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Deleted in colorectal cancer (DCC) is a single pass transmembrane glycoprotein that was originally identified in humans as a candidate tumor suppressor (Fearon et al., 1990). DCC belongs to the immunoglobulin superfamily, and the extracellular fragment is composed of four immunoglobulin-like (Ig-like) domains followed by six fibronectin type III (FN) domains. DCC plays a pivotal role in axon guidance by mediating a combination of attractive and repulsive effects through interactions with the diffusible guidance cue, netrin-1 (Hong et al., 1999) and draxin (Ahmed et al., 2011). We have recently reported the crystal structure of DCC membrane-proximal domains FN5 and FN6 (DCCFN56) in complex with netrin-1. The structure revealed two distinct binding sites located between DCCFN56 and netrin-1 (Finci et al., 2014). Netrin-1 consists of a laminin-like domain (LN), followed by three EGF domains (EGF1, EGF2, and EGF3), and a C-terminal netrin-like domain (NTR) (Kennedy et al., 1994). The designated binding site 1 in our crystal structure is DCC-specific, exclusively involving the FN5 domain of DCC and the EGF3 domain of netrin-1, whereas the designated site 2 is a unique and highly conserved anion-dependent binding site that involves both the FN5 and the FN6 domains of DCC engaging the EGF1-EGF2 domains of netrin-1. Another structure determined in parallel utilized a similar netrin-1 construct, from chicken, along with a different DCC construct, from mouse, containing the FN4-FN5 domains (DCCFN45) (Xu et al., 2014). Both structures share the same binding site 1 via the FN5 domain and the EGF3 domain of netrin-1. In addition, their structure reveals another binding site on netrin-1’s LN domain interacting with the DCC FN4 domain, DCCFN4 (referred to here as binding site 0). The two structures are complementary to each other, encompassing FN4, FN5, and FN6 domains of DCC, interacting with the netrin-1 N-terminal LN domain and all three of the netrin-1 EGF domains (Finci et al., 2015). Located between the functional DCCFN4 and DCCFN5 domains is an 8–28 amino acid linker, which is contingent on the particular DCC isoform. The DCC gene has two alternatively spliced isoforms. Cooper and colleagues demonstrated that the expression of alternatively spliced isoforms of mouse DCC is developmentally regulated. Their analyses on mRNA expression revealed that in D9.5 and D10.5 embryos, the DCCshort isoform (with 8-residue linker) was predominantly expressed, whereas between D10.5 and D11.5 of embryogenesis, the full-length DCClong isoform (with 28-residue linker) mRNA was upregulated (Cooper et al., 1995). A recent study by Leggere et al. shows that the RNA-binding family protein NOVA controls the alternative splicing of DCC. They conclude that the two DCC isoforms are functionally distinct, and the Netrin-1/DCClong interaction functions for axon outgrowth and guidance (Leggere et al., 2016). The linker must be important for the two isoforms to perform their function. An intriguing question yet to be addressed is how the 28-residue long flexible linker of an unliganded receptor eludes degradation in the extra-cellular environment? Here we have undertaken a structural and functional analysis of the DCC construct DCCFN456, with a 22-residue long “artificial” linker composed of Ser-Gly-Gly (SGG) repeats (herein referred to as SGG-22) between the FN4 and FN5 domains. We have crystallized and analyzed the structure of this DCCFN456 construct. From the structure, we discuss how a consolidated FN5 can serve as the key netrin-1 binding domain, and how a closed configuration, where the FN4 domain “lies” on the rod-like FN5-FN6 domains, can be proposed as a resting state of the DCC receptor for stability. It has been acknowledged that the FN4, FN5, and FN6 domains of the DCC receptor are all structurally and functionally required for netrin-1 binding (Finci et al., 2014; Xu et al., 2014). Another netrin-1 receptor, neogenin has a high sequence identity (around 46%) to that of DCC, and yet is most divergent in the FN4-FN5 linker region down to about 17% identity between the two receptors (Cooper et al., 1995). How does this long flexible and less homologous linker play its biological function? In order to dissect the relevance of the sequence and the length of the linker, we first utilized the cell-binding assay to probe the function of DCCFN456 with different linkers of different length between FN4 and FN5. The cell-binding assay has been previously utilized to describe interactions between DCC and netrin-1 in the binding site 1 and binding site 2 (Finci et al., 2014). We show here that when the linker was completely deleted, the binding was obliterated (ΔLinker4-5, in Fig. 1A). Next, we tested different constructs, replacing the wild-type linker with

Structure of unliganded membrane-proximal domains FN4-FN5-FN6 of DCC