The E3 region is not required for viral replication; nevertheless, it plays a critical role in Ad pathogenesis (15
). The importance of this region is underscored by the fact that the first generation of Ad gene therapy vectors which contained large E3 deletions were ultimately deemed unsafe (14
). E3 genes encode integral membrane proteins that regulate a variety of host cell functions involved in innate immunity and inflammatory responses. The ability of these proteins to modify host cell function is due in part to cytosolic tail sequences that interact with sorting machinery and target membrane proteins to specific intracellular compartments. The E3 protein RIDα was originally identified because of its ability to downregulate the EGFR (7
) and other related receptor tyrosine kinases (35
). In this study, we have demonstrated that RIDα residues 71-AYLRH comprise a binding site for AP complexes and that Tyr72 is required for RIDα localization to endosomes and its ability to downregulate the EGFR. These findings support previous studies concluding that RIDα acts by targeting EGFRs undergoing constitutive recycling to the plasma membrane (10
). The fact that 71-AYLRH is precisely conserved in all Ad serotypes that have been sequenced except for Ad12 suggests its fundamental importance in a majority of Ad-induced diseases (7
Although the mutation of Tyr72 to an alanine residue leads to a clear reduction in AP binding in vitro (Table ), 71-AYLRH does not conform to classical tyrosine-dependent YXXØ motifs that have a preference for hydrophobic residues with bulky side chains at the Ø position (39
). Instead, it has a histidine residue that is mildly basic and hydrophilic. However, many other factors contribute to sorting signal recognition, including the position of the signal relative to the membrane and to the carboxyl terminus and the presence of amino acid residues in areas adjacent to the signal. We have reported previously that 71-AYLRH exists in an amphipathic helix that is stabilized by interactions with a membrane-mimetic phospholipid micelle surface based on data obtained using nuclear magnetic resonance spectroscopic methods (45
). This close degree of membrane association could have an important role in regulating the availability of the signal for interaction with APs. Thus, 71-AYLRH may be masked when the cytoplasmic tail is intimately associated with membrane, while cellular events that result in its translocation into the cytosol could make it available for binding APs. Some examples that could bring about such a change include modulation by another membrane protein, changes in the endosomal pH or ionic environment as RIDα traverses different cellular compartments, posttranslational modification, or a dramatic shift in the tilt or membrane placement of the adjacent transmembrane helix. For example, we have recently demonstrated that RIDα function is highly dependent on reversible palmitoylation at a residue in the cytosolic tail (N. L. Cianciola and C. Carlin, unpublished data), suggesting that the association of the RIDα cytosolic tail with membranes is tightly regulated in cells (17
). The possibility that 71-AYLRH availability is regulated by a transmembrane mechanism is particularly intriguing since the RIDα loop domain connecting its two membrane spanning domains resides in compartmental lumens. Thus, the transmembrane domain could act as a conduit to fine-tune 71-AYLRH recognition at specific subcellular organelles.
Even though 71-AYLRH recognizes two different classes of APs, we have demonstrated that AP-1 and AP-2 do not compete for binding to RIDα in vitro (Fig. ) and that mutation of adjacent residue Tyr79 leads to increased binding of AP-2 but diminished binding to AP-1 (Table ). Thus, it seems likely that AP-1 and AP-2 recognize distinct but overlapping sets of tyrosine-based sorting signals in RIDα. The Y72A mutation traps RIDα in the TGN (Fig. ) but not the plasma membrane (Fig. ) suggesting the Ad protein encounters AP-1 first in the biosynthetic pathway. AP-2 is known to interact with a majority of tyrosine-based signals identified for other molecules, in agreement with studies showing that most naturally occurring signals mediate internalization (37
). The broad specificity of AP-2 recognition implies that it serves a quality control function to retarget membrane proteins to their correct intracellular location that escape to the plasma membrane (2
). This would suggest that the plasma membrane may not be an obligatory membrane transport destination for RIDα and that a majority of RIDα is delivered directly to endosomes (see summary model in Fig. ). Accordingly, even though RIDα can be detected on the plasma membrane, this localization correlates with high levels of protein expression and constitutes a relatively minor fraction of the total protein in the cell (10
). This is entirely consistent with the quality control role for AP-2 that has been proposed for membrane proteins that leak to the plasma membrane.
FIG. 8. Summary model. Data presented in this study suggest that newly synthesized RIDα is delivered directly from the TGN to endosomes (open arrows) where it encounters EGFRs undergoing constitutive recycling to the plasma membrane (dashed arrows). Since (more ...)
In addition to the Tyr72-based sorting motif, RIDα also has a potential dileucine-based motif located at residues 87-LL (Fig. ). Although another laboratory has published a study saying that these residues constitute an AP-2 binding site (21
), those results were not substantiated in the present study. We did observe that AP binding was remarkably insensitive to pH or salt (Fig. ), supporting a role for hydrophobic interactions either within the signal itself or in adjacent regions. It is possible that the dialanine substitution at 87-LL analyzed in reference 21
lowers the overall strength of AP binding. Interestingly, 87-LL is part of a larger motif that is precisely conserved in the EGFR (26
), suggesting its involvement in cargo selection and/or targeting EGFRs to lysosomes. This conjecture is supported by evidence that this sequence in the EGFR is necessary for ligand-induced trafficking to lysosomes (32
) and also RIDα-mediated diversion of recycling EGFRs to lysosomes (44
). Thus, although 87-LL may not be directly involved in AP recognition, it undoubtedly has an important role in RIDα function at least as it relates to EGFR downregulation. The 87-LL motif shared with the EGFR is found in group C Ad2 and Ad5; however, this region is not precisely conserved in other serotypes (7
). Thus, different Ads may vary in their ability to specifically target the EGFR.
RIDα has been associated with other activities besides EGFR downregulation. For example, the RID complex (comprised of RIDα and RIDβ) downregulates death receptors, including TNFR1 and FAS (9
). However, mutagenesis studies support a model where the RID complex acts on TNFR1 at the plasma membrane, in contrast to FAS, where the functional interaction occurs intracellularly. These seemingly paradoxical results are best understood by considering the many steps involved in receptor downregulation. These include cargo selection, sorting to specific endocytic compartments involved in transport to lysosomes, and coupling to microtubules necessary for transporting MVB intermediates to the perinuclear region (19
). We have already discussed the idea that the molecular basis for RIDα-mediated EGFR cargo selection likely involves the dileucine motif that is conserved in EGFR and RIDα encoded by Ad2 and Ad5. In addition, we have recently discovered that RIDα interacts with Rab7 effectors, including RILP and ORP1L, which are necessary for microtubule-dependent transport (Shah et al., submitted). Thus, it is likely that RIDα regulates EGFR downregulation at multiple levels. Other cargoes could require accessory molecules to deliver specific receptors to endosomes once the maturation process is under way. Thus, RIDβ may promote TNFR1 uptake to endosomes, whereupon they are then sorted to lysosomes by a mechanism involving RIDα-dependent endosomal maturation. This conjecture is consistent with the observation that RIDβ binds AP-2 and that AP-2 is required for RID-mediated downregulation of TNFR1 but not FAS (9
). The ability to “mix and match” different aspects of RID function may have evolved to allow Ads to fine-tune RID activity in different cell types or during acute versus persistent infections.