, 2007). These data, together with structural studies, led us to propose that modular matching selleck chemicals llc at all three variable domains (Ig2:Ig2, Ig3:Ig3, and Ig7:Ig7) gives rise to exquisite homophilic binding specificity ( Meijers et al., 2007, Sawaya et al., 2008, Wojtowicz et al., 2004 and Wojtowicz et al., 2007). Genetic studies support the notion that Dscam1-mediated homophilic recognition plays a key role in neural circuit assembly by providing the molecular basis for self-avoidance
(Hattori et al., 2007, Hattori et al., 2009, Hughes et al., 2007, Matthews et al., 2007, Soba et al., 2007, Wang et al., 2002, Zhan et al., 2004 and Zhu et al., 2006). Self-avoidance refers to the tendency of neurites of the same cell to avoid each other (Kramer and Kuwada, 1983). Analysis of mutants encoding reduced numbers of isoforms established that thousands of isoforms are required for self-avoidance (Hattori et al., 2007 and Hattori selleck inhibitor et al., 2009). Expression data from several neuronal cell types are consistent with each cell expressing a unique combination of Dscam1 isoforms, thereby endowing each neuron with a distinct cell-surface identity (Neves et al., 2004 and Zhan et al., 2004).
Based on these studies, we proposed that self-neurites express the same isoforms, bind to each other, and are subsequently repelled. By contrast, because neurites of different neurons express different isoforms, Dscam1 does not mediate interactions between them (Hattori et al., 2008). Although isoform-specific homophilic recognition is the linchpin of models for Dscam1 function, whether Resminostat this biochemical property is required in vivo is unknown. In this paper, we use a combined biochemical and genetic approach to address this issue. To directly address the importance of binding specificity in vivo, we sought to generate pairs of Dscam1 isoforms that exhibit interallelic complementation; each
protein would not bind to itself but would bind heterophilically to another isoform. If isoform-specific recognition were critical for self-avoidance, then expression of each homophilic binding-deficient isoform would not rescue the mutant phenotype, whereas expression of the two complementary isoforms in the same cell would. This is analogous to forward genetic approaches in bacteria to identify proteins interacting in vivo through the isolation of allele-specific extragenic suppressor mutations (Hartman and Roth, 1973). To generate pairs of isoforms with altered binding specificities, we focused on the Ig2 interface, because it is the most extensively characterized of the three variable domain interfaces (Meijers et al., 2007, Sawaya et al., 2008 and Wojtowicz et al., 2007). Each specificity interface of the Ig2 domains comprises a different 8-amino-acid β-strand segment (positions 107–114). These unique sequences align in a 2-fold symmetric fashion with a symmetry center and two identical complementary networks that fit together by shape and charge complementarity (Figure 1A).