Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking.
Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli-expressed glutathione-S-transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein-GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.
Liu
Yingmiao
Y
Department of Surgery, Duke University Medical Center, Durham, North Carolina 27710, USA.
Sun
Qi-An
QA
Chen
Qiang
Q
Lee
Tong H
TH
Huang
Yangzhong
Y
Wetsel
William C
WC
Michelotti
Gregory A
GA
Sullenger
Bruce A
BA
Zhang
Xiuwu
X
D000595:Amino Acid Sequence; D000818:Animals; D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000494:pharmacology*; D019921:Biotinylation / Q000379:methods; D002478:Cells, Cultured; D002540:Cerebral Cortex; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004622:Embryo, Mammalian; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D018091:Receptors, AMPA / Q000187:drug effects / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D014162:Transfection
D052157:Aptamers, Nucleotide; D017868:Cyclic AMP-Dependent Protein Kinases; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D018091:Receptors, AMPA; D012694:Serine