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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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Related in: MedlinePlus

Native GluA4 AMPA receptors interact with SAP97.Whole brain detergent extracts prepared from (A) wild-type (WT, GluA1+/+) and (B) GluA1 knockout mice (GluA1−/−) were subjected to immunoprecipitation. Immunoprecipitating antibodies are indicated on top; whereas antibodies used for detection of the immunocomplexes are shown on the left.
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pone-0008715-g003: Native GluA4 AMPA receptors interact with SAP97.Whole brain detergent extracts prepared from (A) wild-type (WT, GluA1+/+) and (B) GluA1 knockout mice (GluA1−/−) were subjected to immunoprecipitation. Immunoprecipitating antibodies are indicated on top; whereas antibodies used for detection of the immunocomplexes are shown on the left.

Mentions: Having confirmed the potential capability of proline-deleted GluA4 for PDZ interactions with SAP97, we next wished to determine whether native GluA4 receptors associate with SAP97 or with related PDZ proteins. Extracts from whole brain homogenates of adult mice were immunoprecipitated with anti-SAP97 or anti-PDZ, a broadly specific antiserum recognizing all four postsynaptic density-95 (PSD-95) family membrane-associated guanylate kinase homologs (Maguks) (Figure S2). The corresponding preimmune sera served as specificity controls. Immunoblot analysis, performed with antibody specific for GluA4 N-terminal domain (anti-Dx [25]) showed the presence of GluA4 in both anti-SAP97 and anti-PDZ immunoprecipitates (Figure 3A). GluA1, employed as a positive control, coimmunoprecipitated with SAP97 as expected. Substantial coimmunoprecipitation of GluA4 and GluA1 was also noticed, consistent with the existence of GluA1/4 heteromeric receptors (Figure 3A). In principle, coassembly with GluA1 subunit would lead to physical association of GluA4 subunits with SAP97 without any direct interaction [14]. To determine if this is the case, immunoprecipitations were also performed from mutant mice lacking GluA1 expression (GluA1−/−; [9]). Immunoreactive GluA4 was prominently present in anti-SAP97 and anti-PDZ immunoprecipitates prepared from brains of GluA1−/− mice, indicating that GluA4 can associate with SAP97 independently of GluA1 (Figure 3B). Conversely, SAP97 was present in the GluA4 immunoprecipitates prepared from both wild-type and GluA1 knockout mice (Figure 3A,B).


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)

Native GluA4 AMPA receptors interact with SAP97.Whole brain detergent extracts prepared from (A) wild-type (WT, GluA1+/+) and (B) GluA1 knockout mice (GluA1−/−) were subjected to immunoprecipitation. Immunoprecipitating antibodies are indicated on top; whereas antibodies used for detection of the immunocomplexes are shown on the left.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806832&req=5

pone-0008715-g003: Native GluA4 AMPA receptors interact with SAP97.Whole brain detergent extracts prepared from (A) wild-type (WT, GluA1+/+) and (B) GluA1 knockout mice (GluA1−/−) were subjected to immunoprecipitation. Immunoprecipitating antibodies are indicated on top; whereas antibodies used for detection of the immunocomplexes are shown on the left.
Mentions: Having confirmed the potential capability of proline-deleted GluA4 for PDZ interactions with SAP97, we next wished to determine whether native GluA4 receptors associate with SAP97 or with related PDZ proteins. Extracts from whole brain homogenates of adult mice were immunoprecipitated with anti-SAP97 or anti-PDZ, a broadly specific antiserum recognizing all four postsynaptic density-95 (PSD-95) family membrane-associated guanylate kinase homologs (Maguks) (Figure S2). The corresponding preimmune sera served as specificity controls. Immunoblot analysis, performed with antibody specific for GluA4 N-terminal domain (anti-Dx [25]) showed the presence of GluA4 in both anti-SAP97 and anti-PDZ immunoprecipitates (Figure 3A). GluA1, employed as a positive control, coimmunoprecipitated with SAP97 as expected. Substantial coimmunoprecipitation of GluA4 and GluA1 was also noticed, consistent with the existence of GluA1/4 heteromeric receptors (Figure 3A). In principle, coassembly with GluA1 subunit would lead to physical association of GluA4 subunits with SAP97 without any direct interaction [14]. To determine if this is the case, immunoprecipitations were also performed from mutant mice lacking GluA1 expression (GluA1−/−; [9]). Immunoreactive GluA4 was prominently present in anti-SAP97 and anti-PDZ immunoprecipitates prepared from brains of GluA1−/− mice, indicating that GluA4 can associate with SAP97 independently of GluA1 (Figure 3B). Conversely, SAP97 was present in the GluA4 immunoprecipitates prepared from both wild-type and GluA1 knockout mice (Figure 3A,B).

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