Binding of the GABA(A) receptor-associated protein (GABARAP) to microtubules and microfilaments suggests involvement of the cytoskeleton in GABARAPGABA(A) receptor interaction.
GABA(A) receptor-associated protein (GABARAP) was isolated on the basis of its interaction with the gamma2 subunit of GABA(A) receptors. It has sequence similarity to light chain 3 (LC3) of microtubule-associated proteins 1A and 1B. This suggests that GABARAP may link GABA(A) receptors to the cytoskeleton. GABARAP associates with tubulin in vitro. However, little is known about the mechanism for the interaction, and it is not clear whether the interaction occurs in vivo. Here, we report that GABARAP interacts directly with both tubulin and microtubules in a salt-sensitive manner, indicating the association is mediated by ionic interactions. GABARAP coimmunoprecipitates with tubulin and associates with both microtubules and microfilaments in intact cells. The cellular distribution is altered by treatment with taxol, nocodazole, and cytochalasin D. The tubulin binding domain was located at the N terminus of GABARAP by using synthetic peptides and deletion constructs and is marked by a specific arrangement of basic amino acids. The interaction between GABARAP and actin might be mediated by other proteins. These results demonstrate the GABARAP interacts with the cytoskeleton both in vitro and in cells and suggest a role of GABARAP in the interaction between GABA(A) receptors and the cytoskeleton. Such interactions are presumably needed for receptor trafficking, anchoring, and/or synaptic clustering. The structural arrangement of the basic amino acids present in the tubulin binding domain of GABARAP may aid in recognition of the potential of tubulin binding activity in other known proteins.
Wang
H
H
Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California, USA.
Olsen
R W
RW
D008841:Actin Cytoskeleton / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism / Q000648:ultrastructure; D014162:Transfection; D014404:Tubulin / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C116741:GABARAP protein, human; D008869:Microtubule-Associated Proteins; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D014404:Tubulin