Phospholipase C-related inactive protein is implicated in the constitutive internalization of GABAA receptors mediated by clathrin and AP2 adaptor complex.
A mechanism for regulating the strength of synaptic inhibition is enabled by altering the number of GABA(A) receptors available at the cell surface. Clathrin and adaptor protein 2 (AP2) complex-mediated endocytosis is known to play a fundamental role in regulating cell surface GABA(A) receptor numbers. Very recently, we have elucidated that phospholipase C-related catalytically inactive protein (PRIP) molecules are involved in the phosphorylation-dependent regulation of the internalization of GABA(A) receptors through association with receptor beta subunits and protein phosphatases. In this study, we examined the implications of PRIP molecules in clathrin-mediated constitutive GABA(A) receptor endocytosis, independent of phospho-regulation. We performed a constitutive receptor internalization assay using human embryonic kidney 293 (HEK293) cells transiently expressed with GABA(A) receptor alpha/beta/gamma subunits and PRIP. PRIP was internalized together with GABA(A) receptors, and the process was inhibited by PRIP-binding peptide which blocks PRIP binding to beta subunits. The clathrin heavy chain, mu2 and beta2 subunits of AP2 and PRIP-1, were complexed with GABA(A) receptor in brain extract as analyzed by co-immunoprecipitation assay using anti-PRIP-1 and anti-beta2/3 GABA(A) receptor antibody or by pull-down assay using beta subunits of GABA(A) receptor. These results indicate that PRIP is primarily implicated in the constitutive internalization of GABA(A) receptor that requires clathrin and AP2 protein complex.
Kanematsu
Takashi
T
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research, Kyushu University, Fukuoka, Japan.
Fujii
Makoto
M
Mizokami
Akiko
A
Kittler
Josef T
JT
Nabekura
Junichi
J
Moss
Stephen J
SJ
Hirata
Masato
M
D033962:Adaptor Protein Complex 2 / Q000502:physiology*; D000818:Animals; D002461:Cell Line, Transformed; D002966:Clathrin / Q000502:physiology*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation / Q000379:methods; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D056918:Nuclear Receptor Coactivators; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014162:Transfection / Q000379:methods
D033962:Adaptor Protein Complex 2; D002966:Clathrin; D047908:Intracellular Signaling Peptides and Proteins; C486559:NCOA6 protein, human; D056918:Nuclear Receptor Coactivators; D021122:Protein Subunits; D011963:Receptors, GABA-A