A recently described protein module consisting of 35-40 semiconserved residues, termed the WW domain, has been identified in a number of diverse proteins including dystrophin and Yes-associated protein (YAP). Two putative ligands of YAP, termed WBP-1 and WBP-2, have been found previously to contain several short peptide regions consisting of PPPPY residues (PY motif) that mediate binding to the WW domain of YAP. Although the function(s) of the WW domain remain to be elucidated, these observations strongly support a role for the WW domain in protein-protein interactions. Here we report the isolation of three novel human cDNAs encoding a total of nine WW domains, using a newly developed approach termed COLT (cloning of ligand targets), in which the rapid cloning of modular protein domains is accomplished by screening cDNA expression libraries with specific peptide ligands. Two of the new genes identified appear to be members of a family of proteins, including Rsp5 and Nedd-4, which have ubiquitin-protein ligase activity. In addition, we demonstrate that peptides corresponding to PY and PY-like motifs present in several known signaling or regulatory proteins, including RasGAP, AP-2, p53BP-2 (p53-binding protein-2), interleukin-6 receptor-␣, chloride channel CLCN5, and epithelial sodium channel ENaC, can selectively bind to certain of these novel WW domains.The recognition and elucidation in recent years of various modular protein domains, along with their specific peptide ligands, have spawned a remarkable progress in our understanding of their role in signal transduction and other fundamental cellular processes (1, 2). Analysis of the SH (Src homology) domains, SH2 and SH3, present in a wide variety of proteins involved in cellular signaling and transformation has been particularly fruitful. The ligand specificity of many different SH2 and SH3 domains has been defined using combinatorial peptide libraries. SH2 domains bind with high affinity to phosphotyrosine residues within a specific sequence context (3). In contrast, SH3 domains bind to proline-rich peptides that share a conserved PXXP motif (4, 5).A newly described protein module, termed the WW domain, has been reported (6 -8). The WW domain consists of 35-40 amino acids and is characterized by four well conserved aromatic residues, two of which are tryptophan. The secondary structure of the WW domain has recently been determined and consists of a slightly bent three-stranded antiparallel -sheet (9). This domain has been reported in a wide variety of proteins of yeast, nematode, and vertebrate origin, including Rsp5, Yesassociated protein (YAP), 1 human and murine Nedd-4, FE65, Pin1, and a human . Although the precise physiological role of the WW domain remains undetermined, its presence in diverse proteins involved in signaling, regulatory, and cytoskeletal functions, as well as its rapidly emerging role in signaling mechanisms that underlie several human diseases, clearly underscores its importance (15, 16). Two ligand proteins for the YAP WW domain, WBP-1...