# Presentation: 079885

## The novel role of Q/R-editing in 

**AMPA receptor trafficking**

**Ingo Greger**
- MRC Laboratory of Molecular Biology
- Cambridge, England

## Glutamate receptors

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

**Notes:**
Let me say a few general words about GRs to set the stage

## Q/R

- Functional properties of AMPAR-heteromers depend
- on the subunit composition

**Q/R**

## 1

- by subunit compositions

_*regulated*__ trafficking_
- (long C-tails - GluR1)

- PDZ

**1**

**1**

**2**

**2**

## Receptor assembly takes place in the ER

**ER**

- Golgi

- TGN

- Plasma
- membrane

- Secretory pathway

## Receptor assembly takes place in the ER

- EndoH sensitive
- (immature)

- EndoH resistant
- (mature)

**ER**

- Golgi

- TGN

- Plasma
- membrane

- Secretory pathway

- EP

**Notes:**
this is also regulated by the ec pathway which together with  the sp regulates density.
The location of proteins passing through the sp can be determined by its glycosl status. Proteins with immat.glycans reside in the early part and are sensitive to eh, maturely glycosylat pr have reached later compartments beyond the med. Golgi and are Eh sensitive

## Specifically GluR2 resides in intracellular compartments 

**GluR1**

**GluR2**

**Calnexin**

**NMDAR1**

*****

- Subcellular rat brain fractions

**1.**

- Specifically GluR2 resides in intracellular compartments

## Specifically GluR2 resides in intracellular compartments 

- Specifically GluR2 resides in intracellular compartments

**PNS (EndoH+):**

**GluR1**

**GluR2**

- 38

- 80

- % immature:

- mature

- immature

**2.**

**GluR1**

**GluR2**

**Calnexin**

**NMDAR1**

- Subcellular rat brain fractions

**1.**

## GluR2 exits the ER inefficiently

- 0

- 2

- 4

- 6

- 9

- 12

- chase t (hrs):

- mature

- immature

**GluR1**

## GluR2 exits the ER inefficiently

- 0

- 2

- 4

- 6

- 9

- 12

- chase t (hrs):

- mature

- immature

**GluR1**

## Identifying the GluR2 ER-retention motif

- N

- C

## RNA editing

- enzymatic modification of ribonucleotides (in metazoa)
** ****C**ytidine to **U**ridine (C/U)
-  **A**denine to **I**nosine (A/I)

## The Q/R site is a key regulator of ion flux

- P. Seeburg

## The Q/R site determines GluR2 ER-retention

## The Q/R site determines GluR2 ER-retention

## Summary:

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

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

-  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.

## AMPAR assembly occurs in 2-steps

- Dimer

- Dimerization
-       (via N-termini)

- N  N

## Q/R-editing affects AMPAR sedimentation

**P1**

**P2**

- Q

- R

- 5 hr

- Chase t:

- sedimentation

## P1 consists of assembly intermediates, 

- P1 consists of assembly intermediates,
- P2 of AMPAR tetramers

- 6

- 6

- 7

- 7

- 11

- Fraction#:

**R**

**Q**

- M

- D

- T

- 5

- 11

- 5

- 11+ SDS

- Blue-Native PAGE:

**P1**

**P1**

**P2**

**P2**

- GluR2(R) is blocked at the step of tetramerisation

## Endogenous GluR2 is largely unassembled - 

- % of Total

- GluR1

- GluR2

- 6

- 7

- 5

- 10

**GluR1**

**GluR2**

- 11

- 12

**P1**

**P2**

- Endogenous GluR2 is largely unassembled -
- contrasting with GluR1

**1**

- 0

- 10

- 20

- 30

- 40

- 4

- 8

- 12

- 6

- 14

- 10

- Fraction #

- P1

- P2

- Cultured neurons

- sedimentation

## Arg607 is located at subunit interfaces

## Slide 22

## Arg607 is located at subunit interfaces

## 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.

## Edited to Arg

**Edited to Arg**

**Not edited to Arg**

- ER

- Synapse

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

## Slide 26

## Acknowledgements

**Acknowl****edgements**
**Ed**** ZIFF**
**Latika ****KHATRI**
**Xiangp****eng KONG**
**H****HMI**

- Yimi Amarillo