Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain.
hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ-protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2-mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.
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Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.
Brandin
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Chan
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D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D029721:Drosophila Proteins; D004910:Erythrocyte Membrane / Q000737:chemistry / Q000235:genetics; D016147:Genes, Tumor Suppressor / Q000235:genetics*; D006801:Humans; D007301:Insect Hormones / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009479:Neuropeptides; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000502:physiology; D017434:Protein Structure, Tertiary; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000502:physiology; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism; D013912:Threonine / Q000502:physiology; D025521:Tumor Suppressor Proteins; D014633:Valine / Q000502:physiology; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D007301:Insect Hormones; D008565:Membrane Proteins; D009479:Neuropeptides; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D012694:Serine; D013912:Threonine; D025521:Tumor Suppressor Proteins; D014633:Valine; D000099149:Ezrin; C028841:erythrocyte membrane band 4.1 protein; C120557:erythrocyte membrane protein band 4.1-like 1; C070350:dlg1 protein, Drosophila