A Membrane Setting for the Sorting Motifs Present in the Adenovirus E3-13.7 Protein Which Down-regulates the Epidermal Growth Factor Receptor

Abstract

The adenovirus E3-13.7 protein interferes with endosomal protein sorting to down-regulate the epidermal growth factor receptor and related tyrosine kinase receptors. The cytoplasmic C terminus of this protein contains three protein sorting motifs which are related to the function of E3-13.7. In this study, the structure of a 23-residue polypeptide corresponding to this domain was examined using solution NMR and CD spectroscopic methods. The peptide was observed to exist in a mostly random structural state in aqueous solution but underwent high affinity association with dodecylphosphocholine micelles, where it adopted an ordered structure. The affinity of this peptide for the micellar surface and the structure of the bound peptide were independent of pH variation, surface charge, or attachment of a myristoyl anchor to the N-terminal. Studies with phospholipid vesicles suggested that the micellar structural results can be extrapolated to a true lipid bilayer. On the micellar surface all three sorting motifs are closely associated with the water/apolar interface: 72-YLRH and 87-LL lie within interfacial amphipathic helices, while 76-HPQY is non-helical and dimples just above the surface. These results contribute to the development of an understanding of the basis for specificity in recognition of sorting motifs by components of the cellular protein trafficking machinery.A 13.7-kDa protein expressed from the "Early 3" genome region of adenovirus (E3-13.7) down-regulates the EGFR 1 and related tyrosine kinase receptors during infection of epithelial cells and fibroblasts (1, 2). For EGFR it has been shown that E3-13.7 exerts its role by altering sorting at the endosomal level (3) such that the ratio of receptor destined for lysosomal degradation relative to receptor destined for recycling to the plasma membrane surface is increased. The down-regulatory action of E3-13.7 is believed to be associated with the constitutive recycling pathway of EGFR and does not require ligand stimulation of the receptor to be operative. An enhanced understanding of the down-regulatory mechanism of E3-13.7 is desirable because of light it will shed both upon the cell biology of protein trafficking pathways and upon the molecular biology of adenoviral infections.As outlined in Fig. 1, E3-13.7 is an integral membrane protein comprised of an N-terminal transmembrane helix followed by an extracellular/lumenal loop, a second transmembrane segment, and a C-terminal cytoplasmic domain (1). While the process by which E3-13.7 down-regulates tyrosine kinase receptors is incompletely understood, it is known that the Cterminal cytoplasmic domain (E3-CyD) is critical to its function. Within this 23-residue domain there are three different sequence motifs (Fig. 1) known to be broadly relevant to the cellular trafficking of membrane proteins via clathrin-mediated internalization pathways (4 -6). One of these is the "dileucine" (87-LL) motif (Refs. 7-9 and references therein). While a functional role for this motif in E3-13.7 has yet to be established, ...

A novel dileucine lysosomal-sorting-signal mediates intracellular EGF-receptor retention independently of protein ubiquitylation

“…The side-chains of the two key leucine residues are largely buried in the micelle surface. We have reported previously that the corresponding LL sequence in the E3-13.7 protein exhibits similar micellebinding and helical properties (Vinogradova et al, 1998). Interestingly, the 679-LL-stabilized helical structure represented in Fig.…”

“…The adenovirus protein diverts receptors in a basal internalization-recycling pathway to MVB internal vesicles without activating EGFR or increasing its internalization rate (Hoffman and Carlin, 1994;Hoffman et al, 1992b). Interestingly, E3-13.7 has an exact copy of a five amino acid sequence including 679-LL found in EGFR that is required for its biological activity (Table 1c), suggesting E3-13.7 can support a normal cellular function (Carlin et al, 1989;Crooks et al, 2000;Hoffman et al, 1992a;Vinogradova et al, 1998). Thus, E3-13.7 provides a novel model for studying the molecular basis of EGFR sorting to lysosomes without ligand-induced saturation of this transport pathway (Piper and Luzio, 2001;Wiley and Burke, 2001).…”

Characterization of the Neuron-Specific L1-CAM Cytoplasmic Tail: Naturally Disordered in Solution It Exercises Different Binding Modes for Different Adaptor Proteins

“…The disappearance of specific peaks has previously been observed in dynamic regions of globular proteins (31), when intermediate conformational exchange results in significant broadening of the spectra, often beyond the experimental detection. In addition to conformational exchange, temperature increase in partially disordered systems (32) stimulates exchange between non-protected amides and water protons, resulting in further broadening of the spectra. We have embedded L1-CT in dodecylphosphocholine (DPC) micelles to mimic the interactions with membrane surface, which could enforce and stabilize the structural rearrangements in otherwise unstructured cytoplasmic tails of integral membrane proteins (33).…”

A Tyrosine-Based Signal Plays a Critical Role in the Targeting and Function of Adenovirus RIDα Protein

“…2) are highlighted in gray or black, respectively. Nuclear magnetic resonance studies have shown that residues 87-LLRIL comprise an amphipathic helix stabilized by interactions with membrane mimetic micelles (45). Two additional residues are presented in the exocytic loop connecting the two membrane spanning helices: Ala23, comprising the amino terminus of the 11.3-kDa cleaved form of the protein produced cotranslationally by signal peptidase, and Cys31, which forms intermolecular disulfide bonds (27).…”