Neurons are polarized cells, with dendrites specialized in receiving and axons in transmitting signals. In cell culture conditions, neurons initially extend multiple neurites. By chance, one neurite elongates faster than the others and is specified to become an axon, while the other neurites remain short and become dendrites. However, such axonal polarization behavior has not been observed in vivo . We have previously reported that loss of function in
syd-1 causes axonal specializations to be formed improperly in dendritic processes of the ventral cord type D motor neurons (Hallam et al., 2002). SYD-1 is localized to presynaptic terminals. In
syd-1 mutants, presynaptic proteins such as SYD-2/liprin, synaptobrevin, CAMKII are mislocalized, suggesting that
syd-1 likely functions upstream of synaptic assembly by either directing the presynaptic components to the correct site or by restricting presynaptic components from diffusing into improper domains. By protein homology search, SYD-1 contains a PDZ domain at the N-terminal, a C2 and a GTPase activating domain at the C-terminal. Functional studies have implicated that all three domains are required for the precise localization and function of SYD-1. We are interested how SYD-1 synaptic localization is regulated. We are examining both known mutants and are performing unbiased genetic screens. In mutants for
unc-10 ,
unc-16 ,
unc-116 ,
unc-104 ,
unc-14 and
unc-51 , SYD-1 localization is grossly normal. To elucidate the molecular signaling pathway involving SYD-1, we attempt to identify proteins that interact with each domain of SYD-1. In a yeast two-hybrid screen using the PDZ domain of SYD-1 as bait, we isolated a neurexin-like protein, NRX-1. Neurexins are a family of cell adhesion molecules that are expressed in neurons and have been shown to be able to promote synapse formation in cultured cells (Dean, et al., 2003). The predicted full length NRX-1 shows an overall homology to alpha-NRX: both have three LNS (Laminin A G domain, Neurexins, and Sex hormone-binding globulin)/LG(Laminin G)-EGF/EGF like-LNS/LG (the domains after the dashes are the domains in NRX-1) repeats in the extracellular parts. It was reported that
nrx-1 exhibited developmentally alternative splicing and that some isoforms were localized at synapses (Soutschek, et al., European Worm Meeting,1998). The C-terminal of NRX-1 contains a PDZ domain binding motif. We have confirmed the NRX-1 and SYD-1 interaction by in vitro GST-pull down assay. The binding of NRX-1 and SYD-1 is mediated through the PDZ domain and the PDZ binding site. We are testing the possibility whether NRX-1 has a role in localizing SYD-1 to precise subcellular positions during axon formation or wherther SYD-1 may transduce NRX-1 signaling. We have also isolated candidate binding proteins for the GAP domain of SYD-1, and the progress will be presented at the meeting.