Ligand-mediated internalization, recycling, and downregulation of the epidermal growth factor receptor in vivo.

Abstract

Abstract. EGF receptor internalization, recycling, and downregulation were evaluated in liver parenchyma as a function of increasing doses of injected EGE The effect of ligand occupancy in vivo on the kinetics and extent of internalization was studied with changes in the receptor content of isolated plasmalemma and endosome fractions evaluated by direct binding, Scatchard analysis, and Western blotting. For all doses of injected EGF, receptor was lost from the plasmalemma and accumulated in endosomes in a time-and dosedependent fashion. However, at doses of injected EGF equivalent to <50% surface receptor occupancy (i.e., <1 #g/100 g body weight), receptor levels returned by 120 min to initial values. This return was resistant to cycloheximide and therefore did not represent newly synthesized receptor. Neither was the return due to replenishment by an intracellular pool of low-affinity receptors as such a pool could not be detected by Scatchard analysis or Western blotting. Therefore, receptor return was due to the recycling of previously internalized receptor.At doses of injected EGF >50% receptor occupancy, net receptor loss-i.e., downregulation-was observed by evaluating the receptor content of total particulate fractions of liver homogenates. At the higher saturating doses of injected EGF (5 and 10 #g/100 g body weight), the majority of surface receptor content was lost by 15 min and remained low for at least an additional 105 min. As the kinetics of ligand clearance from the circulation and liver parenchyma were similar for all doses of EGF injected, then the ligandmediated regulation of surface receptor content and downregulation were not a result of a prolonged temporal interaction of ligand with receptor. Rather, the phenomena must be a consequence of the absolute concentrations of EGF interacting with receptor at the cell surface and/or in endosomes.

Down-Regulation of Cell Surface Receptors Is Modulated by Polar Residues within the Transmembrane Domain

“…It is interesting that one of the proteins identified in the search, P-selectin, was also a protein in which a cytoplasmic tail lysosomal targeting signal was difficult to localize (Straley et al, 1998). And finally in support of our proposed model, it is now clear that receptor down-regulation requires multiple rounds of recycling before lysosomal delivery occurs (Lai et al, 1989;Felder et al, 1992), suggesting that down-regulation, rather than being a distinctive all-or-nothing event, may simply reflect a loss of recycling fidelity (Weissman et al, 1986).…”

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection

“…dissociation of EGFR signal elements from caveolar domains), internalisation of raft-localised EGFR may thus be fundamental to signal transduction (Balbis and Posner, 2010). Data collectively support activity-dependent EGFR internalisation, with low-level agonism triggering clathrin-dependent/cholesterolindependent recycling to the cell membrane (Sigismund et al, 2005;Sigismund et al, 2008), whereas higher level agonism results in clathrin-independent/cholesteroldependent EGFR internalisation and trafficking to late endosomes (Lai et al, 1989).…”

Ligand substitution of receptor targeted DNA complexes affects gene transfer into hepatoma cells

“…It is possible that the C1315 ligand traffics differently from C105Y, accelerating degradation of complex associated with it. Other receptors such as the transferrin 18 and epidermal growth factor 19 receptors have been shown to traffic different ligands differently. This may explain why maximal expression was observed at different days after transfection for different conjugate preparations (ie random substitution versus dilution experiments).…”

“…For the EGF receptor, for example, low concentrations of ligand promote endosomal recycling but high concentrations of ligand promote routing to lysosomes. 19 The abundance of SEC-Rs in lung, liver, and brain, 14 all of which might be potential target tissues for therapeutic gene transfer in common inherited (eg ␣ 1 -antitrypsin deficiency) or acquired (eg Alzheimer's disease) disorders, has made it a desirable target for receptormediated gene therapy. The development of optimal complexes that produce high-level gene expression for shorter or longer periods of time, based on substitution rate or choice of ligand, will be useful in achieving optimal therapeutic effects.…”