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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

Deletion of proline-902 exposes a functional PDZ motif in GluA4 and confers binding to SAP97.(A) Expression of wild-type or mutant GluA4, with or without co-expressed myc-tagged SAP97 in HEK293 cells. Upper panels show expression of all proteins; lower panels show co-immunoprecipitation of GluA4ΔP, but not full-length GluA4 with SAP97. Immunoblotting antibodies are indicated on right. (B) Transiently expressed GluA4ΔP can co- immunoprecipitate with endogenous SAP97 from HEK293 cells. Upper panel shows similar expression levels of transfected GFP-tagged constructs. Lower panel show immunoprecipitation with anti-SAP97 specific antibody. Both blots were probed with anti-GFP IgG. The extreme carboxyterminal sequences of the expressed proteins are shown below.
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pone-0008715-g002: Deletion of proline-902 exposes a functional PDZ motif in GluA4 and confers binding to SAP97.(A) Expression of wild-type or mutant GluA4, with or without co-expressed myc-tagged SAP97 in HEK293 cells. Upper panels show expression of all proteins; lower panels show co-immunoprecipitation of GluA4ΔP, but not full-length GluA4 with SAP97. Immunoblotting antibodies are indicated on right. (B) Transiently expressed GluA4ΔP can co- immunoprecipitate with endogenous SAP97 from HEK293 cells. Upper panel shows similar expression levels of transfected GFP-tagged constructs. Lower panel show immunoprecipitation with anti-SAP97 specific antibody. Both blots were probed with anti-GFP IgG. The extreme carboxyterminal sequences of the expressed proteins are shown below.

Mentions: A previous study using bacterially expressed protein domains demonstrated that removal of the extreme C-terminal proline resulted in avid binding of GluA4 CTD to PDZ domains of SAP97 under in vitro conditions with purified proteins [13]. To investigate the potential functionality of GluA4 cryptic PDZ motif in living mammalian cells, full-length wild-type (wt) and mutated flag-tagged GluA4 were expressed in transfected HEK293 cells together with myc-tagged SAP97. Coimmunoprecipitation from detergent extracts of cell homogenates showed no association between wt GluA4 and SAP97, whereas the GluA4 mutant lacking proline-902 (GluA4ΔP) immunoprecipitated as a complex with SAP97 (Figure 2A). Next, we wished to determine if GluA4ΔP also associated with the endogenously expressed SAP97 in HEK293 cells, and whether the interaction shows features typical of class I PDZ interactions. Thus, HEK293 cells were transfected for expression of N-terminally GFP-tagged GluA4 constructs, followed by immunoprecipitation with anti-SAP97 sera. Consistent with the above results GFP-tagged GluA4ΔP, but not the wild-type receptor, co-precipitated with SAP97. Disruption of the exposed PDZ interaction motif, either by further deletion of the carboxyl-terminal Leu-901 (GluA4ΔLP) or by mutation of Ser-899 and Leu-901 to alanine [GluA4(SL→AA)ΔP], abolished the interaction (Figure 2B). This is in agreement with the requirement for a large aliphatic side chain at the carboxy-terminal position for class I PDZ interactions.


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)

Deletion of proline-902 exposes a functional PDZ motif in GluA4 and confers binding to SAP97.(A) Expression of wild-type or mutant GluA4, with or without co-expressed myc-tagged SAP97 in HEK293 cells. Upper panels show expression of all proteins; lower panels show co-immunoprecipitation of GluA4ΔP, but not full-length GluA4 with SAP97. Immunoblotting antibodies are indicated on right. (B) Transiently expressed GluA4ΔP can co- immunoprecipitate with endogenous SAP97 from HEK293 cells. Upper panel shows similar expression levels of transfected GFP-tagged constructs. Lower panel show immunoprecipitation with anti-SAP97 specific antibody. Both blots were probed with anti-GFP IgG. The extreme carboxyterminal sequences of the expressed proteins are shown below.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806832&req=5

pone-0008715-g002: Deletion of proline-902 exposes a functional PDZ motif in GluA4 and confers binding to SAP97.(A) Expression of wild-type or mutant GluA4, with or without co-expressed myc-tagged SAP97 in HEK293 cells. Upper panels show expression of all proteins; lower panels show co-immunoprecipitation of GluA4ΔP, but not full-length GluA4 with SAP97. Immunoblotting antibodies are indicated on right. (B) Transiently expressed GluA4ΔP can co- immunoprecipitate with endogenous SAP97 from HEK293 cells. Upper panel shows similar expression levels of transfected GFP-tagged constructs. Lower panel show immunoprecipitation with anti-SAP97 specific antibody. Both blots were probed with anti-GFP IgG. The extreme carboxyterminal sequences of the expressed proteins are shown below.
Mentions: A previous study using bacterially expressed protein domains demonstrated that removal of the extreme C-terminal proline resulted in avid binding of GluA4 CTD to PDZ domains of SAP97 under in vitro conditions with purified proteins [13]. To investigate the potential functionality of GluA4 cryptic PDZ motif in living mammalian cells, full-length wild-type (wt) and mutated flag-tagged GluA4 were expressed in transfected HEK293 cells together with myc-tagged SAP97. Coimmunoprecipitation from detergent extracts of cell homogenates showed no association between wt GluA4 and SAP97, whereas the GluA4 mutant lacking proline-902 (GluA4ΔP) immunoprecipitated as a complex with SAP97 (Figure 2A). Next, we wished to determine if GluA4ΔP also associated with the endogenously expressed SAP97 in HEK293 cells, and whether the interaction shows features typical of class I PDZ interactions. Thus, HEK293 cells were transfected for expression of N-terminally GFP-tagged GluA4 constructs, followed by immunoprecipitation with anti-SAP97 sera. Consistent with the above results GFP-tagged GluA4ΔP, but not the wild-type receptor, co-precipitated with SAP97. Disruption of the exposed PDZ interaction motif, either by further deletion of the carboxyl-terminal Leu-901 (GluA4ΔLP) or by mutation of Ser-899 and Leu-901 to alanine [GluA4(SL→AA)ΔP], abolished the interaction (Figure 2B). This is in agreement with the requirement for a large aliphatic side chain at the carboxy-terminal position for class I PDZ interactions.

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