Ligand-mediated internalization of glucagon receptors in intact rat liver.
The ligand-induced internalization of the hepatic glucagon receptor has been studied in rats in vivo using cell fractionation. Injection of glucagon (11 nmol/100 g BW) led to a 2- to 3-fold increase in glucagon-binding activity in Golgi-endosomal (GE) fractions along with a 10-20% decrease in binding activity in plasma membrane (PM) fractions. These changes were time and dose dependent, reaching a maximum by 12-24 min and undergoing reversal in 2 h. Glucagon injection also caused a 20% decrease in glucagon binding to the total particulate fraction, which did not occur when binding was measured in the presence of the detergent octylglucoside. The change in glucagon-binding activity in PM and GE fractions resulted mainly from a change in receptor number; affinity remained unaffected (apparent Kd, 0.5 and 5 nM, respectively). A 5- to 10-fold increase in the glucagon content of GE fractions was observed in glucagon-treated rats. Neither the distribution of PM and Golgi marker enzymes nor that of the asialoglycoprotein receptor was affected by glucagon treatment. Regardless of glucagon treatment, glucagon receptors in GE fractions were less sensitive to GTP than receptors in PM fractions with respect to both inhibition of steady state binding and dissociation of prebound ligand. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, glucagon-receptor complexes formed in PM and GE fractions and subsequently cross-linked showed the same apparent mol wt (57 kilodaltons). In addition, they were identically sensitive to N-glycanase treatment, with two major species of lower mol wt generated. However, only cross-linked complexes associated with PM fractions showed detectable GTP sensitivity. GE fractions displayed a GTP-sensitive adenylate cyclase activity that was about 12 times lower than that of PM fractions. In both fractions, activity was stimulated by the addition of forskolin (8-fold) and, to a lesser extent, glucagon (3-fold). In vivo glucagon treatment led to an increase in activity in GE, but not PM, fractions. These results are consistent with the view that upon acute occupancy, hepatic glucagon receptors are rapidly and specifically internalized along with their ligand. During this process, receptor retained structural integrity and uncouple, albeit partially, from other components of the adenylate cyclase system.
Authier
F
F
INSERM U.30, Hôpital des Enfants-Malades, Paris, France.
Desbuquois
B
B
De Galle
B
B
D000262:Adenylyl Cyclases / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004139:Dinitrofluorobenzene; D004591:Electrophoresis, Polyacrylamide Gel; D005934:Glucagon / Q000378:metabolism / Q000494:pharmacology*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008297:Male; D008970:Molecular Weight; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D011919:Rats, Inbred Strains; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018027:Receptors, Glucagon
D003432:Cross-Linking Reagents; D011964:Receptors, Gastrointestinal Hormone; D018027:Receptors, Glucagon; D006160:Guanosine Triphosphate; D005934:Glucagon; D004139:Dinitrofluorobenzene; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000262:Adenylyl Cyclases