​ELife︱The sorting protein SNX27 regulates the transport of AMPA receptors through the synaptoadhesin LRFN2

Written by Wang Peixiang, edited by Wang Peixiang, Wang Sizhen The endosome network plays a key role in regulating cell surface functions, and this role is mainly achieved through the sorting mechanism of endocytic proteins, that is, the endocytosed integrin can be transported Degraded by the lysosome, it can also be transported to the plasma membrane for reuse through the circulation route [1]
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The sorting proteins that have been identified include nexins, retromers, retrievers, and complexes such as WASH, CCC, and ESCPE-1.
The mutations of these sorting proteins are closely related to neurological diseases, neurological disorders, metabolic abnormalities and pathogen infections
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 nexin-27 (SNX27 for short) is a unique member of the sorting protein family nexin.
The amino terminus of this molecule contains a PDZ domain, so it has a dual function of regulating the interaction of two mutually exclusive proteins: It can be combined with inhibitory retromer [2, 3], and can be combined with the PDZ domain binding motif at the carboxyl end of integrin [4-6]
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Through this interaction, SNX27 can regulate the endocytic integrin containing the binding motif of the PDZ domain, which depends on the recovery of retromer, and can also promote the circulation of the integrin back to the plasma membrane
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SNX27 plays an important role in regulating the endosomal circulation of AMPA receptors
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The current mainstream view is that the sorting of AMPA receptors mediated by SNX27 depends on the combination of the PDZ binding motif at the carboxyl end of AMPA and the PDZ domain of SNX27
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However, Clairfeuille et al.
used isothermal titration calorimetry and did not detect the direct binding of SNX27 to AMPA receptor subunits GluA1 and GluA2 [7]
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These data suggest that the molecular details of SNX27-mediated AMPA receptor endosome sorting need to be reconsidered
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The deregulation of SNX27 expression in Down syndrome can lead to synaptic dysfunction
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This in turn suggests that the identification of neuronal integrins that depend on SNX27 will provide insights into the complex etiology of SNX27-related pathologies
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Recently, Kirsty J McMillan (first article), Kevin A Wilkinson, and Paul J Banks of the University of Bristol, UK, published a new study in eLife magazine under the title Sorting nexin-27 regulates AMPA receptor trafficking through the synaptic adhesion protein LRFN2.
It reveals the mechanism by which SNX27 indirectly controls AMPA receptor-mediated synaptic transmission and plasticity by regulating LRFN2 sorting, and provides insights into the functional abnormalities of SNX27 and LRFN2 in a series of neurological diseases
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 First, in order to identify cargo proteins that rely on SNX27 for transportation, using unbiased proteomics, the researchers identified 212 highly reliable proteins that interact with SNX27 in rat primary cortical neurons.

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Among them, 16 proteins contain type I PDZ binding motifs, which include the best acidic amino acids required for high-affinity binding to SNX27
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And 7 of these 16 are integrins, including high-affinity glutamate transporter SLC1A3, sodium bicarbonate cotransporter SLC4A7, sodium ion-dependent transporter LRFN2, outward rectifier potassium channel KCNT2, etc.

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However, it is interesting that these 212 members do not have any AMPA receptor subunits (including GluA1 and GluA2), which indicates that there is a weak, low-abundance association between SNX27 and GluA2, which may be indirect.
Association mechanism
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 Then, in order to verify this idea, the author conducted an independent experiment
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Through immunoprecipitation experiments and co-immunoprecipitation experiments, the researchers found that the positive controls SLC1A3 and SLC4A7 can effectively bind to SNX27, but no interaction between SNX27 and AMPA receptor subunit GluA1/2/3/4 was detected
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Therefore, these data once again confirmed that the PDZ binding motif of the AMPA receptor lacks the optimal sequence required for high-affinity binding to SNX27, and also verifies the credibility of the proteomics data
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 In order to further search for "bridge" molecules that mediate the interaction between SNX27 and AMPA receptors, the authors further analyzed the SNX27 interacting protein group
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They focused on a molecule involved in synapse aggregation, namely leucine-rich repetitive sequences and type III fibronectin domain protein 2, referred to as LRFN2
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 The LRFN family, also called synaptic adhesion-like molecules (SALMs), consists of 5 single-spanning proteins, LRFN1/2/3/4/5, each of which contains 6 leucine-rich repetitive sequences ( LRR), an immunoglobulin (Ig) domain and an extracellular domain composed of a type III fibronectin domain, but the difference is that only the cytoplasmic carboxy-terminal tail of LRFN1, LRFN2 and LRFN4 contains type I PDZ binding motif
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Using immunoprecipitation experiments, the researchers found that only LRFN1, LRFN2, and LRFN4 containing PDZ binding motifs can bind to endogenous SNX27, and the interaction between LRFN2 and SNX27 depends on the glutamate and valine at the hydroxyl end of the LRFN2 PDZ binding motif.
Amino acid
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The researchers also used isothermal titration calorimetry to prove that the SNX27 PDZ domain directly binds to the LRFN2 PDZ binding motif
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These data indicate that LRFN2 directly binds to the PDZ domain of SNX27 through the PDZ binding motif at the carboxyl end
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What role does the combination of SNX27 and LRFN2 play? Is it related to SNX27-mediated membrane transport? Laser confocal microscopy showed that endosome-related SNX27 and LRFN2 co-localized in the entire rat primary hippocampal neurons, including dendrites and cell bodies
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After SNX27 was knocked down, the level of LRFN2 in the whole cell was significantly reduced
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Not only that, the knockdown of SNX27 also resulted in a decrease in the levels of GluA1, GluA2 and LRFN2 on the cell surface
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Further research found that the inhibition of SNX27 would make LRFN2 enter the lysosomal pathway more
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These data indicate that as an integrin, LRFN2 needs to return to the cell surface through the SNX27-mediated membrane transport cycle mechanism.
In other words, the membrane transport of integrin LRFN2 depends on SNX27
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For LRFN2, research mainly focuses on its ability to accumulate NMDA receptors
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So does LRFN2 also regulate AMPA receptor trafficking? Immunofluorescence experiments showed that LRFN2 co-localized with GluA1 and GluA2 in the dendrites of neurons
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Immunoprecipitation detection revealed that LRFN1, LRFN2 and LRFN4 can bind to GluA1 and GluA2, and LRFN2 mainly mediated its binding to GluA1 and GluA2 through its extracellular LRR domain and Ig domain
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SNX27 knockdown leads to a decrease in the levels of GluA1 and GluA2 on the cell surface, while LRFN2 knockdown only leads to a decrease in GluA2 levels
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When LRFN2 or SNX27 is inhibited, GluA2 endocytosis increases and enters endosomes and lysosomes
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These data indicate that the inhibition of SNX27 or LRFN2 disrupts the circulation of GluA2 and increases its residence time in the lysosomal system
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In short, all evidences indicate that AMPA receptor subunit GluA2 is dependent on SNX27 for membrane transport and has a specific biochemical and functional relationship with LRFN2
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Finally, functionally, in vitro slice electrophysiological experiments showed that in the rat hippocampus, inhibition of SNX27 and LRFN2 significantly reduced excitatory synaptic transmission, and inhibition of LRFN2 significantly reduced glutamatergic synaptic transmission than inhibition of SNX27 ; Secondly, LRFN2 inhibition will slightly slow down the induction of long-term synapse (LTP) in the hippocampus
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Previous studies have shown that SNX27 inhibition can damage the induction of hippocampal LTP [8]
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LTP is generally regarded as one of the main molecular mechanisms that form the basis of learning and memory
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 Article pattern: Sorting protein SNX27 regulates AMPA receptor transport through synaptic adhesion protein LRFN2 (picture quoted from: McMillan, Banks, et al.
eLife 2021;10: e59432) Article conclusion and discussion, inspiration and outlook In other words, this study used unbiased proteomics technology to define the proteome that interacts with SNX27 in rat primary cortical neurons, and identified new SNX27-dependent cargo proteins
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In particular, they functionally verified a specific cargo LRFN2
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SNX27 directly binds to the carboxy terminal PDZ binding motif of LRFN2, while the amino terminal region of LRFN2 binds to the AMPA receptor
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SNX27 is necessary for the circulation of LRFN2 and AMPA receptors on the cell surface
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Knockdown of LRFN2 resulted in decreased surface AMPA receptor expression, decreased synaptic activity, and decreased hippocampal long-term potentiation
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Therefore, the researchers proposed a model in which LRFN2 plays a role in the indirect connection between SNX27 and AMPA receptors, and regulates synaptic transmission by regulating SNX27-mediated endosomal sorting of AMPA receptors (above)
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2018.