5106 Research Article Introduction PDZ domains were originally identified in the postsynaptic density protein PSD-95, the Drosophila tumor suppressor discs-large and the tight junctional protein ZO-1, but they are now known to be present singly or in multiple tandemly repeated copies in a diverse set of proteins (Hung and Sheng, 2002). These domains either bind to a consensus motif that is present at the C-terminus of target proteins or interact with internal peptide fragments of such proteins. They function as a protein-protein interaction module that mediates the clustering of signaling molecules and contributes to the organization of protein networks. Among PDZ domain-containing proteins, members of the LNX (ligand of NUMB-protein X)/PDZRN (PDZ domain- containing RING finger) family contain an N-terminal RING finger and two or four PDZ domains (Katoh and Katoh, 2004). LNX1 (also known as PDZRN2 or SEMCAP1) and LNX2 (PDZRN1) contain four PDZ domains, whereas LNX3 (PDZRN3 or SEMCAP3) and LNX4 (PDZRN4 or SEMCAP3L) contain two PDZ domains. LNX1 binds to NUMB (Dho et al., 1998), an intrinsic inhibitor of the conserved Notch signaling pathway that contributes to the control of cell fate and to signal integration during development (Artavanis-Tsakonas et al., 1999; Conboy and Rando, 2002). NUMB is asymmetrically distributed in dividing neural precursors and becomes segregated into one daughter cell, where it inhibits Notch signaling, thereby allowing the two daughter cells to adopt distinct fates (Zhong et al., 1996). LNX1 functions as an E3 ubiquitin ligase that mediates the ubiquitination and degradation of NUMB (Nie et al., 2002). It is therefore thought to augment Notch signaling by reducing the abundance of NUMB. By contrast, LNX3 and LNX4, which contain two PDZ domains instead of the four found in LNX1, have not been characterized at the protein level. Each of these two proteins also possesses a consensus- binding motif for class I PDZ domains at its C-terminus. We have now examined the properties and functions of LNX3 (PDZRN3). We show that the PDZRN3 gene is expressed in a variety of organs and tissues including the heart, skeletal muscle and liver. In skeletal muscle, the expression of PDZRN3 was found to be developmentally regulated. Furthermore, the protein is essential for differentiation of myoblasts into myotubes. Results Molecular structure of PDZRN3 We performed a yeast two-hybrid screen of a human heart cDNA library with the three PDZ domains of rat PSD-95 as the bait to identify PDZ domain-binding proteins. One of the 17 positive clones obtained by screening a total of 510 5 clones was found to contain an insert of ~3 kb that includes a putative open reading frame of 1506 bp. The final three amino acids at the C-terminus of the protein encoded by the insert were found to be Thr-Thr-Val, which match the consensus- binding motif for class I PDZ domains. A search of the GenBank database revealed that the insert corresponded to the partial sequence of KIAA1095, which was isolated in a random cloning strategy (Kikuno et al., 1999). The predicted protein PDZRN3 contains a RING-finger motif in its N-terminal region, two PDZ domains in its central region and a consensus-binding motif for PDZ domains at its C- terminus. It was identified in silico as a homolog of the protein known as LNX1 or SEMCAP1, which possesses ubiquitin ligase activity and binds the membrane protein Semaphorin 4C. However, PDZRN3 itself has not previously been characterized. We have now evaluated the properties and functions of PDZRN3. The PDZRN3 gene was shown to be expressed in various human tissues including the heart, skeletal muscle and liver and its expression in mouse skeletal muscle was developmentally regulated. Both the differentiation of C2C12 mouse skeletal myoblasts into myotubes and injury-induced muscle regeneration in vivo were found to be accompanied by up- regulation of PDZRN3. The differentiation-associated increase in the expression of PDZRN3 in C2C12 cells follows that of myogenin and precedes that of myosin heavy chain. Depletion of PDZRN3 by RNA interference inhibited the formation of myotubes as well as the associated up-regulation of myosin heavy chain in C2C12 cells. Our data suggest that PDZRN3 plays an essential role in the differentiation of myoblasts into myotubes by acting either downstream or independently of myogenin. Key words: PDZRN3, Myogenic differentiation, Skeletal muscle, C2C12 cell Summary PDZRN3 (LNX3, SEMCAP3) is required for the differentiation of C2C12 myoblasts into myotubes Ji-Ae Ko 1 , Yoshihiro Kimura 1 , Kenji Matsuura 1 , Hisato Yamamoto 1 , Toshikazu Gondo 2 and Makoto Inui 1, * 1 Department of Pharmacology, Yamaguchi University Graduate School of Medicine and 2 Department of Surgical Pathology, Yamaguchi University Hospital, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan *Author for correspondence (e-mail: minui@yamaguchi-u.ac.jp) Accepted 27 September 2006 Journal of Cell Science 119, 5106-5113 Published by The Company of Biologists 2006 doi:10.1242/jcs.03290 Journal of Cell Science