Endocytosis and Lysosomal Targeting of Epidermal Growth Factor Receptors Are Mediated by Distinct Sequences Independent of the Tyrosine Kinase Domain

Abstract

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) leads to accelerated receptor degradation. Two models have been proposed to explain this. In the first model, induced internalization expands the intracellular pool of receptors, leading to enhanced lysosomal targeting. The second model proposes that activation of intrinsic receptor kinase activity induces inward vesiculation of endosomes, thus interrupting receptor recycling. To test these models, we created EGFR mutants that lack the conserved tyrosine kinase domain, but retain different parts of the distal carboxyl terminus regulatory region. Mutants lacking all distal regulatory sequences underwent slow internalization (0.02 min-1) and turnover (t1/2 approximately 24 h), similar to unoccupied, holo-EGFR. Mutant receptors that lacked the kinase domain, but retained the entire distal regulatory domain, were constitutively internalized and targeted to lysosomes, even in the absence of EGF. The turnover of these receptors (t1/2 approximately 11 h) was similar to that of occupied, kinase-active holo-EGFR (t1/2 approximately 9.5 h). These results show that receptor tyrosine kinase activity is not required for the targeting of EGFR to lysosomes. Receptor mutants which expressed previously identified endocytic sequences underwent rapid internalization. Unexpectedly, enhanced turnover of EGFR mutants required additional sequences located between residues 945 and 991 in the holo-EGFR. Thus, internalization and lysosomal targeting of EGFR are separate processes mediated by distinct sequences. Our results indicate that induced internalization is necessary, but not sufficient, for enhanced EGFR degradation. Instead, down-regulation requires exposure of previously cryptic internalization and lysosomal targeting sequences. Occupied EGFR thus appear to be handled by the endocytic machinery in the same fashion as other constitutively internalized or lysosomally targeted receptors.

Evidence that downregulation of the M-CSF receptor is not dependent upon receptor kinase activity

“…It has previously been suggested that EGF receptor degradation is initiated by structural changes of the receptor that are stabilized by autophosphorylation and expose cryptic internalization and degradation sites (Opresko et al, 1995). Our results for the M-CSF receptor are largely consistent with this model except that autophosphorylation was not found to be critical, it may also not be critical for downregulation of the EGF receptor (Livneh et al, 1987).…”

“…The binding of growth factors to receptors usually causes the internalization and degradation of the receptor-ligand complex, a process referred to as receptor downregulation (Opresko et al, 1995;Seaman et al, 1996). Receptor downregulation is important because it attenuates signalling, as shown by the ®nding that mutant receptors for EGF and PDGF that are resistant to ligand-induced degradation have ampli®ed mitogenic activity (Wells et al, 1990;Mori et al, 1993).…”

“…It has been suggested that downregulation of the M-CSF receptor is dependent upon substrate phosphorylation (Carlberg et al, 1991). A di erent model is that autophosphorylation of the EGF receptor stabilizes a change in its structure that exposes cryptic internalization and degradation sites (Opresko et al, 1995).…”

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…Endocytosis of the growth-factor receptors requires intrinsic tyrosine kinase activity [185,186]. Inactivation of the receptor kinase activity by a point mutation almost eliminates agonistinduced internalization of the epidermal-growth-factor receptor [187]. However, deletion of the entire tyrosine kinase domain, leaving the remaining C-terminal tail with its endocytic motifs intact, results in constitutive receptor endocytosis.…”

“…Thus exposure to agonists depletes the cell of surface receptors and accelerates receptor degradation, reflected by a 8-10-fold decrease in halflife [185]. The importance of the endogenous tyrosine kinase domain for endocytosis and trafficking of the epidermal growth factor receptor is controversial, and has been a topic of recent investigation [185,187,213]. The unoccupied epidermal growth factor receptor is internalized at a very low basal rate, and activation by agonist is necessary for rapid internalization and degradation.…”