esting that this domain cannot be directly involved in the intracellular trimeric G-protein signaling cascade. Murine nyctalopin is predicted to have as many as 5 amino acids within the cytoplasm; so in theory these could interact with other components of the cascade. However, given human nyctalopin is thought to be GPI anchored, and therefore lacking this region, we suggest it is unlikely that the intracellular amino acids in murine nyctalopin have any function. Experiments such as truncation of these amino acids and subsequent functional analyses would, however, be needed to confirm this point. Further, our recent data and that of others indicate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22189963 that nyctalopin interacts directly with TRPM1 and is required to localize TRPM1 to the tips of DBCs. LRR domains have been shown to be involved in proteinprotein interactions in several SLRP family members and other proteins. That this domain is critical to function in nyctalopin is highlighted by the fact that mutations in the LRR domain of nyctalopin in humans cause CSNB1. These data, combined with our observation that nyctalopin is required for the localization of TRPM1 to the dendritic tips of DBCs, suggest several possible mechanisms of action. Functional TRP channels are homo or hetero tetramers. Therefore, it is possible that nyctalopin is required for stabilization of the tetrameric structure in the membrane. In support of this, decorin, which is a close relative of nyctalopin, has been shown to interact with epidermal growth factor receptor, causing dimerization and subsequent internalization of the EGF. The detailed mechanism of action of nyctalopin will require detailed dissection of the protein protein interaction domains as well as the predicted glycosylation of nyctalopin, although its functional form does not appear to be a dimer, at least in yeast. In this report we have shown that murine nyctalopin is a transmembrane protein. In contrast human nyctalopin is anchored to the membrane by a GPI moiety. Further experimental analyses will be required to determine which combinations of amino acid substitutions in murine nyctalopin compared to human Topology of Murine Nyctalopin nyctalopin result in a transmembrane anchor. These findings do suggest that how nyctalopin is anchored to the membrane is not critical to its function; rather it may be the structure of the extracellular domain. Future studies to elucidate the proteins with which nyctalopin interacts, and the critical regions of nyctalopin involved in these interactions will likely shed light not only on nyctalopin, but also the role of LRR domains in many other proteins. For mutagenesis experiments, NycDTM3-Cub was created as a deletion variant of Nyc-Cub. This construct had nyctalopin amino acids 455476 deleted. NycL463R-Cub had a single amino acid substitution in predicted transmembrane domain 1. Constructs used in the in vitro Transcription/Translation Experiments SNycLuc was made by inserting a full length nyctalopin cDNA into the BamHI site of the T7 Luciferase Control vector. This arrangement fused luciferase to the 
Cterminus of nyctalopin. For the SLucNyc, the T7 luciferase BIX-02189 site vector was re-engineered so that the nyctalopin signal sequence was fused to luciferase, which was fused to amino acids 23476 of nyctalopin. This arrangement put luciferase at the N-terminus of nyctalopin between the nyctalopin signal sequence and the rest of nyctalopin. Methods Yeast Strains and Growth Media Yeast strains used in this study are NY
