Recognition of the 300-kDa mannose 6-phosphate receptor cytoplasmic domain by 47-kDa tail-interacting protein.

Tail-interacting 47-kDa protein (TIP47) binds the cytoplasmic domains of the cation-dependent (CD) and cation-independent (CI) mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the Golgi complex. TIP47 recognizes a phenylalanine-tryptophan signal in the CD-MPR. We show here that TIP47 interaction with the 163-residue CI-MPR cytoplasmic ...
domain is highly conformation dependent and requires CI-MPR residues that are proximal to the membrane. CI-MPR cytoplasmic domain residues 1-47 are dispensable, whereas residues 48-74 are essential for high-affinity binding. However, residues 48-74 are not sufficient for high-affinity binding; residues 75-163 alone display weak affinity for TIP47, yet they contribute to the presentation of residues 48-74 in the intact protein. Independent competition binding experiments confirm that TIP47 interacts with the membrane-proximal portion of the CI-MPR cytoplasmic domain. TIP47 binding is competed by the binding of the AP-2 clathrin adaptor at (and near) residues 24-29 but not by AP-1 binding at (and near) residues 160-161. Finally, TIP47 appears to recognize a putative loop generated by the sequence PPAPRPG and other hydrophobic residues in the membrane-proximal domain. Although crystallography will be needed to define the precise interaction interface, these data provide an initial structural basis for TIP47-CI-MPR association.
Mesh Terms:
Amino Acid Sequence, Binding Sites, Cytoplasm, DNA-Binding Proteins, Intracellular Signaling Peptides and Proteins, Pregnancy Proteins, Protein Binding, Receptor, IGF Type 2, Recombinant Fusion Proteins
Proc. Natl. Acad. Sci. U.S.A.
Date: Aug. 01, 2000
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