Fate of injected glucagon taken up by rat liver in vivo. Degradation of internalized ligand in the endosomal compartment

Abstract

The uptake and processing of glucagon into liver endosomes were studied in vivo by subcellular fractionation. After injection of [[125I]iodo-Tyr10]glucagon and [[125I]iodo-Tyr13]glucagon to rats, the uptake of radioactivity into the liver was maximum at 2 min (6% of the dose/g of tissue). On differential centrifugation, the radioactivity in the homogenate was recovered mainly in the nuclear (N), microsomal (P) and supernatant (S) fractions, with maxima at 5, 10 and 40 min, respectively; recovery of radioactivity in the mitochondrial-lysosomal (ML) fraction did not exceed 6% and was maximal at 20 min. On density-gradient centrifugation, the radioactivity associated first (2-10 min) with plasma membranes and then (10-40 min) with Golgi-endosomal (GE) fractions, with 2-5-fold and 20-150-fold enrichments respectively. Subfractionation of the GE fractions showed that, unlike the Golgi marker galactosyltransferase, the radioactivity was density-shifted by diaminobenzidine cytochemistry. Subfractionation of the ML fraction isolated at 40 min showed that more than half of the radioactivity was recovered at lower densities than the lysosomal marker acid phosphatase. Throughout the time of study, the [125I]iodoglucagon associated with the P, PM and GE fractions remained at least 80-90% trichloroacetic acid (TCA)-precipitable, whereas that associated with other fractions, especially the S fraction, became progressively TCA-soluble. On gel filtration and h.p.l.c., the small amount of degraded [125I]iodoglucagon associated with GE fractions was found to consist of monoiodotyrosine. Chloroquine treatment of [125I]iodoglucagon-injected rats caused a moderate but significant increase in the late recovery of radioactivity in the ML, P and GE fractions, but had little effect on the association of the ML radioactivity with acid-phosphatase-containing structures. Chloroquine treatment also led to a paradoxical decrease in the TCA-precipitability of the radioactivity associated with the P and GE fractions. Upon h.p.l.c. analysis of GE extracts of chloroquine-treated rats, at least four degradation products less hydrophobic than intact [125I]iodoglucagon were identified. Radio-sequence analysis of four of these products revealed three cleavages, affecting bonds Ser2-Gln3, Thr5-Phe6 and Phe6-Thr7. When GE fractions containing internalized [125I]iodoglucagon were incubated in iso-osmotic KCl at 30 degrees C, a rapid generation of TCA-soluble products was observed, with a maximum at pH 4. We conclude that endosomes are a major site at which internalized glucagon is degraded, endosomal acidification being required for optimum degradation.

Negative Regulation of Epidermal Growth Factor Signaling by Selective Proteolytic Mechanisms in the Endosome Mediated by Cathepsin B

“…high (glucagon) (16,17), moderate (insulin) (4,30) and low (EGF and prolactin) (31). Using an alternate approach in which hydrolysis was measured by the generation of degradation products from unlabeled peptides, we have confirmed that EGF and insulin hydrolysis, but not glucagon hydrolysis, appears to be limited.…”

“…Therefore, we prepared endosomes at 15 min after EGF injection and the intact vesicles were incubated for various times and pH at 37°C in an isotonic buffer mimicking the intracellular milieu (4,16,17) (Fig. 11).…”

“…The cytosolic fraction was isolated from homogenates in 0.25 M sucrose by differential centrifugation as described previously (14,16,19,20). The endosomal fraction was isolated by discontinuous sucrose gradient centrifugation and collected at the 0.25 M sucrose-1.0 M sucrose interface (4, 14, 16 -19).…”

Endosomal proteolysis of internalized proteins

“…1). Subsequent investigations have implicated endosomal proteases in processing polypeptide hormones such as glucagon [3][4][5][6] and PTH [7], growth factors such as EGF [8], plant and bacterial toxins such as ricin toxin [9,10] and cholera toxin [11]. The conclusion that degradation of internalized ligands occurs or is, at least in part, initiated in endocytic vesicles is based on: (i) the high recovery of internalized ligands in purified endosomes by subcellular fractionation studies, while little association was observed with lysosomes [12]; (ii) the major accumulation of internalized hormones [3,12] and toxins [11] induced by acidotropic agents (i.e.…”

“…Subsequent investigations have implicated endosomal proteases in processing polypeptide hormones such as glucagon [3][4][5][6] and PTH [7], growth factors such as EGF [8], plant and bacterial toxins such as ricin toxin [9,10] and cholera toxin [11]. The conclusion that degradation of internalized ligands occurs or is, at least in part, initiated in endocytic vesicles is based on: (i) the high recovery of internalized ligands in purified endosomes by subcellular fractionation studies, while little association was observed with lysosomes [12]; (ii) the major accumulation of internalized hormones [3,12] and toxins [11] induced by acidotropic agents (i.e. chloroquine), while effecting a minor accumulation of these ligands in lysosomes; (iii) the extraction from endosomes of polypeptide hormone fragments of insulin [13], glucagon [3,4,6] and EGF [8] using a reverse-phase high performance liquid chromatography procedure corresponding to the in vivo sites of hormone hydrolysis; (iv) the degradation of endosomal ligands in cell-free endosomes incubated at low pH [1,2,4,8,14]; (v) the identification using morphological [15], biochemical [16] and immunological criteria [1,6,17,18] of soluble and membrane-associated endosomal hydrolases; and (vi) the demonstration using iodinated protein ligands and 0014-5793/96/$12.00 © 1996 Federation of European Biochemical Societies.…”