PDZ interaction sites in integrin α subunits -: TIP-2/GIPC binds to a type I recognition sequence in α6a/α5 and a novel sequence in α6B

We used published peptide library data to identify PDZ recognition sequences in integrin a subunit cytoplasmic domains and found that the alpha (6)A and a. 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 B-6, although the alpha B-6 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 B-6 defines a new subclass of type I PDZ interaction site, characterized by a non-aliphatic residue at position 0.