Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033
ON BINDING OF GROWTH FACTORS,
growth factor receptors in the plasma membrane can trigger a variety of
intracellular signaling pathways, promoting responses ranging from
differentiation to proliferation and cell migration (21).
A principal mechanism for modulating cellular responses to growth
factors is control of their accessibility to receptors by dynamically
regulating receptor distribution between plasma and intracellular
membrane compartments. The trafficking of the epidermal growth factor
(EGF) receptor, a member of the ErbB family of protein-tyrosine kinases (21), has been perhaps the most extensively studied of any
of the growth factor receptors. Binding of EGF to EGF receptor
initiates the EGF-mediated intracellular signaling cascade
(21) while concomitantly initiating the rapid clearance of
EGF-EGF receptor complexes from the plasma membrane to endosomes by
clathrin-mediated endocytosis (17, 19). Unlike other types
of receptors such as nutritional receptors (e.g., transferrin
receptors) that are rapidly recycled to the plasma membrane after
release of ligand in endosomes (10), the EGF-EGF receptor
complex is routed to lysosomes for degradation (1). This
phenomenon is referred to receptor downregulation, and it is a key
element in controlling the magnitude and duration of EGF signaling.
Although a pool of EGF receptors may recycle constitutively,
endocytosis of EGF receptors is enhanced by EGF binding and the resulting increase in its intrinsic receptor kinase activity
(22). Work by Opresko et al. (14) with
different EGF receptor mutants lacking the conserved tyrosine kinase
domain but retaining distal carboxy terminus sequences has extended
this proposed model of ligand-dependent EGF receptor phosphorylation
and internalization, suggesting that receptor phosphorylation elicits a
conformational change responsible for unmasking of cryptic targeting
signals within the cytoplasmic domain that cue endocytosis and
lysosomal targeting.
What sorts of targeting signals might cue EGF receptor internalization
and downregulation? The increased availability of protein sequence
information has led to the identification of different amino acid
motifs present in the cytoplasmic tails of a variety of membrane
receptors of all types (signaling, nutritional, housekeeping) that
interact with components of the membrane trafficking machinery to
promote selective targeting to specific membrane compartments. These
motifs include those containing tyrosine residues such as NPXY (X
representing any amino acid; Refs. 4, 6) and
YXX Several sequences within the EGF receptor cytoplasmic tail have been
implicated in the lysosomal targeting of ligand-occupied EGF receptor
that is critical for its downregulation, including the tyrosine-based
sorting signal YLVI, which resembles the YXX Before this work, the 954YLVI site within the EGF receptor
had been implicated in lysosomal targeting only indirectly. Mutagenesis studies previously demonstrated that a region located between residues
945 and 991 that included this motif was required for EGF receptor
downregulation (14). Furthermore, a unique sorting nexin,
SNX1, was identified through a yeast two-hybrid screen using the
cytoplasmic tail of the EGF receptor as bait (11). The
motif within the EGF receptor responsible for binding to SNX-1 was
subsequently narrowed down to a 15-amino acid domain spanning residues
943-957 containing 954YLVI, whereas overexpression of
SNX-1 in CV-1 cells significantly increased EGF receptor downregulation.
The work by Jones et al. (6a) provides the first direct evidence that
this particular targeting motif confers lysosomal targeting of EGF
receptor. However, two additional sorting motifs have been identified
within the cytoplasmic tail region of the EGF receptor: an LL motif
(679LL) and a c-Cbl recognition site for ubiquitination
(1045Y). Like 954YLVI, mutagenesis studies of
679LL showed that this motif is essential for
ligand-induced receptor degradation (9) and further
suggested that this effect is associated with the ability of this motif
to sequester EGF-EGF receptor complexes into multivesicular endosomes
(8). Other investigations have implicated
autophosphorylation of 1045Y in c-Cbl binding and
phosphorylation, followed by recruitment of ubiquitin-activating and
-conjugating enzymes that participate in receptor ubiquitination for
enhanced degradation (13). As for 954YLVI and
679LL, mutation of 1045Y impairs EGF receptor
downregulation (12).
By directly demonstrating the involvement of 954YLVI in
lysosomal targeting of EGF receptor, this study by Jones et al. (6a) has raised several new questions. Clearly, a major issue is the relationship between these different sorting motifs present in the EGF
receptor, each of which appears to be critical for EGF receptor
downregulation. On the basis of their data and those of other
investigators in this field, Jones et al. (6a) propose a novel but
speculative model suggesting that that these different motifs may act
in a concerted manner. They suggest that ligand binding and the
resultant autophosphorylation of 1045Y lead to rapid
recruitment of c-Cbl that stabilizes and ubiquitinates the cytoplasmic
region of the EGF receptor in such a way as to expose the
954YLVI and 679LL lysosomal targeting motifs.
They then posit that these two domains may form a bipartite binding
site for recruitment of SNX-1 and other factors responsible for
escorting this complex to lysosomal compartments.
If these two motifs actually form a bipartite domain after receptor
phosphorylation and c-Cbl-mediated ubiquitination, we would expect that
this mechanism would be conserved at minimum across other species
expressing the EGF receptor. Interestingly, the analysis by Jones et
al. (6a) of the sequence conservation of 954YLVI reveals
that it is highly conserved in EGF receptors from other species and in
other ErbB family members. In contrast, the 679LL dileucine
motif is not conserved in EGF receptors from other species and,
furthermore, is not shared among other ErbB human family members. Other
studies have suggested that one leucine within the LL motif can be
replaced by isoleucine, valine, alanine, or methionine
(6), although none of these "modified" LL motifs is
apparent in the sequence alignment for the LL motif chosen by Jones et
al. (6a) for nonhuman EGF receptors. It is also conceivable that a
related hydrophobic motif such as the newly identified phenylalanine-isoleucine (FI) motif present in furin (18)
may function in place of LL to form a bipartite binding site for
SNX-based sorting machinery in other species. Alternatively,
679LL and 954YLVI may represent discrete sites
for interaction of different membrane trafficking proteins that are
each essential for accurate lysosomal transit.
Just as changes in EGF receptor expression levels have been linked to
changes in cellular proliferation associated with cell transformation,
modulation of cellular responsiveness to EGF can be achieved through
changes in its trafficking. Recent work has shown that an oncogenic
form of c-Cbl, v-Cbl, is able to prevent EGF-induced EGF receptor
downregulation, directing EGF receptor through recycling rather than
degradative pathways (12). The adenoviral protein
E3-13.7 was recently localized to early and multivesicular
endosomes in infected cells; this protein mediates the selective
ligand-independent downregulation of EGF receptor by rerouting
constitutively recycling receptor to lysosomes (2). Intriguingly, this protein appears to interact with a region of the EGF
receptor containing the 679LL motif and itself contains a
LL motif. Insights obtained from such models of viral pathogenesis as
well as extension of EGF receptor trafficking work into additional
physiological model systems may prove important in dissecting further
intricacies of EGF receptor sorting.
ARTICLE
TOP
ARTICLE
REFERENCES
(
representing a bulky hydrophobic amino acid; Refs.
5, 6), as well as those containing LL, with
or without a cluster of acidic amino acid residues (acidic cluster;
Refs. 3, 6, 7, 15). Other motifs in the cytoplasmic tails of receptors have been identified that specify interactions with regulatory elements such as the ubiquitination machinery (16, 20).
lysosomal sorting
signal identified for the resident lysosomal protein, LAMP1
(5). In the current article in focus (Ref.
6a; see p. C420 in this issue), Jones et al. present data
substantiating the involvement of this tyrosine-based sorting signal,
954YLVI, in the targeting of human EGF receptor to
lysosomes. These investigators generated two mutant human EGF
receptors by replacing the 954YL with either AA or VD and
then examined the consequences of these replacements to EGF receptor
endocytosis and lysosomal targeting in transiently or stably
transfected HEK 293 cells, which have extremely low endogenous levels
of EGF receptor. Fluorescence detection of wild-type and mutant EGF
receptors with Texas red EGF in transiently transfected cells revealed
that the patterns of cell surface distribution and internalization to
early endosomes (marked by rab5-GFP) were comparable. However,
biochemical analyses revealed that these mutations significantly
impaired EGF-induced downregulation of EGF receptor without affecting
EGF-induced substrate phosphorylation. Further analysis in cell lines
stably expressing wild-type and mutant EGF receptors substantiated the
observations from transiently transfected cells, showing that the
mutations completely blocked EGF-induced EGF receptor downregulation.
Furthermore, immunofluorescence microscopy revealed that wild-type but
not mutant EGF receptors could be colocalized with LAMP1 after exposure to EGF for 60 min.
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ACKNOWLEDGEMENTS |
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This work was supported by National Eye Institute Grant EY-11386.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. F. Hamm-Alvarez, USC School of Pharmacy, 1985 Zonal Ave., Los Angeles CA 90033 (E-mail: shalvar{at}hsc.usc.edu).
10.1152/ajpcell.00550.2001
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