| Abstract No.: | C-B3053 |
| Country: | Canada |
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| Title: | CYTOPLASMIC MOTIFS INVOLVED IN THE DENDRITIC TARGETING OF NLG-1
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| Authors/Affiliations: | 1 Katka Gazdik*;
1 SFU, Burnaby, BC, Canada
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| Content: | Introduction: Cell polarity is a remarkable feature of neurons that underlies most aspects of their function in the nervous system. Several ion channels, cytoskeletal proteins, and cell adhesion molecules are localized exclusively to the axonal or somato-dendritic domain of neurons thereby enabling them to propagate action potentials and carry out synaptic transmission. Appropriate polarized targeting is particularly relevant in the case of proteins that contribute to synapse formation. Neuroligin-1 (NLG-1) mediates heterophilic adhesion with the axonal cell adhesion molecule -neurexin (NRX), and NLG-1 - NRX interactions can trigger the assembly of functional presynaptic terminals in axons. A prerequisite for such a role in synapse formation is that NLG-1 should be exclusively targeted to the somato-dendritic domain and excluded from axons. Previous work demonstrated that the dendritic targeting of NLG-1 relies on a cytoplasmic motif of 32 amino acids. Objectives: This study is geared towards identifying the specific amino acids present in NLG-1 cytoplasmic domain that target NLG-1 to the dendritic membrane.
Materials and Methods: The targeting of NLG-1, and various mutants was analyzed in primary cultured hippocampal neurons by quantitative immunofluorescence microscopy.
Results: Using truncations and point mutants of NLG-1 we demonstrate that three discrete, yet collinear sequences contribute to polarity. Specifically, a tyrosine motif (YTLA), a dihydrophobic motif (VL), and an acidic couplet (DD) are found within the 32aa domain and are all required for the polarized sorting of NLG-1. When all three motifs were replaced with a series of alanine residues, NLG-1 was localized in the axon at levels to that of uniformly distributed membrane proteins. Deletion of any one of theses sequences resulted in an intermediate polarity. Furthermore, when the uniform membrane protein CD-8 is appended with the minimal NLG-1 sorting sequence, this chimera is now polarized at levels comparable to wild type NLG-1. Conclusion: These results demonstrate a tripartite sorting signal is necessary and sufficient for the dendritic targeting of NLG-1. Based on the similarity of these sorting sequences to other previously identified dendritic sorting signals, for example in the Kv4.2 potassium channel and the transferrin receptor, there may exist a common, as yet undiscovered, cellular mechanism for dendritic targeting. These results are also relevant to understanding the cell biological mechanisms that contribute to synapse formation, by ensuring the correct localization of key synaptic organizers such as NLG-1. Experiments are now geared towards understanding the cellular machinery required for dendritic localization, for example vesicle populations that transport NLG-1 and adaptor proteins that may contribute to NLG-1 sorting.
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