PDZ interaction sites in integrin alpha subunits. T14853, TIP/GIPC binds to a type I recognition sequence in alpha 6A/alpha 5 and a novel sequence in alpha 6B.
We used published peptide library data to identify PDZ recognition sequences in integrin alpha subunit cytoplasmic domains and found that the alpha(6)A and alpha(5) subunits contain a type I PDZ binding site (TSDA*) (asterisk indicates the stop codon). The alpha(6)A cytoplasmic domain was used for screening a two-hybrid library to find interacting proteins. The bulk of the captured cDNAs (60%) coded for TIP-2/GIPC, a cytoplasmic protein with one PDZ domain. The interaction of TIP-2/GIPC with different integrin subunits was tested in two-hybrid and in vitro binding assays. Surprisingly, TIP-2/GIPC bound strongly to the C terminus of both alpha(6)A and alpha(6)B, although the alpha(6)B sequence (ESYS*) is not suggestive of a PDZ binding site because of its polar C-terminal residue. For high affinity interaction with TIP-2/GIPC, at least one of the residues at positions -1 and -3 must be negatively charged. An aliphatic residue at position 0 increases the affinity of but is not required for this interaction. The alpha(5) integrin subunit also bound to TIP-2/GIPC. The alpha(6) integrin and TIP-2/GIPC co-localize in retraction fibers in carcinoma cells plated on laminin, a finding suggesting a functional interaction in vivo. Our results demonstrate that both splice variants of alpha(6) integrin contain a conserved PDZ binding site that enables interaction with TIP-2/GIPC. The binding site in alpha(6)B defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.
Tani
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Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Mercurio
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D048868:Adaptor Proteins, Signal Transducing; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000737:chemistry*; D001665:Binding Sites; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002448:Cell Adhesion; D002478:Cells, Cultured; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D039482:Integrin alpha5; D039503:Integrin alpha6; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry* / Q000378:metabolism*; D019151:Peptide Library; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D002352:Carrier Proteins; C492009:GIPC1 protein, human; D039482:Integrin alpha5; D039503:Integrin alpha6; D009479:Neuropeptides; D019151:Peptide Library; D012333:RNA, Messenger; D011994:Recombinant Proteins; D005982:Glutathione Transferase