| Abstract: |
Insulin receptors in rat liver plasma membranes contain two α- and two β-subunits held together by interchain disulphide bonds ([αβ]2 receptors). Affinity-labelled receptors were digested with chymotrypsin or elastase and then exposed to dithiothreitol before solubilization from membranes and SDS/polyacrylamide-gel electrophoresis. This resulted in partial reduction and isolation of M(r)-225,000 αβ, M(r)-200,000 α1β M(r)-165,000 αβ1 and M(r)-145,000 α1β1 receptor halves containing intact (α, β) or degraded (α1, β1) subunits. The ability to identify half-receptor complexes containing intact or degraded subunits made it possible to assay each subunit simultaneously for insulin-induced proteolysis in isolated plasma membranes or during perfusion of rat liver in situ with insulin. In liver membranes, insulin binding increased the fraction of receptors containing degraded α-subunits to about one-third of the total population during 2 h of incubation at 23°C, β-Subunit proteolysis increased only minimally during this time. Plasma membranes isolated from liver perfused with insulin at 37°C contained degraded α-subunits but only intact β-subunits, showing that insulin induced cell-surface proteolysis of the binding, but not the kinase, domain of its receptor. Since previous observations [Lipson, Kolhatkar & Donner (1988) J. Biol. Chem 263, 10495-10501] have shown that receptors containing degraded α-subunits are internalized but do not recycle, it is possible that cell-surface degradation may play a role in the regulation of insulin-receptor number in hepatic tissue. Proteolysis of the β-subunit is not a likely mechanism by which receptor-kinase activity may be attenuated under physiological conditions. |
