Glucagon‐mediated internalization of serine‐phosphorylated glucagon receptor and Gsα in rat liver

Abstract

To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.

Glucagon receptors

“…The change in glucagonbinding activity in plasma membrane and endosomal fractions results from a change in receptor number, with receptor affinity remaining unaffected [23]. A time-dependent increase in GR content has also been demonstrated in hepatic endosomes at 5-45 min after glucagon injection using an antibody directed against the distal end of the intracellular C-terminal tail of GR [95,151]. The loss of both [ 125 I]iodoA C H T U N G T R E N N U N G glucagon binding [23] and immunoreactive GR [151] from plasma membranes is comparably partial, indicating that the changes in the subcellular distribution of GR induced by its ligand are quantitatively modest.…”

“…A time-dependent increase in GR content has also been demonstrated in hepatic endosomes at 5-45 min after glucagon injection using an antibody directed against the distal end of the intracellular C-terminal tail of GR [95,151]. The loss of both [ 125 I]iodoA C H T U N G T R E N N U N G glucagon binding [23] and immunoreactive GR [151] from plasma membranes is comparably partial, indicating that the changes in the subcellular distribution of GR induced by its ligand are quantitatively modest. In acutely glucagon-treated rats, no loss of hepatic GR is observed [23], whereas chronically hyperglucagonemic rats and isolated hepatic cells exposed to glucagon in vitro display a net decrease in total glucagonbinding activity [4].…”

“…Moreover, a mutant receptor containing a total of seven Ser-to-Ala mutations in the C-terminal 47 amino acids displays a dramatically decreased glucagon-dependent internalization and phosphorylation, suggesting a predominant role for phosphorylation of the C-terminal tail in GR endocytosis [72]. The post-endosomal fate of internalized GR and its lysosomal association has been evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection into rats [151]. A partial endolysosomal transfer of internalized GR occurred with no immunoreactive intermediate degradative products generated from the GR at the endosomal and lysosomal locus [151].…”

“…Internalization and endosomal fate of glucagon-GR complex in rat hepatocytes. Following binding of glucagon to its cell surface receptor, the occupied GR is rapidly internalized and Ser phosphorylated both at the plasma membrane and within endosomes [151]. Coincident with glucagon-GR complex endocytosis, both the 45-and 47-kDa Gsa proteins are massively internalized.…”

“…The glucagon-processing cathepsins induce proteolytic modifications in the C terminus, internal and N terminus of the hormone, producing more than 30 endosomal metabolites [139]. No study has been directed towards the cell-mediated metabolism of glucagon analogs except for the desHis 1 -[Glu 9 ]glucaA C H T U N G T R E N N U N G gon antagonist [151]. In vitro proteolysis of the antagonist and wild-type glucagon using endosomal lysates were comparable at acidic pH (pH 4), but a reduced rate of degradation was observed for des-His 1 -[Glu 9 ]glucaA C H T U N G T R E N N U N G gon (less than 65 % of that of glucagon) at neutral pH.…”