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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