The Drosophila melanogaster Dock protein — identified several years ago for its crucial function in photoreceptor axon guidance, does not act alone. Reporting in the Journal of Biological Chemistry, Dixon, Worby and colleagues have identified a Drosophila sorting protein, which, when tyrosine phosphorylated, can interact with this adaptor protein.

To unmask Dock's gang of accomplices, the authors stably expressed the src-homology-2 (SH2) domain of Dock as a tagged protein in Drosophila S2 cells and analysed proteins that complexed with it by affinity chromatography. Of the five protein bands that were visible, a protein of 63 kDa was found to contain an amino-terminal SH3 domain, an internal Phox homology (PX) domain, a proline-rich motif and a predicted coiled-coil domain. Based on its homology with human sorting nexin 9, also known as SH3PX1, the 63-kDa protein was re-named DSH3PX1. The interaction between Dock and DSHPX1 — which the authors confirmed by co-immunoprecipitation — occurs mainly through the SH2 domain of Dock.

As other sorting nexins can associate with some cell-surface receptors, Dixon and colleagues then investigated whether DSH3PX1 could interact with another of the five proteins found in the Dock complex, called Dscam — the Drosophila orthologue of the human Down Syndrome cell-adhesion molecule (DSCAM). Not only did they find an interaction, but they also showed that Dock wasn't needed for the association by using RNA interference to eliminate expression of Dock. Further analysis showed that DSH3PX1 can also interact with AP-50, a member of the AP-2 complex that recognizes proteins that are destined to be endocytosed in clathrin-coated pits. So, DSH3PX1 might function to bridge Dscam to proteins in these pits.

Dscam is known to be required for embryonic axonal guidance, so Dixon et al. reasoned that by investigating the interactions of DSH3PX1, they might get an insight into the mechanisms by which Dscam mediates this effect. So, using full-length DSH3PX1, they carried out a two-hybrid screen. Among the interacting proteins was DSH3PX1 itself, indicating that this protein could dimerize. More significantly, however, Wasp — a regulator of the actin cytoskeleton, with a known function in endocytosis — was also identified as a positive binding partner. Wasp contains several protein–protein interaction domains (a Cdc42/Rac-binding (CRIB) domain, a proline-rich domain, two verprolin homology domains, a cofilin homology domain, a pleckstrin homology domain and an IQ domain), but the interaction with DSH3PX1 occurs through DSH3PX1's SH3 domain. This is consistent with previous observations that Wasp binds to several SH3-domain-containing proteins through its proline-rich sequence. Moreover, no competition was seen between the binding of Wasp and Dock to DSH3PX1, indicating that DSH3PX1 can interact with Wasp through its SH3 domain, whereas Dock uses an SH2-domain interaction.

So it is quite possible to imagine a scenario in which axonal-guidance cues induce tyrosine phosphorylation of DSH3PX1, which thereby enables it to associate with Dock. Dock, in turn, is bound to Dscam, so that, in conjunction with AP-50 and Wasp, DSH3PX1 might be involved in the cytoskeletal rearrangements that are required to endocytose Dscam. In this way, guidance receptors on the plasma membrane would undergo timely removal — a critical aspect of growth-cone remodelling.