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A role for sorting nexin 27 in AMPA receptor trafficking.

Loo LS, Tang N, Al-Haddawi M, Dawe GS, Hong W - Nat Commun (2014)

Bottom Line: Mechanistically, SNX27 interacts with K-ras GTPase via the RA domain; and following chemical LTP stimuli, K-ras is recruited to SNX27-enriched endosomes through a Ca(2+)/CaM-dependent mechanism, which in turn drives the synaptic delivery of homomeric GluA1 receptors.Impairment of SNX27 prevents LTP and associated trafficking of AMPARs.These results demonstrate a role for SNX27 in neuronal plasticity, provide a molecular explanation for the K-ras signal during LTP and identify SNX27 as the PDZ-containing molecular linker that couples the plasticity stimuli to the delivery of postsynaptic cargo.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Molecular and Cell Biology, Singapore 138673, Singapore [2] Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 637553, Singapore.

ABSTRACT
Sorting nexin 27 (SNX27), a PDZ domain-containing endosomal protein, was recently shown to modulate glutamate receptor recycling in Down's syndrome. However, the precise molecular role of SNX27 in GluA1 trafficking is unclear. Here we report that SNX27 is enriched in dendrites and spines, along with recycling endosomes. Significantly, the mobilization of SNX27 along with recycling endosomes into spines was observed. Mechanistically, SNX27 interacts with K-ras GTPase via the RA domain; and following chemical LTP stimuli, K-ras is recruited to SNX27-enriched endosomes through a Ca(2+)/CaM-dependent mechanism, which in turn drives the synaptic delivery of homomeric GluA1 receptors. Impairment of SNX27 prevents LTP and associated trafficking of AMPARs. These results demonstrate a role for SNX27 in neuronal plasticity, provide a molecular explanation for the K-ras signal during LTP and identify SNX27 as the PDZ-containing molecular linker that couples the plasticity stimuli to the delivery of postsynaptic cargo.

No MeSH data available.


Related in: MedlinePlus

AMPAR binding properties of SNX27.(a) Endogenous coimmunoprecipitation with SNX27 antibody. SNX27 interacts with GluA1 receptor, but not GluA2 receptor. (b) Reverse coimmunoprecipitation with GluA2 antibody to verify interaction of GluA2 with GluA1. GluA2 interacts with GluA1, but not SNX27. (c) Immunoprecipitation of HEK293 cells expressing GFP-GluA1 c-terminal tail and Myc-SNX27 or Myc-SNX27 without the PDZ domain (Myc-SNX27ΔPDZ). (d) Time dependence of enhanced K-ras and GluA1 binding to SNX27 following Gly stimulation (upper). Quantitative analysis of time course of K-ras and GluA1 binding to SNX27 following Gly stimulation (lower). The density bands at each time point for GluA1 and K-ras were normalized against the respective SNX27 density band at the same time point. This result would give the normalized intensity ratio on the y axis and these were plotted against time on the x axis. n=3, ** indicates P≤0.01, unpaired t-test; error bars=s.e.m. (e) Recruitment of GluA1 by SNX27 is inhibited by CaM kinase inhibitor (KN93) and farnesyltransferase inhibitors (FTI-276 and FTI-277). The GluA1 density bands were normalized against the respective SNX27 density band. This result would give the normalized intensity ratio on the y axis. n=3, ** indicates P≤0.05, unpaired t-test; error bars=s.e.m.
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f8: AMPAR binding properties of SNX27.(a) Endogenous coimmunoprecipitation with SNX27 antibody. SNX27 interacts with GluA1 receptor, but not GluA2 receptor. (b) Reverse coimmunoprecipitation with GluA2 antibody to verify interaction of GluA2 with GluA1. GluA2 interacts with GluA1, but not SNX27. (c) Immunoprecipitation of HEK293 cells expressing GFP-GluA1 c-terminal tail and Myc-SNX27 or Myc-SNX27 without the PDZ domain (Myc-SNX27ΔPDZ). (d) Time dependence of enhanced K-ras and GluA1 binding to SNX27 following Gly stimulation (upper). Quantitative analysis of time course of K-ras and GluA1 binding to SNX27 following Gly stimulation (lower). The density bands at each time point for GluA1 and K-ras were normalized against the respective SNX27 density band at the same time point. This result would give the normalized intensity ratio on the y axis and these were plotted against time on the x axis. n=3, ** indicates P≤0.01, unpaired t-test; error bars=s.e.m. (e) Recruitment of GluA1 by SNX27 is inhibited by CaM kinase inhibitor (KN93) and farnesyltransferase inhibitors (FTI-276 and FTI-277). The GluA1 density bands were normalized against the respective SNX27 density band. This result would give the normalized intensity ratio on the y axis. n=3, ** indicates P≤0.05, unpaired t-test; error bars=s.e.m.

Mentions: Synaptic accumulation of AMPARs during LTP requires interaction between GluA1 and a yet to be defined Type I PDZ domain-containing protein1314. Therefore, we hypothesized that SNX27 interacts with GluA1 via its PDZ domain and drives AMPARs to dendritic spines following LTP. Consistent with our hypothesis, endogenous coimmunoprecipitation of endogenous proteins showed that SNX27 forms a complex with GluA1, but not GluA2 (a Type II PDZ domain interacting protein), in native hippocampal lysate (Fig. 8a). This interaction is independent of scaffolding proteins of the postsynaptic density (SAP97 and PSD95) (Fig. 8a). Since previously it was reported that SNX27 interacts with GluA2 via in vitro recombinant pull-down and coimmunoprecipitation from HEK cells2, we verified our endogenous immunoprecipitation results by performing a reverse endogenous coimmunoprecipitation, a highly stringent and less promiscuous method for detecting real (and most likely direct) protein interaction in vivo. Reverse co-immunoprecipitation with GluA2 antibody revealed that GluA2 interacts with GluA1, but not SNX27 (Fig. 8b), thus further verifying that GluA2 does not interact with SNX27.


A role for sorting nexin 27 in AMPA receptor trafficking.

Loo LS, Tang N, Al-Haddawi M, Dawe GS, Hong W - Nat Commun (2014)

AMPAR binding properties of SNX27.(a) Endogenous coimmunoprecipitation with SNX27 antibody. SNX27 interacts with GluA1 receptor, but not GluA2 receptor. (b) Reverse coimmunoprecipitation with GluA2 antibody to verify interaction of GluA2 with GluA1. GluA2 interacts with GluA1, but not SNX27. (c) Immunoprecipitation of HEK293 cells expressing GFP-GluA1 c-terminal tail and Myc-SNX27 or Myc-SNX27 without the PDZ domain (Myc-SNX27ΔPDZ). (d) Time dependence of enhanced K-ras and GluA1 binding to SNX27 following Gly stimulation (upper). Quantitative analysis of time course of K-ras and GluA1 binding to SNX27 following Gly stimulation (lower). The density bands at each time point for GluA1 and K-ras were normalized against the respective SNX27 density band at the same time point. This result would give the normalized intensity ratio on the y axis and these were plotted against time on the x axis. n=3, ** indicates P≤0.01, unpaired t-test; error bars=s.e.m. (e) Recruitment of GluA1 by SNX27 is inhibited by CaM kinase inhibitor (KN93) and farnesyltransferase inhibitors (FTI-276 and FTI-277). The GluA1 density bands were normalized against the respective SNX27 density band. This result would give the normalized intensity ratio on the y axis. n=3, ** indicates P≤0.05, unpaired t-test; error bars=s.e.m.
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f8: AMPAR binding properties of SNX27.(a) Endogenous coimmunoprecipitation with SNX27 antibody. SNX27 interacts with GluA1 receptor, but not GluA2 receptor. (b) Reverse coimmunoprecipitation with GluA2 antibody to verify interaction of GluA2 with GluA1. GluA2 interacts with GluA1, but not SNX27. (c) Immunoprecipitation of HEK293 cells expressing GFP-GluA1 c-terminal tail and Myc-SNX27 or Myc-SNX27 without the PDZ domain (Myc-SNX27ΔPDZ). (d) Time dependence of enhanced K-ras and GluA1 binding to SNX27 following Gly stimulation (upper). Quantitative analysis of time course of K-ras and GluA1 binding to SNX27 following Gly stimulation (lower). The density bands at each time point for GluA1 and K-ras were normalized against the respective SNX27 density band at the same time point. This result would give the normalized intensity ratio on the y axis and these were plotted against time on the x axis. n=3, ** indicates P≤0.01, unpaired t-test; error bars=s.e.m. (e) Recruitment of GluA1 by SNX27 is inhibited by CaM kinase inhibitor (KN93) and farnesyltransferase inhibitors (FTI-276 and FTI-277). The GluA1 density bands were normalized against the respective SNX27 density band. This result would give the normalized intensity ratio on the y axis. n=3, ** indicates P≤0.05, unpaired t-test; error bars=s.e.m.
Mentions: Synaptic accumulation of AMPARs during LTP requires interaction between GluA1 and a yet to be defined Type I PDZ domain-containing protein1314. Therefore, we hypothesized that SNX27 interacts with GluA1 via its PDZ domain and drives AMPARs to dendritic spines following LTP. Consistent with our hypothesis, endogenous coimmunoprecipitation of endogenous proteins showed that SNX27 forms a complex with GluA1, but not GluA2 (a Type II PDZ domain interacting protein), in native hippocampal lysate (Fig. 8a). This interaction is independent of scaffolding proteins of the postsynaptic density (SAP97 and PSD95) (Fig. 8a). Since previously it was reported that SNX27 interacts with GluA2 via in vitro recombinant pull-down and coimmunoprecipitation from HEK cells2, we verified our endogenous immunoprecipitation results by performing a reverse endogenous coimmunoprecipitation, a highly stringent and less promiscuous method for detecting real (and most likely direct) protein interaction in vivo. Reverse co-immunoprecipitation with GluA2 antibody revealed that GluA2 interacts with GluA1, but not SNX27 (Fig. 8b), thus further verifying that GluA2 does not interact with SNX27.

Bottom Line: Mechanistically, SNX27 interacts with K-ras GTPase via the RA domain; and following chemical LTP stimuli, K-ras is recruited to SNX27-enriched endosomes through a Ca(2+)/CaM-dependent mechanism, which in turn drives the synaptic delivery of homomeric GluA1 receptors.Impairment of SNX27 prevents LTP and associated trafficking of AMPARs.These results demonstrate a role for SNX27 in neuronal plasticity, provide a molecular explanation for the K-ras signal during LTP and identify SNX27 as the PDZ-containing molecular linker that couples the plasticity stimuli to the delivery of postsynaptic cargo.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Molecular and Cell Biology, Singapore 138673, Singapore [2] Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 637553, Singapore.

ABSTRACT
Sorting nexin 27 (SNX27), a PDZ domain-containing endosomal protein, was recently shown to modulate glutamate receptor recycling in Down's syndrome. However, the precise molecular role of SNX27 in GluA1 trafficking is unclear. Here we report that SNX27 is enriched in dendrites and spines, along with recycling endosomes. Significantly, the mobilization of SNX27 along with recycling endosomes into spines was observed. Mechanistically, SNX27 interacts with K-ras GTPase via the RA domain; and following chemical LTP stimuli, K-ras is recruited to SNX27-enriched endosomes through a Ca(2+)/CaM-dependent mechanism, which in turn drives the synaptic delivery of homomeric GluA1 receptors. Impairment of SNX27 prevents LTP and associated trafficking of AMPARs. These results demonstrate a role for SNX27 in neuronal plasticity, provide a molecular explanation for the K-ras signal during LTP and identify SNX27 as the PDZ-containing molecular linker that couples the plasticity stimuli to the delivery of postsynaptic cargo.

No MeSH data available.


Related in: MedlinePlus