Worm Breeder's Gazette 14(3): 30 (June 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Stanford University School of Medicine, Stanford, CA 94305
The activity of LIN-2, LIN-7 and the receptor tyrosine kinase LET-23 are required for the vulval precursor cell P6.p to receive and transduce the anchor cell signal that induces the 1! vulval cell fate. P6.p, like the other Pn.p cells, is a polarized epithelial cell; its basal surface faces the anchor cell and its apical surface faces the cuticle. In wild-type animals, a LET-23::GFP fusion protein is found in a lateral ring that is co-localized with and basal to the vulval precursor cell junctions. LIN-2 and LIN-7 are necessary for subcellular localization of LET-23 (the putative receptor for the inductive ligand), since LET-23::GFP is mislocalized and distributed across the apical surface of the Pn.p cells in lin-2 and lin-7 mutants (Simske et al., (1996) Cell 85, 195-204). We are interested in the mechanism of LET-23 localization, and are testing whether there might be direct protein interactions between LET-23 and LIN-2 or LIN-7. LIN-2 is a member of a family of proteins called MAGUKs (for Membrane-Associated GUanylate Kinase) (Hoskins et al., (1996) Development 122, 97-111). MAGUKs have been found to specifically localize to cellular junctions or to regions of cell-cell contact. MAGUKs contain at least one PDZ domain, which are domains known to mediate protein-protein interactions. LIN-7 contains a PDZ domain, but does not contain other domains typically found in MAGUKs. To test if LIN-2, LIN-7 or LET-23 interact with each other, we used the yeast two-hybrid system and in vitro binding experiments. We found that full-length LIN-7 interacts with both a 200 amino acid region spanning the PDZ of LIN-2 and with the intracellular region of LET-23 in the yeast two-hybrid system. However, we did not detect an interaction between LIN-2 and LET-23 in yeast. in vitro binding experiments were used to confirm the yeast two-hybrid results, and to delineate the regions of LIN-7 that bind to LET-23 and LIN-2. We tested the ability of different glutathione S-transferase (GST)::LIN-7 fusion proteins, purified from bacteria and immobilized on glutathione agarose beads, to specifically bind to LET-23 or LIN-2 from cell lysates. First, we found that GST::LIN-7 fusion proteins containing either full-length LIN-7 or only the PDZ of LIN-7 (amino acids 171-297) could bind full-length LET-23 in a cell lysate from Sf9 cells expressing LET-23 from a baculovirus vector (kindly provided by Andy Golden). Second, we observed that GST::LIN-7 fusion proteins containing either full-length LIN-7 or the N-terminus (amino acids 1-125) of LIN-7 could bind LIN-2 expressed from transiently transfected Cos7 cells. We did not observe any binding between the PDZ of LIN-7 and LIN-2. Therefore it seems there are two distinct regions of LIN-7 that mediate the interactions with LIN-2 and LET-23 (see illustration). The N-terminus of LIN-7 (amino acids 1-125) binds to the PDZ domain of LIN-2, and the C-terminus of LIN-7 (amino acids 171-297) binds to the intracellular region of LET-23. The above data suggest the possibility that LIN-2, LIN-7 and LET-23 form a ternary complex. To test this idea, we used a yeast "three" hybrid system. As reported above, we did not see an interaction between LIN-2 (fused to the GAL4 activation domain, ACT::LIN-2) and the intracellular region of LET-23 (fused to the GAL4 DNA binding domain, DBD::LET-23) in the yeast two-hybrid system. Interestingly, when we expressed LIN-7 tagged with an SV40 nuclear localization signal in the presence of ACT::LIN-2 and DBD::LET-23, it appeared that the three proteins were able to interact. This ternary interaction was specific to the combination of LIN-2, LIN-7, and LET-23 as we saw no protein interactions with the combination of DBD::LET-23, LIN-7 and the yeast protein ACT::SNF4. These data suggest that LIN-2 binds LIN-7 and LIN-7 binds LET-23 to form a ternary complex. Experiments are in progress to test if these interactions are required for vulval development in vivo.