The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2.
A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.
Barritt
D S
DS
The Burnham Institute, Cancer Research Center, La Jolla, California 92037, USA.
Pearn
M T
MT
Zisch
A H
AH
Lee
S S
SS
Javier
R T
RT
Pasquale
E B
EB
Stallcup
W B
WB
D000595:Amino Acid Sequence; D000941:Antigens / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D000099216:Chondroitin Sulfate Proteoglycan 4
D000941:Antigens; D002352:Carrier Proteins; D018076:DNA, Complementary; D008024:Ligands; D008565:Membrane Proteins; D011509:Proteoglycans; D000099216:Chondroitin Sulfate Proteoglycan 4; C111442:MPDZ protein, human