In this study we performed proteomic screening of AMPA-R-associated proteins by combining medium-scale co-IP of AMPA-R and MS. Through this study we developed several methodologies that can isolate sets of components from AMPA-R complexes in vivo, resulting in the identification of several novel components of the AMPA-R complex as well as some previously identified AMPA-R interactors (summarized in B).
The sensitivity of the LC-MS/MS is high enough to detect a protein even in the femtomolar range. For this high sensitivity, many proteomic analyses using the LC-MS/MS have successfully detected many cytoplasmic proteins even in a small amount of cell lysate. However, proteomic analysis of low abundant integral membrane proteins, such as receptors and ion channels, is still a challenging process for two main reasons: low abundance and high hydrophobicity. In the mixture of proteins from most animal tissues, the relative amount of receptors and ion channels is too low to be detected, as major peptide signals in a mass spectrometry analysis. Isolation of target proteins through the IP process was introduced to overcome this low abundance. However, the IP system should be improved to be compatible with MS because the IP system itself introduces significant contamination through nonspecific binding of proteins as shown in a previous study (22
) and our data (–).
To extract hydrophobic proteins from the membrane, detergents are required but may cause problems in both IP and MS. A successful IP of a membrane protein complex is possible by keeping a balance between extraction efficiency and preservation of protein interactions. Strong detergents (e.g. ionic detergent) in high concentrations can extract integral membrane proteins with high efficiency but disrupts protein interactions. To preserve protein interaction during IP, either a low percentage of a strong ionic detergent or a mild non-ionic detergent has been used for solubilization of membrane protein complexes. In this limited detergent condition, only a small portion of integral membrane proteins was extracted from the membrane.
As shown in a recent study (22
) as well as our data (see lane labeled Pep
+ in –), the major problem of the IP system is nonspecific binding to the IP-matrix, which includes Sepharose/agarose beads, protein A/G, and non-antigen binding sites of antibodies. Preclearing of solubilized brain lysate with protein A-Sepharose beads reduces only a portion of this nonspecific binding. Therefore, in our IP, the nonspecific bindings were always monitored by running a negative control group side-by-side in which synthetic peptides block the binding of antibody to AMPA-R (peptide block, Pep
+ in –). By analyzing the difference in the protein bands between two groups (Pep + and Pep −), proteins that specifically bind to the antibody can be distinguished from proteins that bind to the IP matrix non-specifically. This peptide block is a more cost-effective negative control compared with the knock-out animal as a negative control.
As a way of overcoming the problems in solubilization and IP as described above, we introduced an enrichment process before IP. In the limited detergent conditions as described above, only a small percentage of integral membrane proteins could be extracted out of the membrane, which was improved by an enrichment process with lectin affinity chromatography. As shown in supplemental Fig. 3
, membrane proteins (e.g.
AMPA-R and NMDA receptor) were effectively enriched using chromatography with WGA. This enrichment process also removed a significant pool of nonspecific protein binding to IP-matrix (supplemental Fig. 2
Based on our study, it is possible that more than one methodology is necessary for proteomic analysis of AMPA-R complexes. Solubilization with different detergent conditions isolated different AMPA-R complexes. GRIP1 and Stargazin were associated with AMPA-R only in D2 and D1, respectively (, A
). GEF-H1 and LARGE association with AMPA-R was also detected only in D1 and D2, respectively (). Certain detergent conditions could disrupt some protein interactions in the AMPA-R complex. Different detergents may solubilize different pools of AMPA-R in certain membrane (e.g.
postsynaptic membrane) that are associated with distinct sets of interacting proteins. In addition, the binding of two proteins to AMPA-R could be competitive in vivo
. As shown in our data (supplemental Fig. 3
), both AMPA-R and NMDA receptors were significantly enriched through WGA chromatography, resulting in the increase in the yield of our proteomic screening of AMPA-R interactors. However, the association of Zizimin1 with AMPA-R was decreased through the lectin chromatography (compare A
). Similarly, through the elution with peptides in IP, two additional protein bands (i.e.
Cx_6 and Cx_7) were resolved by LC-MS/MS. However, due to a low yield of peptide elution, some protein bands were hard to be visualized by silver staining. Therefore, it is possible that only a certain set of components of AMPA-R complex could be isolated through a combination of the proteomic processes. Further optimization of each process in protein sample preparation described above could minimize the numbers of protocols that can cover a wide range of protein interactions in the AMPA-R complex.
Functional studies of the novel components of the AMPA-R complex identified through this study will enhance our understanding of the neurobiological function of the AMPA-R complex. For instance, our functional studies of two of these novel interactors, GEF-H1 and LARGE, have demonstrated important functions of these proteins in structural (28
) and functional4
synaptic plasticity, respectively. Furthermore, through our immunostaining and biochemical studies of Zizimin1, ZO-1, and ERC2 side by side, we analyzed their subcellular localization in the brain and cultured neurons. This could give us insights into their potential function in the brain as well as supportive data for their specific interaction with AMPA-R in the brain and neurons ( and ).
Zizimin1 is highly expressed in the brain including the hippocampus and cerebral cortex (39
). Mediated by its pleckstrin homology and CZH1 domain, Zizimin1 is localization at the membrane (39
), which was consistent with our subcellular fractionation data (A
). Both in soma and dendrite, Zizimin1 puncta were bigger than those of ERC2 and ZO-1 (). In dendrite and soma (), only part of the big puncta or small puncta of Zizimin1 were co-localized with GluA1. However, in spines, Zizimin1 puncta localized throughout almost the entire spine and fully overlapped with GluA1 puncta, although Zizimin1 was not in all spines. Zizimin1 (also known as Dock9) regulates dendrite growth in hippocampal neurons by regulating CDC42 activity (39
). Our immunostaining demonstrating Zizimin1 in the spine as well as in the dendrite suggested that Zizimin1 could regulate spine development and/or dynamics. Actually, the developmental profile of Zizimin1 expression in cultured hippocampal neurons demonstrates that its expression is kept high after 13 days in vitro
), when neurons form spines on dendrites (40
). Zizimin1 interacts and modulates CDC42 activity to regulate filopodia induction in NIH-3T3 cells (31
), suggesting its role in the regulation of actin cytoskeleton dynamics, a key mechanism of spine dynamics (42
). GEF-H1, another GEF indentified in our proteomics, regulates spine development depending on AMPA-R activity (28
). Together, further analysis of Zizimin1 function in spine development and dynamics is warranted.
Although electron microscopy showed that ERC2 is mainly localized in the active zone (43
), ERC2 was originally found in PSD fraction due to its low solubility with mild detergent (e.g.
Triton X-100) (43
). Consistently, in our subcellular fractionation of the brain, ERC2 was enriched not in presynaptic but in PSD fraction (B
). The low solubility of ERC2 by mild detergents could be due to its tight association with cytoskeletons in the synaptosomal membrane complexes (43
). ERC2 is known as a presynaptic scaffolding protein as a member of the active zone-associated structural protein (CAST), which was consistent to our double-staining of ERC2 with GluA1 showing the typical overlapping feature between presynaptic and postsynaptic proteins (double arrowheads
). This suggested that ERC2 interacts with AMPA-R through trans-synaptic interaction probably mediated by structural components of synaptic protein complexes. The interaction between ERC2 and AMPA-R could play a role for stabilization of active synapses considering a previous study, the critical role of GluA4 N-terminal region for synaptogenesis (44
). In a high percentage of neurons, a significant pool of ERC2 was in the nucleus (B
). ERC2 was also enriched in P1 fraction, which included nuclear proteins (A
). A recent study demonstrated that Elongator protein 3 controls active zone morphology by acetylating the Bruchpilot, a member of ELKS/CAST family proteins (45
) that includes ERC (46
). Given that the Elongator protein 3 orthologs are largely nuclear (45
), the nuclear localization of ERC2 is not surprising but actually interesting. Further study of ERC2 subcellular localization could give us a novel insight of ERC2 function in the neurons, such as ERC2 roles in the trafficking of synaptic proteins between cytoplasm and nucleus. ELKS/CAST family proteins have various functions including exocytosis of insulin, microtubule stabilization, and vesicle transport (46
Although we found ZO-1 as a component of GluA4-containing AMPA-R complexes in the cerebellum, we analyzed ZO-1 in the cerebral cortex and cortical neuron culture as ZO-1 is mainly expressed in the cerebral cortex (mRNA in situ
images from Allen Institute for Brain Science). In the soma of cultured neurons, ZO-1 was mainly localized in small puncta that seemed to be arranged in a regular pattern (e.g.
not randomly defused but arranged with regular space). Considering ZO-1 function in intercellular junctions such as tight and adherens junctions (47
), the organized pattern of somatic ZO-1 puncta could suggest ZO-1 function in formation and/or regulation of a neuronal junction, the synapse. ZO-1 is a member of the membrane-associated guanylate kinase (MAGUK) homologue family proteins and contains three PSD-95/discs-large/Zonula occludens-1 (PDZ) domains (47
), a main protein-protein interaction motif for many synaptic scaffolding proteins, including AMPA-R interacting proteins (41
). In our study ZO-1 was enriched in synaptosomal fractions (B
) and significantly co-localized with GluA1 puncta in neurons (). Thus, ZO-1 might play a role in anchoring AMPA-R in the synapse through its association with AMPA-R complexes.
As described above, the potential functions of these novel interactors of the AMPA-R are very interesting and exciting. Therefore, additional functional studies of these novel interactors will give us a better understanding of the regulation of AMPA-R activity and trafficking and, thus, its role in synaptic transmission and plasticity. Furthermore, these functional studies may significantly contribute to the development of new therapies for the treatment of neurological and psychiatric disorders such as mental retardation, dementia, schizophrenia, and epilepsy, which are caused by abnormal synaptic transmission and/or plasticity.