The scaffold protein PDZK1 undergoes a head-to-tail intramolecular association that negatively regulates its interaction with EBP50.

An essential aspect of cell organization is that proteins have to be properly localized through specific and tightly regulated associations. One of the major contributors to achieving this state is through scaffold proteins, which are modular molecules often made up of multiple protein-protein interaction domains that serve to precisely link other molecules together to regulate their functions. Among the most common protein-protein interaction modules in scaffold proteins is the PDZ (Post-Synaptic Density-95, Discs Large, Zonula Occludens-1) domain. These domains are primarily known for their ability to interact with carboxyl-terminal tails of target proteins, although some internal binding motifs have also been found (1–3). By interacting with the tails of proteins, PDZ-containing molecules can mediate the localization of their binding partners to specific subcellular destinations (2). Further, scaffold proteins are themselves regulated in various ways, such as by phosphorylation, degradation or targeting to specific locations. In addition, PDZ motifs can undergo hetero- and homo-dimerization or oligomerization, which may contribute to their functions by allowing the formation of larger, more extensive, scaffolds (4, 5). There is a growing awareness of regulation through intramolecular associations, wherein a motif in a protein interacts with another domain within that same molecule (6, 7). Well-studied examples of regulation by intramolecular associations include the tyrosine kinase Src, the membrane-cytoskeletal linking Ezrin-Radixin-Moesin (ERM) proteins, and the Arp2/3 complex activator Wiskott Aldrich Syndrome protein (WASP) (7, 8). In these and many other cases the intramolecular interaction maintains the protein in an autoinhibited state, which is relieved by some regulatory stimulus. Intramolecular associations may be regulated by binding of outside elements, such as binding of active Cdc42 to WASP, or by modification via covalent linkages, such as ubiquitination, which induces an intramolecular association in the EPS15 endocytic adaptor protein (9, 10). Phosphorylation is also a common regulator of intramolecular associations, where it may serve to decrease such an association, as in the ERM proteins, or stimulate them, as in phosphorylation of Src at tyrosine 527 (7, 11). PDZ-containing proteins have also been found to be regulated by an intramolecular association, specifically between their own tail and one of their PDZ domains. In the case of EBP50/NHERF1, a scaffolding protein with two PDZ domains and a carboxyl-terminal tail that ends in a PDZ-binding motif, the tail interacts with the second PDZ domain, and in so doing reduces access of target molecules to both PDZ domains (12). Another example is the X11α/Mint1 scaffold protein (13). In this study we have analyzed the in vitro properties of PDZK1 (also called CAP70, NHERF3, or NaPi-Cap1), a scaffolding protein with 4 tandem PDZ domains. PDZK1 is a physiologically important molecule implicated in tissue-specific regulation of expression and localization of membrane-associated receptors and directly linked to maintenance of serum cholesterol levels (14–16). We show that PDZK1 is able to undergo both an intermolecular and an intramolecular association. Furthermore, the intramolecular association regulates the ability of its tail to interact with EBP50, thereby regulating the association between these two scaffold proteins.