Down-modulation of the apoptosis receptor Fas and the EGF receptor by the adenovirus E3/10.4-14.5K proteins requires the concerted action of two distinct transport signals

Human adenoviruses (Ads) have evolved elaborate mechanisms to
counteract the host’s antiviral immune response. The early
transcription unit 3 (E3) of the virus is not essential for virus
replication in vitro, but is known to encode proteins with
immunomodulatory functions. The Ad2 E3/10.4-14.5K proteins are both
integral membrane proteins, which form a physical complex and
function together to modulate cell surface expression of the EGFR
and selective members of the TNF/NGF receptor superfamily, namely
Fas/CD95 and TRAIL-R1, whereas TRAIL-R2 modulation additionally
requires E3/6.7K. In a process referred to as receptor
down-regulation, 10.4-14.5K relocates receptor targets from the
cell surface to lysosomes for degradation. The aim of this study
was to characterize functional determinants within the Ad2
10.4-14.5K proteins, that are required for down-regulation of
plasma membrane receptors. In particular, I focussed on the
characterization of potential transport motifs present in the
cytoplasmic tail of both proteins: The Ad2 14.5K tail contains
three YXXF sequence motifs (Y denotes tyrosine, X any amino acid
and F a bulky, hydrophobic residue) while the Ad2 10.4K sequence
displays two consensus elements of the second large class of
transport signals, the dileucine motifs. Both types of motifs are
recognized by cellular adaptor proteins which select cargo for
directed transport in clathrin-coated vesicles. FACS analysis of
stable E3-transfectants expressing 10.4-14.5K mutant proteins
revealed that residues contained within these putative transport
motifs were essential for down-regulation of Fas and the EGFR in
vivo. Receptor expression was restored when either the dileucine
pair (LL87,88) of 10.4K or 14.5K Y74 or Y122 were replaced by
alanine. Whereas loss of function of the 14.5K mutant Y74 can be
explained by its inability to interact with 10.4K, several lines of
evidence suggest that the 10.4K dileucine pair and 14.5 Y122XXF
motif function as transport signals: (i) Surface plasmon resonance
spectroscopy showed that mutation of the two motifs prevents
binding of 10.4K and 14.5K cytoplasmic tail peptides to purified
adaptor protein complexes AP-1 and AP-2 in vitro. (ii) FACS
analysis demonstrated that mutation of these motifs strongly
affects FLAG-14.5K cell surface expression. (iii) In line with the
FACS data, immunofluorescence microscopy revealed that mutant
14.5Y122A accumulates together with 10.4K at the cell surface,
suggesting that the Y122FNL motif normally directs internalization
of 10.4-14.5K. (iv) Substitution of the 10.4K dileucine pair
increased the transport of 10.4-14.5K into lysosomes, resulting in
enhanced degradation of both 10.4K and 14.5K without significantly
disrupting complex formation. (v) The accumulation of mutant
10.4-14.5K at the cell surface upon coexpression of 10.4LL/AA and
14.5Y122A suggests that the dileucine motif acts downstream of Y122
and fulfills a sorting function subsequent to endocytosis. Transfer
of the mutations into Ad2 and infection of primary fibroblasts
revealed a similar defect in trafficking of 10.4LL/AA and 14.5
Y122A mutant proteins. Moreover, in infected cells substitution of
the 10.4K dileucine pair and 14.5K Y122 impaired down-regulation of
Fas, EGFR and both TRAIL-R1 and TRAIL-R2, implying a general role
of these sorting signals for the mechanism of receptor
down-regulation. Thus, two distinct transport signals present in
the different subunits of the 10.4-14.5K complex seem to act in
concert to establish efficient down-regulation of receptor targets.
Alanine replacement mutagenesis of several other strictly conserved
amino acids in 14.5K and FACS analysis of stable E3-transfectants
revealed that those mutants which exhibited an altered FLAG-14.5K
surface expression had defects in Fas and EGFR down-modulation.
Surprisingly, Ad4 was unable to modulate Fas and EGFR expression,
even though the Ad4 14.5K protein contained all the strictly
conserved amino acids. As a first step to identify structural
features that determine target specificity of 10.4-14.5K, I chose
to replace the 10.4-14.5K ORFs in Ad2 by their Ad4 homologues.
Although the Ad4 10.4-14.5K proteins could be detected in
Ad4-infected cells, their expression level was drastically reduced
when encoded by the Ad2 E3 region. This indicated that expression
of Ad4 10.4-14.5K is differently regulated as compared to Ad2,
possibly due to altered splicing. Further exploration of this
system will require a detailed analysis of splicing within the Ad4
E3 region