Abstract
Endocytosis of membrane proteins is typically mediated by signals present in their cytoplasmic domains. These signals usually contain an essential tyrosine or pair of leucine residues. Both tyrosine- and dileucine-based endocytosis signals are recognized by the adaptor complex AP-2. The best understood of these interactions occurs between the tyrosine-based motif, YXXPhi, and the mu2 subunit of AP-2. We recently reported a tryptophan-based endocytosis signal, WLSL, contained within the cytoplasmic domain of the neonatal Fc receptor. This signal resembles YXXPhi. We have investigated the mechanism by which the tryptophan-based signal is recognized. Both interaction assays in vitro and endocytosis assays in vivo show that mu2 binds the tryptophan-based signal. Furthermore, the WLSL sequence binds the same site as YXXPhi. Unlike the WXXF motif, contained in stonin 2 and other endocytic proteins, WLSL does not bind the alpha subunit of AP-2. These observations reveal a functional similarity between the tryptophan-based endocytosis signal and the YXXPhi motif, and an unexpected versatility of mu2 function.
Highlights
Membrane proteins that enter cells at coated pits typically contain endocytosis signals in their cytoplasmic domains
The Cytoplasmic Domain of FcRn Interacts with the 2 Subunit of AP-2—We expressed the cytoplasmic domain of FcRn as an N-terminal glutathione S-transferase (GST) fusion protein and asked whether it could interact with purified His-tagged 2 in a pull-down assay
Using GST pull-down experiments, we found that the tryptophan-based endocytosis signal in FcRn is recognized by the subunit of AP-2
Summary
FcRn, neonatal Fc receptor; AP, adaptor protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco’s modified Eagle’s medium; FcRn internalized at coated pits at the apical plasma membrane of intestinal epithelial cells carries IgG from milk toward the blood circulation of suckling rats [8, 9]. The endocytosis signal that includes Trp-311 of rat FcRn includes Leu-314, but not amino acids 307 to 310, 312, 313, or 315 [7]. The other aromatic amino acids, tyrosine and phenylalanine, can be substituted for Trp-311 [7], and Leu-314 can be replaced with the bulky isoleucine [7] or phenylalanine without loss of function. Interaction of a Tryptophan-based Endocytosis Signal with 2 recognized by AP-2 adaptor proteins at the plasma membrane [43, 44]. This revealed an unexpected versatility in the recognition of signals by 2
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