HEK 293 cells GR utilized both clathrin- and caveolin-mediated endocytosis. beta-arrestin1 and beta-arrestin2 colocalized with GR at the plasma membrane and in the cytosol. Furthermore, glucagon induced beta-arrestin1 and beta-arrestin2 translocation from the cytosol to the perinuclear region. Overexpression of either beta-arrestin1 or beta-arrestin2 promoted GR internalization. Interestingly, both beta-arrestins colocalized with caveolin-1, demonstrating a point of interaction between the caveolar and the clathrin-mediated endocytosis. Internalized GRs recycled to the plasma membrane within 30-60 min of glucagon removal. During recycling, the receptors colocalized with markers of recycling vesicles, Rab4 and Rab11. Disruption of actin cytoskeleton or microtubules reduced the recycling efficiency by 10--15% without any effect on internalization. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol, where they colocalized with GR. Inhibition of vesicular acidification to prevent uncoupling of glucagon from GR, had no effect on either internalization or recycling. Upon prolonged treatment with glucagon (2--5 h), GRs were targeted to lysosomes. At 5 h of treatment, approximately 40% of receptors were degraded. Degradation was blocked by lysosomal inhibitors and unaffected by proteosomal inhibitors.; In conclusion, this study demonstrated that protein kinases GRK2, GRK3, GRK5 and PKCalpha play a role in GR internalization. Upon stimulation with glucagon, GRs internalize either via clathrin mediated endocytosis or via caveolar endocytosis. Internalized receptors colocalize with caveolin-1, actin and beta-arrestins. After internalization, the receptors are either rapidly recycled to the plasma membrane via Rab4 and Rab11-positive vesicles, or degraded in lysosomes. |
