, 2008) and GLR-1::mCherry

in AVA of transgenic worms. We did not observe any BiFC fluorescence in these worms indicating that NMR-2 and SOL-2 do not associate (Figure 3E). This result is consistent with earlier studies showing that neither GLR-1 nor SOL-1 colocalize with NMDARs (Brockie et al., 2001b; Zheng et al., 2004). The BiFC data suggest a model in which SOL-1 and SOL-2 directly interact. To more rigorously test this hypothesis, we asked whether we could detect SOL-1 and GLR-1 after immunoprecipitation of SOL-2. Because of technical limitations (low abundance of auxiliary proteins and receptors in whole worms), we could not reliably detect these proteins in whole worm lysates. Therefore, we coexpressed these proteins in HEK293 cells and used these transfected cells for biochemical studies. We found that SOL-2 was associated with GLR-1 and SOL-1 (Figure S4A) but did not associate with the unrelated transmembrane protein DCC (Keino-Masu et al., 1996). Together, selleck chemicals the biochemical and BiFC data indicate that SOL-1, SOL-2, and GLR-1 physically interact and form a receptor complex. We this website had previously demonstrated that GLR-1 surface expression was not appreciably altered

in sol-1 mutants ( Zheng et al., 2004). Is surface expression of GLR-1 also independent of sol-2? To test this possibility, we double labeled GLR-1 with a HA-epitope (extracellular) and GFP (intracellular) and expressed this functional construct (HA::GLR-1::GFP) in transgenic worms ( Zheng et al., 2004).

We assessed the surface expression of GLR-1 by injecting fluorescently labeled anti-HA antibodies into the pseudocoelomic space of transgenic wild-type worms and sol-2 mutants ( Gottschalk et al., 2005; Zheng et al., 2004). We observed punctate anti-HA antibody fluorescence along the ventral cord in both wild-type worms ( Figure 3F) and sol-2 mutants ( Figure 3G), suggesting that GLR-1 is expressed on the cell surface in sol-2 mutants. We obtained additional evidence to support whatever the claim that GLR-1 surface expression was not appreciably altered in sol-2 mutants by generating a transgenic strain that expressed GLR-1 fused to superecliptic phluorin (SEP, a pH-sensitive variant of GFP) ( Miesenböck et al., 1998). SEP was inserted in the extracellular N-terminal domain four amino acids from the mature N terminus (SEP::GLR-1). In agreement with our antibody studies, we found that the fluorescence intensity of surface-expressed SEP::GLR-1 appeared similar in transgenic sol-2 mutants and wild-type worms ( Figure 3H). Control experiments (acid wash) indicated that the SEP fluorescence signal represented surface receptors ( Figure S4B). Another possible explanation for the reduced glutamate-gated current in sol-2 mutants is reduced surface delivery of SOL-1. However, we also found no appreciable difference in fluorescence intensity when we compared SEP::SOL-1 surface delivery in transgenic wild-type worms and sol-2 mutants ( Figure 3I).