Subunit specificity and interaction domain between GABA(A) receptor-associated protein (GABARAP) and GABA(A) receptors.
GABARAP (GABA(A) receptor-associated protein) interacts with both microtubules and GABA(A) receptors in vitro and in vivo and is capable of modulating receptor channel kinetics. In this study, we use the intracellular loop of 15 GABA(A) receptor subunits to show that the interaction between GABARAP and GABA(A) receptor is specific for the gamma subunits. Pharmacological characterization of proteins purified by GABARAP affinity column indicates that native GABA(A) receptors interact with GABARAP. Quantitative yeast two-hybrid assays were used to identify the interaction domain in the gamma2 subunit for GABARAP binding, and to identify the interaction domain in GABARAP for GABA(A) receptor binding. A peptide corresponding to the GABARAP interaction domain in the gamma2 subunit was used to inhibit the interaction between GABARAP and the gamma2 subunit. In addition, the ability of GABARAP to promote cluster formation of recombinant receptors expressed in QT-6 fibroblasts was inhibited by a membrane-permeable form of this peptide in a time-dependent manner. The establishment of a model for GABARAP-induced clustering of GABA(A) receptors in living cells and the identification of subunit specificity and interaction domains in the interaction between GABARAP and GABA(A) receptors is a step in dissecting the function of GABARAP in GABA(A) receptor clustering and/or targeting.
Nymann-Andersen
Jesper
J
Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California, USA.
Wang
Hongbing
H
Chen
Li
L
Kittler
Josef T
JT
Moss
Stephen J
SJ
Olsen
Richard W
RW
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D001569:Benzodiazepines / Q000378:metabolism; D003370:Coturnix; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D010446:Peptide Fragments / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits; D051381:Rats; D011940:Receptor Aggregation / Q000187:drug effects / Q000502:physiology; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C116741:GABARAP protein, human; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D001569:Benzodiazepines; D005982:Glutathione Transferase