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.
Orsel
J G
JG
Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305-5307, USA.
Sincock
P M
PM
Krise
J P
JP
Pfeffer
S R
SR
D000595:Amino Acid Sequence; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D011485:Protein Binding; D017527:Receptor, IGF Type 2 / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins
D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C038196:PLIN3 protein, human; D000070796:Perilipin-3; D011257:Pregnancy Proteins; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins