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Analysis of the potential role of GluA4 carboxyl-terminus in PDZ interactions.

Coleman SK, Cai C, Kalkkinen N, Korpi ER, Keinänen K - PLoS ONE (2010)

Bottom Line: GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97).Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Division of Biochemistry, Viikki Biocenter, University of Helsinki, Helsinki, Finland.

ABSTRACT

Background: Specific delivery to synapses of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.

Methodology/principal findings: Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.

Conclusion/significance: We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

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Analysis of AMPA receptors with an antibody specific for the exposed PDZ motif in GluA4ΔP.(A) HEK293 cells expressing flag-tagged AMPA receptor subunits with all potential wild-type CTDs and the mutant GluA4ΔP (indicated above) were immunoblotted with the antibodies indicated to the left. Short-tailed isoforms of GluA2 and GluA4 are indicated by SH. The initial antiserum, anti-BDL detects both A2 and A4 long tails isoforms and GluA4ΔP. After the depletion procedure and purification, the anti-ΔP IgG only recognises GluA4ΔP (lower panel). (B) Anti-ΔP IgG labels a single 100 kD band in rat cerebellar tissue; this is specifically blocked by preincubation with 13mer peptide (upper panels). Similarly anti-GluR4 IgG also labels a 100 kDa band. This labelling is blocked by pre-incubation with 14 mer peptide (lower panels). (C) Immunoprecipitation from rat cerebellar extract using independent AMPA receptor antibodies fails to bring down anti-DΔP immunoreactivity (upper panel). An alternative antibody shows GluA4 levels were highly enriched in the immunoprecipitates (lower panel).
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pone-0008715-g004: Analysis of AMPA receptors with an antibody specific for the exposed PDZ motif in GluA4ΔP.(A) HEK293 cells expressing flag-tagged AMPA receptor subunits with all potential wild-type CTDs and the mutant GluA4ΔP (indicated above) were immunoblotted with the antibodies indicated to the left. Short-tailed isoforms of GluA2 and GluA4 are indicated by SH. The initial antiserum, anti-BDL detects both A2 and A4 long tails isoforms and GluA4ΔP. After the depletion procedure and purification, the anti-ΔP IgG only recognises GluA4ΔP (lower panel). (B) Anti-ΔP IgG labels a single 100 kD band in rat cerebellar tissue; this is specifically blocked by preincubation with 13mer peptide (upper panels). Similarly anti-GluR4 IgG also labels a 100 kDa band. This labelling is blocked by pre-incubation with 14 mer peptide (lower panels). (C) Immunoprecipitation from rat cerebellar extract using independent AMPA receptor antibodies fails to bring down anti-DΔP immunoreactivity (upper panel). An alternative antibody shows GluA4 levels were highly enriched in the immunoprecipitates (lower panel).

Mentions: Due to the failure of direct mass spectrometric identification of GluA4 C-terminus, we decided upon an immunochemical approach. The polyclonal anti-BDL antiserum was originally generated by using GluA4 CTD lacking the C-terminal proline as the antigen, and therefore it may contain also antibodies binding specifically to the extreme, processed C-terminus. However, in immunoblots, anti-BDL serum recognizes GluA2L, GluA4 and GluA4ΔP in an equally robust manner (Figure 4A, middle panel), suggesting that antibodies specific for GluA4ΔP are a small minority. To enrich GluA4ΔP-specific antibodies potentially present in anti-BDL preparations, the antiserum was repeatedly adsorbed to purified glutathione S-transferase (GST) fusion protein of wild-type GluA4 CTD, thereby depleting antibodies which bind to epitopes shared by GluA4ΔP CTD and wild-type CTD. The specificity of the final immunoreagent was determined by immunoblotting against the full range of AMPA receptor subunit CTDs. As expected, the depleted antiserum, ‘anti-ΔP’, did not any more recognize the wild-type GluA2L or GluA4 subunits (or any other wild-type subunit), but reacted strongly with GluA4ΔP (Figure 4A, bottom panel). Similar specificity was observed in the immunofluorescence analysis of GluA4 and GluA4ΔP receptors expressed in transiently transfected HEK293 cells: the original anti-BDL stained both GluA4 and GluA4ΔP transfectants, whilst anti-ΔP stained only the proline-deleted receptors (Figure S3A). In peptide competition experiments, binding of anti-ΔP to GluA4ΔP was abolished by a 13mer peptide corresponding to GluA4ΔP carboxyl-terminus, but not by a corresponding 14mer “wild-type” peptide (Figure S3B).


Analysis of the potential role of GluA4 carboxyl-terminus in PDZ interactions.

Coleman SK, Cai C, Kalkkinen N, Korpi ER, Keinänen K - PLoS ONE (2010)

Analysis of AMPA receptors with an antibody specific for the exposed PDZ motif in GluA4ΔP.(A) HEK293 cells expressing flag-tagged AMPA receptor subunits with all potential wild-type CTDs and the mutant GluA4ΔP (indicated above) were immunoblotted with the antibodies indicated to the left. Short-tailed isoforms of GluA2 and GluA4 are indicated by SH. The initial antiserum, anti-BDL detects both A2 and A4 long tails isoforms and GluA4ΔP. After the depletion procedure and purification, the anti-ΔP IgG only recognises GluA4ΔP (lower panel). (B) Anti-ΔP IgG labels a single 100 kD band in rat cerebellar tissue; this is specifically blocked by preincubation with 13mer peptide (upper panels). Similarly anti-GluR4 IgG also labels a 100 kDa band. This labelling is blocked by pre-incubation with 14 mer peptide (lower panels). (C) Immunoprecipitation from rat cerebellar extract using independent AMPA receptor antibodies fails to bring down anti-DΔP immunoreactivity (upper panel). An alternative antibody shows GluA4 levels were highly enriched in the immunoprecipitates (lower panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806832&req=5

pone-0008715-g004: Analysis of AMPA receptors with an antibody specific for the exposed PDZ motif in GluA4ΔP.(A) HEK293 cells expressing flag-tagged AMPA receptor subunits with all potential wild-type CTDs and the mutant GluA4ΔP (indicated above) were immunoblotted with the antibodies indicated to the left. Short-tailed isoforms of GluA2 and GluA4 are indicated by SH. The initial antiserum, anti-BDL detects both A2 and A4 long tails isoforms and GluA4ΔP. After the depletion procedure and purification, the anti-ΔP IgG only recognises GluA4ΔP (lower panel). (B) Anti-ΔP IgG labels a single 100 kD band in rat cerebellar tissue; this is specifically blocked by preincubation with 13mer peptide (upper panels). Similarly anti-GluR4 IgG also labels a 100 kDa band. This labelling is blocked by pre-incubation with 14 mer peptide (lower panels). (C) Immunoprecipitation from rat cerebellar extract using independent AMPA receptor antibodies fails to bring down anti-DΔP immunoreactivity (upper panel). An alternative antibody shows GluA4 levels were highly enriched in the immunoprecipitates (lower panel).
Mentions: Due to the failure of direct mass spectrometric identification of GluA4 C-terminus, we decided upon an immunochemical approach. The polyclonal anti-BDL antiserum was originally generated by using GluA4 CTD lacking the C-terminal proline as the antigen, and therefore it may contain also antibodies binding specifically to the extreme, processed C-terminus. However, in immunoblots, anti-BDL serum recognizes GluA2L, GluA4 and GluA4ΔP in an equally robust manner (Figure 4A, middle panel), suggesting that antibodies specific for GluA4ΔP are a small minority. To enrich GluA4ΔP-specific antibodies potentially present in anti-BDL preparations, the antiserum was repeatedly adsorbed to purified glutathione S-transferase (GST) fusion protein of wild-type GluA4 CTD, thereby depleting antibodies which bind to epitopes shared by GluA4ΔP CTD and wild-type CTD. The specificity of the final immunoreagent was determined by immunoblotting against the full range of AMPA receptor subunit CTDs. As expected, the depleted antiserum, ‘anti-ΔP’, did not any more recognize the wild-type GluA2L or GluA4 subunits (or any other wild-type subunit), but reacted strongly with GluA4ΔP (Figure 4A, bottom panel). Similar specificity was observed in the immunofluorescence analysis of GluA4 and GluA4ΔP receptors expressed in transiently transfected HEK293 cells: the original anti-BDL stained both GluA4 and GluA4ΔP transfectants, whilst anti-ΔP stained only the proline-deleted receptors (Figure S3A). In peptide competition experiments, binding of anti-ΔP to GluA4ΔP was abolished by a 13mer peptide corresponding to GluA4ΔP carboxyl-terminus, but not by a corresponding 14mer “wild-type” peptide (Figure S3B).

Bottom Line: GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97).Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences, Division of Biochemistry, Viikki Biocenter, University of Helsinki, Helsinki, Finland.

ABSTRACT

Background: Specific delivery to synapses of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.

Methodology/principal findings: Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.

Conclusion/significance: We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.

Show MeSH
Related in: MedlinePlus