Microtubule-associated protein light chain 2 is a stargazin-AMPA receptor complex-interacting protein in vivo.
The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, -TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse.
Ives
Jane H
JH
School of Biological and Biomedical Sciences, Science Research Laboratories, University of Durham, South Road, Durham DH1 3LE, United Kingdom.
Fung
Susanna
S
Tiwari
Priyanka
P
Payne
Helen L
HL
Thompson
Christopher L
CL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D051379:Mice; D008817:Mice, Mutant Strains; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D018091:Receptors, AMPA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
C113940:Cacng2 protein, mouse; D015220:Calcium Channels; D017931:DNA Primers; C490547:MAP1A protein, mouse; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D018091:Receptors, AMPA; D011994:Recombinant Proteins; C104722:glutamate receptor ionotropic, AMPA 2