Interaction Proteomics Reveals Brain Region-Specific AMPA

Article pubs.acs.org/jpr

Interaction Proteomics Reveals Brain Region-Specific AMPA Receptor Complexes Ning Chen,† Nikhil J. Pandya,† Frank Koopmans,‡ Violeta Castelo-Székelv,† Roel C. van der Schors,† August B. Smit,†,§ and Ka Wan Li*,†,§ †

Department of Molecular and Cellular Neurobiology, and ‡Department of Functional Genomics, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam, VU University, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands S Supporting Information *

ABSTRACT: Fast excitatory synaptic transmission in the brain is mediated by glutamate acting on postsynaptic AMPA receptors. Recent studies have revealed a substantial number of AMPA receptor auxiliary proteins, which potentially contribute to the regulation of AMPA receptor trafficking, subcellular receptor localization, and receptor gating properties. Here we examined the AMPA receptor interactomes from cortex, hippocampus, and cerebellum by comprehensive interaction proteomics. The study reveals that AMPA receptor auxiliary proteins are engaged in distinct brain region-specific AMPA receptors subcomplexes, which might underlie brain region-specific differential regulation of AMPA receptor properties. Depending on the brain region, an interacting protein can be involved in an AMPA and a non-AMPA receptor complex. KEYWORDS: AMPA receptor, brain, protein complex, proteomics, synapse

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INTRODUCTION AMPA receptors are the principle ionotropic glutamate-gated receptors mediating excitatory synaptic transmission in the brain. They form homo- or heterotetramers containing a combination of two of the GluA1−A4 subunits,1 of which the GluA2 subunitcontaining receptor is the major form comprising more than 90% of the AMPA receptors in the hippocampus.2,3 The channel conductance and number of AMPA receptors in the postsynaptic membrane are dynamically regulated in a synaptic activitydependent manner, leading to alteration of synaptic strength.4−6 This plastic change is considered instrumental to synaptic information storage and processing; it modulates neuronal circuitry function, and in turn it underlies higher cognitive modalities including learning and memory. As such, it is important to understand the molecular mechanisms that govern the AMPA receptor biophysical properties and numbers in the postsynapse. Recent biochemistry and proteomics studies identified a number of proteins stably associated with the AMPA receptor.7−17 Whereas the functional impact on the AMPA receptor of only a few proteins has been examined, divergence and convergence of molecular mechanisms impinged on the receptor by different interacting proteins, namely, cornichons (CNIHs),7−9 transmembrane AMPA receptor regulatory proteins (TARPs),10−14 and CKAMP44/Shisa9,15 have become apparent. These involve (i) the alteration of biophysical properties of the receptor, including current amplitude, desensitization, deactivation rates, and resensitization,14,15,18 and (ii) the biosynthesis and trafficking of the AMPA receptor, © XXXX American Chemical Society

including spatial anchoring/transporting of the AMPA receptor in distinct subcellular domains.8,10,19 In particular, interaction proteomics studies of the AMPA receptor reported a large number of AMPA receptor-interacting proteins;7,15−17 Schwenk et al.16 described 34 proteins as high-confident constituents of AMPA receptor complexes. It was proposed that these receptor complexes contain a common inner core, comprising the GluA tetramer and four auxiliary subunits consisting of various combination of CNIHs, TARPs, and/or GSG1L. Other interacting proteins form the so-called outer core and are a variable peripheral extension of the inner core. This study used whole brain for analysis and gives a global view of AMPA receptor-interacting proteins.16 Thus, the proposed model does not accommodate for potential differences in AMPA receptor properties and plasticity in different brain regions. Furthermore, whereas AMPA receptors are expressed all over the brain, many of the identified interacting proteins show strong brain region-specific expression patterns. This implies that the protein constituents of a given AMPA receptor complex could exhibit brain region specificity, underlying the differences of the AMPA receptor function and localization. To address the brain region specificity of AMPA receptor complexes, we performed quantitative interaction proteomics on the AMPA receptor complex isolated from three brain regions and focused on the most abundant form of AMPA receptors, the GluA2- and/or GluA3-containing receptors. Received: July 3, 2014

A

dx.doi.org/10.1021/pr500697b | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

Article

Using mainly the protein part list as previously defined by Schwenk et al.16 for the whole brain, we detected 21 AMPA receptor-interacting proteins from hippocampus, 20 from cortex, and 18 from cerebellum. Whereas many of the differences reflect the corresponding differences in their brain region-specific expression patterns, several proteins show brain region-specific interactions. For instance, we revealed that PRRT1 preferentially interacts with the AMPA receptor in the hippocampus but interacts preferentially with potassium channel proteins KCNC3/1 in the cerebellum. This study demonstrates the potentially different set of interactions that the AMPA receptor may be involved in across different brain regions versus the exclusive AMPA and non-AMPA receptor interactions that may exist across brain regions for specific AMPA receptor-interacting proteins.

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EXPERIMENTAL PROCEDURES

Sources of Antibodies

Detailed information on the antibodies used is shown in Supporting Information Table s1. The specificities of the newly made polyclonal antibodies against GluA2/3 and PRRT1 were tested on Western blotting and are shown in Supporting Information Figure s1. Preparation of Brain Regions Figure 1. Evaluation of the effect of three different detergents on the isolation efficiency and integrity of AMPA receptor complexes. Protein complexes were extracted from hippocampal P2+microsome fraction using, respectively, 1% NP40, Triton-X 100 (Tx-100), or n-dodecyl β-D-maltoside (DDM), separated by blue native gel electrophoresis, and immunoblotted with antibodies against GluA2/3 or TARP. The locations of the molecular weight marker proteins ranging between 1236 and 66 kDa are shown on the left side of the images.

Adult C57Bl6/J mice (Charles River) were sacrificed by cervical dislocation. The brain regions, cortex, hippocampus, and cerebellum, were dissected and stored at −80 °C until use.20 Frozen brain tissues were homogenized in ice-cold isotonic buffer (0.32 M sucrose, 5 mM HEPES (pH 7.4), and protease inhibitors (Roche Applied Science) with a glass homogenizer (potter S from B. Braun) set at 900 rpm for 12 up−down strokes. The homogenate was centrifuged at 1000g for 10 min. The supernatant was centrifuged at 100 000g for 2 h to obtain the pellet 2 + microsome (P2+M) fraction. For each immunoprecipitation or control experiment, 5 mg of P2+M was used, which was obtained from a pool of 6 mice hippocampi, 5 mice cerebellums, or two-thirds of a single mouse cortex. In total, about 170 mice were used.

the pellet was re-extracted with extraction buffer for 1 h and then centrifuged. The supernatants from the two extractions were combined as an input for IP. The extract was incubated with 10 μg of primary antibody on a rotator at 4 °C overnight. For negative controls, we used empty beads and also peptide antigen blocking, in which 30 μg of peptide antigen was preincubated with 10 μg of antibody for 30 min at 4 °C before they were transferred to the input.21 After overnight incubation, protein A/G PLUS-agarose beads (Santa Cruz) were added and incubated at 4 °C for 1 h. Then the beads were washed four times in washing buffer (0.1% Triton-X 100, 150 mM NaCl, and 50 mM HEPES (pH 7.4)) and eluted with SDS sample buffer for separation by SDS gel electrophoresis.

Evaluation of Detergents on Complex Integrity

We tested three commonly used detergents for AMPA receptor complex extraction, namely, NP40, Triton-X 100, and n-dodecyl β-D-maltoside (DDM). The P2+M fraction (5 mg) was extracted in 1 mL of extraction buffer (1% detergent, 150 mM NaCl, 50 mM HEPES (pH 7.4)) at 4 °C for 1 h. After centrifugation at 20 000g for 20 min, the protein complexes in the extract were separated on linear 3−12% blue native polyacrylamide gradient gels in an Xcell SureLock Mini-Cell (both from Invitrogen) on ice at 150 V for 60 min and then 250 V for 45 min. Proteins were electro-transferred to PVDF membranes (Bio-Rad) at 60 V, 4 °C, for 4 h. After blocking with 5% nonfat dried milk in Tris-buffered saline Tween-20 (TBST) (28 mM Tris, 136.7 mM NaCl, 0.05% Tween-20, pH 7.4) for 2 h, the membrane was incubated with the primary antibody with 3% nonfat dried milk in TBST overnight at 4 °C, followed by the secondary antibody for 1 h at room temperature. Target proteins were detected by ECL (Pierce).

Gel Separation and In-Gel Digestion

The beads with bound protein complexes from 5 mg input fraction were mixed with SDS sample buffer in a total volume of 28 μL and heated to 98 °C for 5 min. To block cysteine residues, 5 μL of 30% acrylamide was added and incubated at room temperature for 30 min.22 Proteins were then resolved on a 10% SDS polyacrylamide gel and stained with colloidal Coomassie Blue. Each sample lane of the IPs was cut into five slices. The gel slices were cut into small fragments with a scalpel and transferred to 1.5 mL tubes (Eppendorf). After destaining with 50% acetonitrile in 50 mM ammonium bicarbonate, the gel particles were dehydrated in 100% acetonitrile and rehydrated in 50 mM ammonium bicarbonate. The destaining cycle was repeated once. Gel particles were dehydrated in 100% acetonitrile and dried in a speedvac. Each sample was incubated in trypsin solution containing 10 μg/mL trypsin (sequence grade;

Immunoprecipitation

Immunoprecipitation was carried out as previously described,21 with the exception that DDM was used for extraction. In brief, 5 mg of P2+M fraction was extracted in 1000 μL of DDM buffer at 4 °C for 1 h. After centrifugation at 20 000g for 20 min, B


C

RAP2B SACM1L

MPP3

MPP2

Shisa6 Shisa9 LRRTM4

PRRT2

OLFM1 OLFM2 OLFM3 PRRT1

DLG1 DLG2 DLG4 GSG1L

CPT1C

CNIH3

CACNG2 (TARPγ2) CACNG3 (TARPγ3) CACNG7 (TARPγ7) CACNG8 (TARPγ8) CNIH2

protein name

glutamate receptor 1 glutamate receptor 2 glutamate receptor 3 glutamate receptor 4 DOMON domaincontaining protein FRRS1L voltage-dependent calcium channel gamma-2 subunit voltage-dependent calcium channel gamma-3 subunit voltage-dependent calcium channel gamma-7 subunit voltage-dependent calcium channel gamma-8 subunit protein cornichon homologue 2 protein cornichon homologue 3 carnitine O-palmitoyltransferase 1, brain isoform disks large homologue 1 disks large homologue 2 disks large homologue 4 germ-cell-specific gene-1-like protein noelin noelin-2 noelin-3 proline-rich transmembrane protein 1 proline-rich transmembrane protein 2 protein Shisa-6 protein Shisa-9 leucine-rich repeat transmembrane neuronal protein 4 MAGUK p55 subfamily member 2 MAGUK p55 subfamily member 3 Ras-related protein Rap-2b phosphatidylinositide phosphatase SAC1

gene name

GRIA1 (GluA1) GRIA2 (GluA2) GRIA3 (GluA3) GRIA4 (GluA4) FRRS1L (CG6)

× × × × × 106 107 107 106 105

IP2 3.6 2.6 1.5 1.9 1.3

× × × × × 106 107 107 106 105

IP1 4.4 4.5 2.6 2.6 6.3

× × × × × 105 106 106 105 104

IP3 1.2 6.9 2.9 8.7 1.6

× × × × × 106 106 106 105 104

IP1

AMPAR IP

5.2 1.9 7.7 4.4 2.8

× × × × × 106 107 106 106 105

IP2

Ab2

5.8 2.5 1.2 4.1 2.4

× × × × × 106 107 107 106 105

IP3

cerebellum

× × × × × 105 106 106 104 104

PB1 2.3 2.2 1.0 5.4 2.0

× × × × × 106 107 107 105 106 4.2 2.5 1.1 4.9 4.0

× × × × × 106 107 107 105 105

IP2 9.4 6.9 3.0 1.1 7.3

× × × × × 105 106 106 105 104

IP3 1.3 1.1 2.8 2.1 2.7

× × × × × 106 107 106 105 105

IP1 2.3 1.3 4.6 1.8 5.0

× × × × ×

106 107 106 105 104

IP2 3.8 1.9 5.8 3.6 3.6

× × × × ×

106 107 106 105 105

IP3

3.1 × 105

3.4 × 104

2.7 × 105

4.4 × 104

8.6 × 103

9.6 × 103

1.2 × 103 1.6 × 104 7.2 × 103

6.2 × 103

3.8 × 105 1.2 × 105 1.4 × 104 5.8 × 104 1.7 × 105 3.1 × 105 7.2 × 103 8.2 × 103

1.0 × 106 5.7 × 104 1.4 × 104 3.5 × 104 2.2 × 105 4.0 × 105 6.9 × 103 5.6 × 103 3.0 × 104 3.1 × 103 7.6 × 103 5 5 1.2 × 10 2.8 × 10 1.4 × 105 2.0 × 104 1.6 × 104

× × × ×

105 9.8 × 104 105 3.4 × 104 104 105 1.7 × 105

3.0 6.9 3.2 6.6

× × × ×

104 103 103 104

× × × ×

103 104 105 105 1.4 × 103

7.8 6.9 1.4 3.1

7.8 2.0 5.5 3.6

× × × ×

104 104 103 105

1.3 1.2 8.8 2.1

× × × ×

105 105 104 105

3.9 × 104 3.6 × 103

9.6 × 103

4.8 × 104

2.8 × 105 1.1 × 105 6.7 × 104 1.0 × 106 2.2 × 105 4.3 × 105 6.5 × 102 2.0 × 104

2.9 × 103

4.5 1.5 1.5 4.9

1.4 × 105 2.7 × 103 7.0 × 103 6.3 × 103 7.4 × 104 4.3 × 104 7.7 × 105 2.0 × 105 5.0 × 104 7.4 × 105 4.6 × 104 5.6 × 105

3.4 × 104 6.1 × 103 1.7 × 103 2.6 × 104 6.0 × 103 8.4 × 103

3.2 × 105 7.3 × 104 2.9 × 104

2.9 × 105

2.7 × 105 8.1 × 104 1.8 × 104 1.7 × 106 4.9 × 104 6.8 × 105

1.0 × 104

3.4 × 106 5.6 × 105 1.5 × 105 5.8 × 105 3.2 × 103 3.3 × 105

3.8 × 105 1.7 × 104 2.9 × 103 3.7 × 104 1.2 × 105 2.0 × 105 7.9 × 104 1.5 × 104 9.7 × 103 1.6 × 104 4.0 × 104 3.2 × 105 1.0 × 103 3.9 × 104 2.1 × 105 2.1 × 105

1.6 × 103

× × × × ×

IP1

Ab2

1.5 × 106 4.6 × 105 1.7 × 105 1.2 × 106 4.2 × 105 1.2 × 106

5.2 3.1 1.6 8.9 1.0

AMPAR IP

1.2 × 105 1.3 × 104 4.6 × 103 1.0 × 104 5.8 × 103 5.0 × 104

4.5 × 103

1.9 × 105 1.2 × 105

BC2 BC3

Ab1

cortex

2.1 × 103 2.3 × 104 1.6 × 104

7.3 × 104

8.2 × 104 2.0 × 104 2.4 × 104

BC1

bead control

105 2.4 × 103 106 3.8 × 103 105 104 103

PB2 1.7 1.5 6.1 3.4 9.7

Ab2

peptide block

5.2 × 106 2.4 × 106 1.3 × 106 7.8 × 105 7.4 × 106 6.3 × 106 5.0 × 105 3.4 × 105

5.7 4.4 2.2 5.1 2.7

Ab1

Table 1. Proteins Identified from AMPA Receptor IPs and Controlsa

Journal of Proteome Research Article


protein name

D

Shisa6 Shisa9

PRRT2

OLFM1 OLFM2 OLFM3 PRRT1

DLG1 DLG2 DLG4 GSG1L

CPT1C

CNIH3

CACNG2 (TARPγ2) CACNG3 (TARPγ3) CACNG7 (TARPγ7) CACNG8 (TARPγ8) CNIH2

protein name

glutamate receptor 1 glutamate receptor 2 glutamate receptor 3 glutamate receptor 4 DOMON domaincontaining protein FRRS1L voltage-dependent calcium channel gamma-2 subunit voltage-dependent calcium channel gamma-3 subunit voltage-dependent calcium channel gamma-7 subunit voltage-dependent calcium channel gamma-8 subunit protein cornichon homologue 2 protein cornichon homologue 3 carnitine O-palmitoyltransferase 1, brain isoform disks large homologue 1 disks large homologue 2 disks large homologue 4 germ-cell-specific gene-1-like protein noelin noelin-2 noelin-3 proline-rich transmembrane protein 1 proline-rich transmembrane protein 2 protein Shisa-6 protein Shisa-9

gene name

monoacylglycerol lipase ABHD6 monoacylglycerol lipase ABHD12

GRIA1 (GluA1) GRIA2 (GluA2) GRIA3 (GluA3) GRIA4 (GluA4) FRRS1L (CG6)

ABHD12

ABHD6

gene name

Table 1. continued

cortex

IP2

3.9 × 103

IP2

Ab2

1.0 × 106

1.3 × 106

7.5 × 105 8.1 × 105

105 105 104 106

1.0 × 106 7.9 × 105

× × × ×

4.3 × 105

6.4 1.0 3.9 6.3 7.2 × 105

105 105 104 106

× × × ×

2.2 × 106

2.8 × 106

4.8 × 103

2.5 × 106

3.4 × 106

1.8 × 104 7.4 × 104

1.1 × 107

1.2 × 107

2.2 × 104

1.4 × 106

2.8 × 106

× × × × × 106 107 106 104 105

IP1

6.9 × 105 5.3 × 105

3.9 × 104 7.7 × 104

1.7 × 103

1.2 × 106

8.0 × 106 4.7 × 105

4.1 × 104

3.5 × 104

4.0 × 105

5.7 × 105

1.3 × 105

8.3 2.0 4.8 5.3 1.8

PB2

6.0 × 105 1.0 × 105

7.9 × 103 2.5 × 105

1.4 × 106

1.9 × 106

2.3 × 106

1.2 × 107

1.6 × 106

2.5 × 106

107 107 107 105 106

× × × × ×

5.3 9.9 2.6 8.4 7.6

IP3

107 108 107 105 106

× × × × ×

4.9 1.1 2.5 9.0 4.6

IP2

107 108 107 106 106

PB1

Ab2

peptide block

AMPAR IP

× × × × ×

Ab1 IP1

1.5 × 106

8.4 1.5 6.6 7.5

IP3

8.8 × 103 7.5 × 103

2.0 × 106

4.6 1.0 3.3 1.0 3.4

IP1

1.6 × 104 7.3 × 104

BC3

IP3

AMPAR IP

2.8 × 103

BC2

bead control

1.6 × 104

BC1

IP1

Ab1

cerebellum

× × × × × 107 107 107 105 105

IP2

× × × ×

105 104 104 106

7.0 × 104 3.7 × 105

4.1 × 105

1.1 2.1 1.2 2.6

4.3 × 105

3.5 × 104

2.0 × 106

3.4 × 106

5.4 × 105

2.8 6.1 1.4 1.3 4.6

Ab2

BC1

× × × × × 107 107 107 105 106

IP3

× × × ×

104 104 104 106

1.5 × 105 5.1 × 105

6.9 × 105

9.5 7.2 6.4 3.4

6.5 × 103 1.0 × 104

5.6 × 105

6.3 × 104

2.9 × 106

9.8 × 106

2.1 × 105

9.9 × 105

3.2 6.8 1.4 2.1 2.2

IP1

106 106 105 103 103

1.1 × 105

1.7 × 104

× × × × ×

PB1 1.1 2.4 5.5 1.4 5.3

hippocampus

BC2 BC3

bead control

× × × × ×

106 106 106 103 104

2.1 × 104

1.3 × 104

2.7 × 103

2.8 × 105

2.7 × 105

7.4 × 103

2.4 6.8 1.3 2.8 4.2

PB2

Ab2

peptide block

IP2

Ab1

1.3 × 104 5.2 × 104 9.3 × 102

PB3

IP3

IP2

Ab2

3.0 × 103

BC2

IP3

8.3 × 103

BC3

9.2 × 103

bead control

9.4 × 102 2.5 × 102

BC1

1.3 × 104

IP1

AMPAR IP

cortex




9.2 × 103 8.3 × 104 5.2 × 104

4.8 × 104 3.9 × 104 4.0 × 104 3.7 × 104

1.8 × 104 2.5 × 105 2.3 × 104 1.8 × 105 2.7 × 105 1.7 × 105

3.4 × 104

3.6 × 104 1.7 × 105

ABHD12

ABHD6

RAP2B SACM1L

MPP3

MPP2

Promega, Madison, WI, USA) in 50 mM ammonium bicarbonate overnight at 37 °C. Peptides from the gel pieces were extracted twice with 200 μL of 50% acetonitrile in 0.1% trifluoroacetic acid. The extracted peptides were dried in a speedvac and stored at −20 °C until mass spectrometric identification.

a Two antibodies (Ab1, Ab2) were used for immunoprecipitation experiments. Proteins that overlap with Schwenk’s part list,16 with the addition of DLG2 and MPP3, are shown in this table. IP: immunoprecipitation experiment; PB: peptide blocking control experiment; BC: bead control experiment. Multiple replicates were performed for the IPs (IP1−IP3) and controls (PB1−PB3; BC1−BC3). The iBAQ intensities of the identified proteins are shown in this table, indicative of absolute protein abundances.

PB2 IP3 IP2 IP2 BC2 protein name gene name

LRRTM4

Table 1. continued

leucine-rich repeat transmembrane neuronal protein 4 MAGUK p55 subfamily member 2 MAGUK p55 subfamily member 3 Ras-related protein Rap-2b Phosphatidylinositide phosphatase SAC1 Monoacylglycerol lipase ABHD6 Monoacylglycerol lipase ABHD12

BC1

bead control

cortex

BC3

IP1

Ab1

IP3

AMPAR IP

IP1

Ab2

hippocampus

PB1

Ab2

peptide block

PB3

BC1

BC2

bead control

BC3

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LC-MS/MS Analysis

Peptides were redissolved in 20 μL of 0.1% acetic acid and loaded on a 5 mm Pepmap 100 C18 (Dionex) column (300 μm i.d., 5 μm particle size) and separated on a 200 mm Alltima C18 homemade column (100 μm i.d., 3 μm particle size) with an Eksigent HPLC system, using a linear gradient of increasing acetonitrile concentration from 5 to 35% in 45 min and to 90% in 5 min. The flow rate was 400 nL/min. The eluted peptides were electro-sprayed into the LTQ-Orbitrap discovery. The mass spectrometer was operated in a data-dependent manner with one MS (m/z range from 330 to 2000) followed by MS/MS on the five most abundant ions. The exclusion window was 25 s. MS/MS spectra were searched against the Uniprot mouse proteome database (version 2014-04) with MaxQuant software (version 1.3.0.5). Methionine oxidation and protein N-terminal acetylation were set as variable modifications, and propionamide (C) was set as fixed modification. The maximum mass deviations of parent and fragment ions after mass recalibration were set to 6 ppm and 0.5 Da, respectively. Trypsin was chosen as the digestion enzyme, and the maximum missed cleavage was set at 2. Each valid protein hit should contain at least one unique peptide. The false discovery rates of both peptides and proteins were set within a threshold value of 0.01. For intensity-based absolute quantification (iBAQ), the summed intensity of all assigned peptides for each protein was divided by the number of theoretically observable peptides.

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RESULTS AND DISCUSSION

Effects of Different Detergents on the Integrity of AMPAR Complexes

A critical issue in the purification of protein complexes that are membrane-receptor-associated is the choice of an extraction buffer that solubilizes the bait complex effectively, while maintaining the integrity of the complex during the whole IP procedure.23 We first evaluated three commonly used detergents with different chemical properties to solubilize AMPA receptor complexes. Protein complexes were size fractionated by blue native gel electrophoresis and detected by immunoblotting. The amount of AMPA receptor recovered from the different extraction detergents was similar; however, the AMPA receptor complex integrity was different (Figure 1). Intact AMPA receptors with apparent MW of 450 kDa that probably did not contain interacting proteins were clearly detected in the NP40 and Tx-100 extraction samples, but at a substantial lower level in the DDM extraction sample. Furthermore, the immunostained band of the AMPA receptor of the DDM sample started at slightly higher mass than those of the NP40 and Tx-100 samples of 720 kDa, likely indicative of additional higher MW complexes. DDM and Tx-100 samples contained a similar amount of TARP-containing protein complexes of >720 kDa, whereas lower immunoreactivity was detected for the NP40 sample. Given the good extraction efficiency, together with the intactness of the AMPA receptor auxiliary protein complexes, DDM was chosen for the subsequent experiments. E



Article

Figure 2. Mean iBAQ abundance (left side) and number of detections (right side) for all high-confident AMPA receptor-interacting proteins found in GluA2/3 IPs (using data from Table 1). Two antibodies were used over three different brain regions. The color scale indicates log10-scaled iBAQ values, with all values above the 0.75 quantile (106) capped to maximum (red) to prevent the bait protein(s) from dominating the color scaling.

Brain Region-Specific Interacting Proteins of AMPA Receptors

GluA2- and/or GluA3-containing AMPA receptors. Peptideblocked IPs and empty bead negative controls were included to substantiate the specificity of the IPs.21 We focused mainly on the high-confident AMPA receptorinteracting proteins that were previously reported by Schwenk et al.16 (Table 1), which were identified in extraction samples of the whole brain. Protein abundances are reported as iBAQ values, as this normalizes protein intensities to account for their number of tryptic peptides, thereby compensating for the low amount of peptides contributing to small proteins (such as is the case for CNIHs). We also identified additional proteins that are enriched in GluA IPs compared to empty beads and peptide blocking controls (Supporting Information Table s2). Whether some of these proteins may represent novel AMPAR interactors remains to be determined.

A number of proteins have been reported to interact with the AMPA receptor and were shown to regulate AMPA receptor function and trafficking.24−27 In addition, the presence of (sets of) stable AMPA receptor-interacting proteins that potentially represent receptor auxiliary subunits have emerged from recent high-context interaction proteomics studies. How AMPA receptor complexes are organized in different brain regions is not known. In the present study, we examined mainly the GluA2 and/or GluA3 subunit-containing receptors, which account for more than 90% of brain AMPA receptors.28 Two different antibodies (i.e., anti-GluA2 (Ab1) and anti-GluA2/3 (Ab2) antibodies) and three independent biological replicates were used, yielding in total 18 independent IPs that captured F



Article

by Western blotting analysis (Figure 3). The major AMPA receptor auxiliary proteins are TARPs.10−14 Consistent with previous reports,10−12,14 TARPγ2 was found to be the main TARP form present in cerebellar AMPA receptors, with a low amount of TARPγ7. TARPγ2 is critical for the functional expression of AMPA receptors in granule neurons.10,11 TARPγ2 represented the main form of TARPs in cortex, as well, with TARPγ3 and TARPγ8 recovered at slightly lower amounts. TARPγ8 in hippocampus was present at several-fold higher amounts than TARPγ2, together with a low amount of TARPγ3. It was reported that, in hippocampal CA1 pyramidal neurons, TARPγ8 is selectively enriched and has a unique role in regulating the pool of extrasynaptic AMPA receptors.38 As AMPA receptor trafficking, gating, and pharmacology are regulated in a TARP sub-type-specific manner, the differential expression of TARPs underlies at least in part the excitatory synapse physiology.39 CNIHs are another type of abundant AMPA receptorinteracting proteins in hippocampus. However, they were found at low level in the cortex and below detection levels in the cerebellum. CNIH has been proposed to regulate AMPA receptor properties and trafficking in ways similar to those of TARPs.7−9,40 In the hippocampus, CNIH binds to GluA1containing AMPA receptor associated with TARPγ8.8 This enables the forward trafficking of GluA1A2, the major form of GluA1-containing receptors, to the neuronal surface. The differential levels of CNIHs enriched in GluA IPs in these brain regions implicate that the contribution of CNIHs to AMPA receptor functioning is highly brain region-specific; it plays an important role in the regulation of hippocampal AMPA receptors; however, it is of insignificance in the cerebellum. Consequently, there are likely alternative mechanisms for forward trafficking of the (GluA1-containing) receptor in different brain regions. We previously described the specific high expression of CKAMP44/Shisa9 in the hippocampus dentate gyrus.15 Here, we confirmed that Shisa9 is present in the IPs of the hippocampus and cortex but is barely detectable in the cerebellum. In accordance, our previous study reported the high expression of Shisa9 in hippocampal dentate gyrus granule cells, where Shisa9 modulates AMPA receptor channel property in shortterm plasticity.15 Another member of the Shisa family, Shisa6, was found in the IP of the hippocampus and cerebellum but not in the cortex. The function of Shisa6 remains to be determined. GSG1L was highly enriched in the IP of the cortex and was also detected in the hippocampus. GSG1L has been found to modulate AMPAR gating.17 OLFMs were found in IPs of all brain regions. The role of these proteins with respect to AMPAR function remains to be established. PRRT1 and PRRT2 are detected at higher level in the hippocampus than in the cerebellum. Besides these, several other proteins were shown to interact with the AMPA receptor with a regional preference, such as MPP2 and MPP3 in the cerebellum, LRRTM4 in the cortex, or RAP2B and ABHD12 in the hippocampus. Could the differences in AMPAR interactomes reflect the differences in their brain region-specific protein expression patterns? The protein levels of AMPA receptor and its interacting proteins in the solubilized brain fractions and co-immunoprecipitated AMPA receptor complex were analyzed by Western blotting (Figure 3). Comparing the protein abundance in the input and the co-immunoprecipitated fractions reveals that, generally, there is positive correlation between the protein expression and their interaction with AMPAR in a brain region-specific manner.

All GluA subunits were identified in IPs of the AMPA receptor in all brain regions examined. AMPA receptor subunit combinations show clear brain region-specific heterogeneity. Generally, the subunit abundance is hippocampus, GluA2 > GluA1 > GluA3 ≫ GluA4; cortex, GluA2 > GluA3 ≥ GluA1 ≫ GluA4; and cerebellum, GluA2 > GluA3 ≥ GluA1 > GluA4. Thus, in accordance with previous studies, we demonstrated that GluA2 is the predominant subunit of brain AMPA receptors. GluA2-containing AMPA receptors are present mainly in the excitatory principal cells of neuronal circuits and are typified by small unitary events and Ca2+ impermeability.29,30 GluA2-lacking AMPA receptors are often found in inhibitory interneurons and glial cells and exhibit Ca2+ permeability and inwardly rectifying behavior.31−34 It should be noted that AMPA receptors lacking GluA2/3 subunits, such as the GluA1 homotetramer in hippocampus and the GluA1/4-type receptor expressed in cerebellar Bergmann glial cells,31,35,36 were not an IP target in this study. In cerebellum, a considerably higher level of GluA4 than that in cortex and hippocampus is detected, although it remains the least abundant AMPA receptor subunit. The higher GluA4 amount recovered from cerebellum may result from its major expression together with the GluA2 subunit in cerebellar granule cells.37 With respect to AMPA receptor-interacting proteins, it is apparent that members of a gene family often show overlapping, though distinct spatial distribution across brain regions (Figure 2; Table 1). These interaction proteomics data are further validated

Figure 3. Western blotting analysis of AMPA receptor-interacting proteins from the inputs (P2+microsome fraction) and the GluA2/3 IPs. CX, cortex; HC, hippocampus; CE, cerebellum. G


gene name

H

GRIA1 (GluA1)

PRRT1

gene name

HOMER3

KCNC3

PPP1CB; PPP1CC; PPP1CA PPP3CA; PPP3CB AHCYL1; AHCYL2 KCNC1

STIM2 ATP6 V1A

CYFIP2

GRIA1 (GluA1) GRIA2 (GluA2) GRIA3 (GluA3) GRIA4 (GluA4) CACNG2 (TARPγ2) CACNG3 (TARPγ3) CACNG8 (TARPγ8) CNIH3 DLG4 Shisa6 RAP2B HPCA; NCALD

PRRT1

protein name

proline-rich transmembrane protein 1 glutamate receptor 1

protein name

proline-rich transmembrane protein 1 glutamate receptor 1 glutamate receptor 2 glutamate receptor 3 glutamate receptor 4 voltage-dependent calcium channel gamma-2 subunit voltage-dependent calcium channel gamma-3 subunit voltage-dependent calcium channel gamma-8 subunit protein cornichon homologue 3 disks large homologue 4 protein Shisa-6 Ras-related protein Rap-2b neuron-specific calcium-binding protein hippocalcin; neurocalcin-delta cytoplasmic FMR1-interacting protein 2 stromal interaction molecule 2 V-type proton ATPase catalytic subunit A serine/threonine-protein phosphatase PP1 catalytic subunit serine/threonine-protein phosphatase 2B catalytic subunit S-adenosylhomocysteine hydrolase-like protein potassium voltage-gated channel subfamily C member 1 potassium voltage-gated channel subfamily C member 3 Homer protein homologue 3

IP3

2.0 × 105 2.3 × 104

1.4 × 105 5.8 × 103

IP2 4.9 × 106 2.9 × 105

IP1 1.4 × 106 2.8 × 105

1.7 × 106

2.3 × 107

IP3

3.7 × 104

2.6 × 104

Ab3

9.7 × 102

2.7 × 103

2.2 × 106

3.4 × 107

IP4

IP1

5.5 × 105

8.8 × 105

IP2

IP2

Ab4

3.7 × 105

IP3

2.7 × 104

8.0 × 105

2.1 × 104

8.1 × 103

4.2 × 104

6.0 × 102

1.6 × 104

1.5 × 104

6.3 × 103 5.1 × 104

1.9 × 107

IP3

4.4 × 106

4.4 × 106

5.0 × 107

IP4

9.0 × 104 hippocampus

3.1 × 107

Ab4

2.5 × 104

1.2 × 104

1.9 × 103

3.3 × 104

1.6 × 104

7.7 × 103

1.6 × 104

3.7 × 103

1.1 × 104

2.5 × 105

1.1 × 103 6.4 × 103

7.7 × 106

5.3 × 106

8.2 × 103

3.6 × 104

1.3 × 103

6.3 × 104

6.1 × 103

3.0 × 103

2.1 × 103

1.4 × 103

5.1 × 106

IP1

PRRT1 IP

1.2 × 105

3.8 × 106

1.0 × 106

1.2 × 104

1.3 × 104

6.9 × 103

3.4 × 103 3.0 × 103

6.4 × 104

2.1 × 104

1.3 × 104

3.2 × 103

2.8 × 105

4.5 × 103

1.6 × 105

2.4 × 104

2.9 × 103

1.0 × 107

PRRT1 IP

4.0 × 103

2.7 × 104

IP2 3.0 × 106

IP1 1.6 × 106

Ab3

cerebellum

PB1 3.7 × 104

PB1

PB2

BC1

PB2 2.4 × 105

Ab4

1.0 × 103

9.8 × 103

1.0 × 104

4.6 × 105

PB1

peptide block

2.1 × 105

7.1 × 105

Ab3

2.0 × 104

3.6 × 104

PB1

Ab4

peptide block Ab3

Table 2. Co-immunoprecipitation Protein List of PRRT1-Interacting Proteins in Cerebellum and Hippocampus Extractsa

BC1

BC2

BC3

8.1 × 103

BC3

bead control

7.8 × 104

1.4 × 104

BC2

bead control



I

1.6 × 104

4.1 × 103

1.7 × 104

1.8 × 104

1.2 × 10 1.7 × 104

1.4 × 104

7.6 × 102

1.6 × 105

2.6 × 104

8.5 × 103

2.2 × 103

4

5.4 × 103

5.4 × 103

4.6 × 104

IP2 4.0 × 105 5.0 × 104

IP1 5.0 × 105 3.6 × 104

IP3

2.0 × 104

6.8 × 104

8.4 × 103

1.1 × 104

1.1 × 105

4.0 × 104 2.4 × 104

2.6 × 104

2.0 × 103

1.7 × 105

4.8 × 105

2.9 × 104

2.1 × 104

4.5 × 106 3.6 × 105

IP4

4.5 × 103

1.4 × 104

5.4 × 103 1.4 × 104

2.4 × 103

2.2 × 104

2.6 × 103

3.3 × 105

1.0 × 106 9.3 × 104

IP1

PRRT1 IP

1.0 × 105

1.5 × 105

1.7 × 103

3.4 × 104

2.2 × 106 2.9 × 105

Ab3 IP2

8.1 × 103

1.4 × 104

2.4 × 103

1.8 × 103 4.3 × 103

5.9 × 102

2.5 × 10 1.9 × 104 4

6.8 × 105 6.6 × 104

8.8 × 104

8.0 × 103

3.9 × 104 2.4 × 104

7.2 × 103

3.1 × 105

1.0 × 105

1.3 × 105

1.2 × 104

4.6 × 104 1.5 × 104

3.8 × 104

1.2 × 104 5.0 × 104 2.3 × 105

3.4 × 104

2.7 × 104

IP4 7.1 × 106 5.1 × 105

IP3 6.6 × 106 8.1 × 105

Ab4

hippocampus

9.4 × 104

PB1

PB2

4.6 × 103

9.4 × 104

PB2

2.2 × 104

1.0 × 104

Ab4 3.1 × 103

PB1

peptide block

1.1 × 104

Ab3 BC1

6.3 × 103

1.4 × 103

BC2

bead control

BC3

Two PRRT1 antibodies (Ab3, Ab4) were used for immunoprecipitation experiments (IP). The antigen sequences are shown in Table s1. Multiple replicates were performed for the IPs (IP1−IP4) and controls (PB1−PB2; PB, peptide blocking control; BC1−BC3; BC, bead control). The iBAQ intensities of the identified proteins are shown in this table, indicative of absolute protein abundances.

a

HOMER3

KCNC3

PPP1CB; PPP1CC; PPP1CA PPP3CA; PPP3CB AHCYL1; AHCYL2 KCNC1

STIM2 ATP6 V1A

CYFIP2

protein name

glutamate receptor 2 glutamate receptor 3 glutamate receptor 4 voltage-dependent calcium channel gamma-2 subunit voltage-dependent calcium channel gamma-3 subunit voltage-dependent calcium channel gamma-8 subunit protein cornichon homologue 3 disks large homologue 4 protein Shisa-6 Ras-related protein Rap-2b neuron-specific calcium-binding protein hippocalcin; neurocalcin-delta cytoplasmic FMR1-interacting protein 2 stromal interaction molecule 2 V-type proton ATPase catalytic subunit A serine/threonine-protein phosphatase PP1 catalytic subunit serine/threonine-protein phosphatase 2B catalytic subunit S-adenosylhomocysteine hydrolase-like protein potassium voltage-gated channel subfamily C member 1 potassium voltage-gated channel subfamily C member 3 Homer protein homologue 3

gene name

GRIA2 (GluA2) GRIA3 (GluA3) GRIA4 (GluA4) CACNG2 (TARPγ2) CACNG3 (TARPγ3) CACNG8 (TARPγ8) CNIH3 DLG4 Shisa6 RAP2B HPCA; NCALD

Table 2. continued




Article

Figure 4. Mean iBAQ abundance (left side) and number of detections (right side) for all high-confident AMPA receptor-interacting proteins found in PRRT1 IPs from respective hippocampus and cerebellum extracts (using data from Table 2). Two antibodies were used. The same color scaling as in Figure 2 was applied.

Nevertheless, SACM1L and FRRS1L that are expressed equally among three brain regions were intensively enriched in the coimmunoprecipitated AMPA receptor complex in the hippocampus.

proteins TARPs and CNIHs (Table 2; Figure 4). The presence of three different GluA subunits in the PRRT1 IP suggests that it interacts with at least two types of AMPA receptor, GluA1A2- and GluA2A3-containing receptors. These multiple AMPA receptor complexes contained TARPs and/or CNIHs. In contrast, PRRT1 IP in the cerebellum yielded a very low amount of AMPA receptor and no other AMPA receptor auxiliary proteins (Tables 1 and 2). In the cerebellum, it is apparent that PRRT1 preferentially interacts with KCNC1 and KCNC3 and HOMER3 (Table 2). Thus, in the hippocampus, a PRRT1 selection mechanism acts that favors interaction of the AMPAR over KCNC1/3. As KCNC1/3 regulates action potential duration in presynaptic terminals, whereas AMPA receptor relays neurotransmission in the postsynapse; it can be envisioned that the PRRT1−AMPA receptor and PRRT1− KCNC1/3 complexes are spatially separated and perform distinct functions. In what way PRRT1 regulates AMPA receptor and KCNC1/3 function remains to be determined.

PRRT1 Associating with AMPA Receptor Specifically in the Hippocampus

PRRT1 is a member of the SynDIG family. Whereas SynDIG1 (TMEM90B) has been reported to interact with AMPAR in juvenile animals and is implicated in synapse formation,41,42 not much is known about PRRT1 (SynDIG4), which is the major SynDIG member interacting with the hippocampal AMPA receptor in adult animals (see Table 1). We therefore focused on this protein and performed interaction proteomics of PRRT1 using hippocampus and cerebellum extracts with two polyclonal antibodies raised to different epitopes of PRRT1. In total, 14 IPs were performed, including four replicates for each antibody in the hippocampus and three replicates for each antibody in the cerebellum. Peptide-blocked IPs and empty beads were used as controls. We considered proteins that were characterized in IPs with both antibodies and that were not present in controls as true PRRT1-interacting proteins. In hippocampus, PRRT1 IP followed by mass spectrometry analysis revealed GluA1−3 and the AMPA receptor auxiliary

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CONCLUSION Previous studies have identified a multitude of AMPA receptorinteracting proteins, but the specific interaction rules are largely unknown. Here, we demonstrated that AMPA receptorinteracting proteins show brain region specificity and can be J



Article

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part of non-AMPA receptor protein assemblies. This points to the existence of distinct regulatory mechanisms of AMPA receptor properties in different brain regions and suggests that AMPA receptor function is more complexly regulated than currently anticipated.

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ASSOCIATED CONTENT

S Supporting Information *

Antibody source and validation. Table s1: antibodies sources. Figure s1: quality control of newly made polyclonal antibodies against GluA2/3 and PRRT1 using Western blotting in conjunction with peptide blocking. Table s2: complete list of proteins enriched in the GluA IPs. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (31) 20 5987107. Fax: (31) 20 5989281. Author Contributions §

A.B.S. and K.W.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS N.C. was funded by a grant from The Netherlands Institute for Systems Biology (NISB (NWO-ALW)). K.W.L., R.C.vdS., and A.B.S. received support from HEALTH-2009-2.1.2-1 EU-FP7 “SynSys” (#242167). N.P. was funded by EU ITN BrainTrain (#238055). F.K. was funded from The Netherlands Organization for Scientific Research (NWO) Complexity project 645.000.003. We thank Sabine Spijker for critical reading of the manuscript.

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ABBREVIATIONS DDM, n-dodecyl β-D-maltoside; IP, immunoprecipitation; P2+M, pellet 2 + microsome; TARP, transmembrane AMPA receptor regulatory proteins

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REFERENCES

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L


Interaction Proteomics Reveals Brain Region-Specific AMPA

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