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
Department of Cancer Biology
Adaptor Proteins, Signal Transducing; Alternative Splicing; Amino Acid Sequence; Antigens, CD; Binding Sites; Blotting, Northern; Carrier Proteins; Cell Adhesion; Cells, Cultured; Glutathione Transferase; Humans; Integrin alpha5; Integrin alpha6; Molecular Sequence Data; Neuropeptides; Peptide Library; Plasmids; Point Mutation; Protein Binding; Protein Structure, Tertiary; RNA, Messenger; Recombinant Proteins; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Two-Hybrid System Techniques
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.
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Citation: J Biol Chem. 2001 Sep 28;276(39):36535-42. Epub 2001 Jul 30. Link to article on publisher's site