Previous studies that used different Doxorubicin assays have shown that these fragments, which encompass the three variable domains, exhibited binding properties indistinguishable from full-length ectodomains (Wojtowicz et al., 2004). Sedimentation equilibrium AUC experiments showed that the wild-type isoforms homodimerized strongly with KD values of 1 μM for Dscam110.27.25 (shorthand nomenclature for the isoform comprising Ig2.10, Ig3.27, and Ig7.25) and 2.1 μM for Dscam13.31.8 (Table 1). By contrast, homodimers were not observed with isoforms containing chimeric Ig2 domains, as indicated by the results of both sedimentation equilibrium (Table 1) and sedimentation
velocity (see Figure S1 available online) AUC experiments. We then measured heterophilic binding between complementary pairs of chimeras in both velocity and equilibrium AUC experiments. As predicted, each pair of complementary chimeras bound to each other with affinities similar to wild-type homodimers; the Ig2.3C-containing isoform bound to the Ig2.4C-containing isoform with a KD of 1 μM, and the Ig2.10C-containing isoform bound to the Ig2.11C-containing isoform with a KD of 4.6 μM. These data argue that the loss of homophilic binding is not due to more global changes in protein conformation,
but rather to an alteration in binding specificity. Our results indicate that matching Ig3 and Ig7 is not sufficient to form dimers within the detection limit for equilibrium Screening Library chemical structure AUC (i.e., <500 μM). These quantitative analytical studies support the view, based on our previous ELISA-based assays, that the vast majority of Dscam1 isoforms show little binding to isoforms with a markedly different interface at only a single variable domain. In summary, isoforms containing chimeric Ig2 domains exhibit altered recognition specificities with a profound loss of homophilic binding that is accompanied by a gain of heterophilic specificity between isoforms containing complementary chimeric Ig2 domains. MTMR9 Some degree of homophilic binding of chimeric
isoforms was observed when isoforms were overexpressed in S2 cells (Figure S2). Whether this reflects a limited ability for interactions when proteins are presented on the cell surface, is a result of overexpression, or both is unknown. The chimeric isoforms with altered binding specificities provided us with a unique opportunity to definitively test the notion that specific recognition between Dscam1 isoforms on sister neurites is necessary to promote self-avoidance. As a first step toward addressing this issue, we sought to knock in the chimeric isoforms into the endogenous locus; expression from the endogenous locus would ensure that the gene is expressed at the same level and spatiotemporal pattern as the wild-type gene.