Nyctalopin Expression in Grm6 Mutant Mice and Membrane Topology Determined in Yeast

Abstract

Purpose: : Signal transmission through depolarizing bipolar cells (DBC) in the retina is dependent on metabotropic glutamate receptor 6 (Grm6) mediated modulation of a non-specific cation channel. Nob mice, which carry a mutation in the Nyx gene that encodes nyctalopin, results in the failure of glutamate to modulate the DBC cation channel. Previous data from our lab established that expression of nyctalopin in DBC dendrites restores visual function in nob mice, providing evidence that nyctalopin is involved in the Grm6 to cation channel cascade. In this study, we determine if expression of Grm6 is required for proper localization of nyctalopin and if Grm6 and nyctalopin interact when expressed in yeast.

Methods: : We generated transgenic mice (TgNyc) expressing an eYFP-nyctalopin fusion protein in DBCs. These mice were crossed to Grm6^nob4 mice to generate Grm6^nob4/TgNyc animals. Immunohistochemistry for eYFP-nyctalopin, Grm6, ribeye, G_O and PKC was performed in retinal sections of Grm6^nob4/TgNyc mice. The membrane split-ubiquitin yeast two-hybrid (MYTH) system was used to analyze nyctalopin’s orientation, homodimerization and possible interaction with Grm6.

Results: : Immunohistochemistry for the eYFP-nyctalopin fusion protein in the absence of Grm6 showed the normal staining pattern of nyctalopin at the tips of the DBC dendrites. All other markers showed staining patterns similar to previously published results. To evaluate whether nyctalopin interacts with Grm6 we used the MYTH system. Full length nyctalopin and Grm6 expressing plasmids were co-expressed in yeast, but there was no genetic evidence of interaction. Co-expression of nyctalopin bait vectors with controls that detect interaction either inside or outside the cell showed that nyctalopin is orientated in the membrane with its amino-terminus extracellular. Additional tests found no evidence that nyctalopin can form homodimers, like other SLRP family members.

Conclusions: : Expression of Grm6 is not required for proper targeting of nyctalopin to the DBC dendrites. Murine nyctalopin is a transmembrane protein with its carboxy-terminus inside the cell and does not form homodimers. Finally, nyctalopin and Grm6 do not interact in yeast.

Keywords: bipolar cells • gene/expression • transgenics/knock-outs