Perspective in which the–2 position side chain is hydrophobic and interacts with a corresponding hydrophobic residue at the base of αB. PDZ domains recognizing S/T or a hydrophobic residue at the–2 position have been termed class I and class II, respectively. This structural model (Fig. 2) would predict that specificity is based primarily on interactions mediated by only two residues within the COOH-terminal ligand, which seems an improbable mechanism for generating binding specificity between a large and growing number of binding partners. This dilemma may be resolved in two ways. First, PDZ domains are always found within larger multidomain proteins. Other domains or sequences may target the protein to a specific membrane domain, such as the lateral membrane surface, thereby limiting the number of potential targets the PDZ may encounter. In some cases, these targeting sequences bind to actin-binding proteins, such as protein 4.1, that already have restricted spatial distributions. Second, the determination of binding specificity is likely to depend on more than just residues at the–2 and 0 positions. Analysis of target specificity using degenerate peptide and phage display library screening methods has demonstrated that many PDZ interactions require additional side chain interactions at the–1,–4, and/or–5 positions (5, 6). Many of the specificities defined by peptide library screening and confirmed by in vivobinding assays are difficult to explain on the basis our present understanding of crystallographic data on class I and II domains. Thus, a further understanding of PDZ domain specificity will require continued definition of novel binding interactions along with crystallographic or nuclear magnetic resonance studies. Adding to the complexity of how PDZ domains create protein networks is the recognition that some bind to non–COOH-terminal target sequences. For example, PDZ domains have also been demonstrated to interact with internal (T/S) XV motifs, such as that described in the Drosophila phototransduction system between the PDZ protein InaD and the transient receptor potential (TRP) store-operated Ca2+ channel (7). PDZ domains have also been demonstrated to bind directly to other PDZ domains, forming homomeric and heteromeric complexes (8). Finally, PDZ interactions have been documented with recognized domains commonly found in other proteins of the cortical cytoskeleton, such as LIM domains or ankyrin and spectrin repeats. The structural basis for these interactions is unknown. It is possible that the PDZ domain is binding to a short polypeptide with standard consensus embedded within the larger motif, similar to the interaction between InaD and TRP. Alternatively, these target sequences may interact with a unique surface on the PDZ domain. The latter hypothesis is supported by the observation that some PDZ domains can dimerize while simultaneously binding to the COOH-terminal motif of a third protein and that deletions within the PDZ domain that eliminate COOH-terminal binding can have no effect on PDZ dimerization (9). It is unclear whether all PDZ domains share multiple protein-binding mechanisms or whether these examples represent interactions unique to specific PDZ domains. In any case, they point to the extremely complex way in which PDZ domains are used to connect transmembrane proteins with the cortical protein network. Recent observations suggest that the interaction of PDZ domains with transmembrane binding partners can also be directly regulated by posttranslational modification or by receptor activation. For example, binding of the inwardly rectifying K+ channel Kir 2.3 to the second PDZ …