The novel role of QRediting in

1
The novel role of Q/R-editing in AMPA receptor
trafficking
Ingo Greger MRC Laboratory of Molecular
Biology Cambridge, England
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Glutamate receptors

• metabotropic (G-protein coupled)
• ionotropic (Ion channels)

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Functional properties of AMPAR-heteromers depend
on the subunit composition
Q/R
4
Trafficking modes of AMPARs are determined by
subunit compositions
regulated trafficking (long C-tails - GluR1)
2
2
1
1
PDZ
5
Receptor assembly takes place in the ER
Plasma membrane
TGN
Secretory pathway
Golgi
ER
6
Receptor assembly takes place in the ER
Plasma membrane
EP
TGN
EndoH resistant (mature)
Secretory pathway
Golgi
EndoH sensitive (immature)
ER
7
Specifically GluR2 resides in intracellular
compartments
1.
Subcellular rat brain fractions

GluR2
GluR1
NMDAR1
Calnexin
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Specifically GluR2 resides in intracellular
compartments
1.
Subcellular rat brain fractions
GluR2
GluR1
NMDAR1
Calnexin
2.
PNS (EndoH)
GluR1
GluR2
mature
immature
immature
38
80
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GluR2 exits the ER inefficiently
chase t (hrs)
0
2
4
6
9
12
mature
GluR1
immature
10
GluR2 exits the ER inefficiently
chase t (hrs)
0
2
4
6
9
12
mature
GluR1
immature
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Identifying the GluR2 ER-retention motif
N
C
12

• RNA editing
• enzymatic modification of ribonucleotides (in
  metazoa)
• Cytidine to Uridine (C/U)
• Adenine to Inosine (A/I)

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The Q/R site is a key regulator of ion flux
P. Seeburg
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The Q/R site determines GluR2 ER-retention
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The Q/R site determines GluR2 ER-retention
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Summary

• GluR2 is largely intracellular (ER), GluR1 is
  predominantly post-ER
• (cell surface).

• GluR2 is stably retained in the ER where it
  forms an intracellular pool.
• GluR1 exits from the ER within 9-12 hr.

• GluR2 ER-exit is controlled by the Q/R site. The
  edited R-form is
• ER-retained, the unedited Q-form exits the
  ER efficiently.

• Unedited GluR2(Q) accumulates at the cell
  surface/synapses.

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AMPAR assembly occurs in 2-steps
N N

• Dimerization
• (via N-termini)

Dimer
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Q/R-editing affects AMPAR sedimentation
sedimentation
P1
P2
Chase t
Q
5 hr
R
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P1 consists of assembly intermediates, P2 of
AMPAR tetramers
Blue-Native PAGE
R
Q
11 SDS
6
6
7
7
11
Fraction
5
11
5
T
D
M
P1
P1
P2
P2
GluR2(R) is blocked at the step of tetramerisation
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Endogenous GluR2 is largely unassembled -
contrasting with GluR1
Cultured neurons
1
sedimentation
P1
P2
GluR1
GluR2
6
7
5
10
11
12
P2
40
GluR1
P1
GluR2
30
of Total
20
10
0
4
8
12
6
14
10
Fraction
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Arg607 is located at subunit interfaces
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Arg607 is located at subunit interfaces
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Summary

• Arg607 restricts GluR2 ER-exit at the level of
  channel assembly.

• GluR2(R) forms dimers, assembly is blocked at
  the step of tetramerization.

• GluR2(Q) tetramerizes efficiently.

• Endogenous GluR2 is largely unassembled, GluR1
  is mostly tetrameric.

• Other alterations in the pore loop, apart from
  editing to Arg607, affect
• traffic/assembly, suggesting that the
  overall conformation of the P-loop
• is critical.

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Edited to Arg
Not edited to Arg
Synapse
ER

• Channel stoichiometry - microscopic properties
• Synaptic channel abundance - macroscopic currents

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Acknowledgements Ed ZIFF Latika
KHATRI Xiangpeng KONG HHMI
Yimi Amarillo