Summary Membrane-associated guanylate kinases (MAGUKs) are the major family of scaffolding proteins at the postsynaptic density. The PSD-MAGUK subfamily, which includes PSD-95, PSD-93, SAP97 and SAP102, is well accepted to be primarily involved in the synaptic anchoring of numerous proteins, including N-methyl-D-aspartate receptors (NMDARs). Notably, the synaptic targeting of NMDARs depends on the binding of the PDZ ligand on the GluN2B subunit to MAGUK PDZ domains as disruption of this interaction dramatically decreases NMDAR surface and synaptic expression. We recently reported a secondary interaction between SAP102 and GluN2B, in addition to the PDZ interaction. Here, we identify two critical residues on GluN2B responsible for the non-PDZ binding to SAP102. Strikingly, either mutation of these critical residues or knock-down of endogenous SAP102 can rescue the defective surface expression and synaptic localization of PDZ binding-deficient GluN2B. These data reveal an unexpected, non-scaffolding role for SAP102 in the synaptic clearance of GluN2B-containing NMDARs.

SUMMARYG-protein-coupled receptors (GPCRs) constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER) to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.

G protein–coupled receptors (GPCRs) are important modulators of synaptic functions. A fundamental but poorly addressed question in neurobiology is how targeted GPCR trafficking is achieved. Rab GTPases are the master regulators of vesicle-mediated membrane trafficking, but their functions in the synaptic presentation of newly synthesized GPCRs are virtually unknown. Here, we investigate the role of Rab43, via dominant-negative inhibition and CRISPR–Cas9–mediated KO, in the export trafficking of α 2 -adrenergic receptor (α 2 -AR) and muscarinic acetylcholine receptor (mAChR) in primary neurons and cells. We demonstrate that Rab43 differentially regulates the overall surface expression of endogenous α 2 -AR and mAChR, as well as their signaling, in primary neurons. In parallel, Rab43 exerts distinct effects on the dendritic and postsynaptic transport of specific α 2B -AR and M3 mAChR subtypes. More interestingly, the selective actions of Rab43 toward α 2B -AR and M3 mAChR are neuronal cell specific and dictated by direct interaction. These data reveal novel, neuron-specific functions for Rab43 in the dendritic and postsynaptic targeting and sorting of GPCRs and imply multiple forward delivery routes for different GPCRs in neurons. Overall, this study provides important insights into regulatory mechanisms of GPCR anterograde traffic to the functional destination in neurons.

Chlorpyrifos (CPF) is an extensively used organophosphorus pesticide that has recently come under increasing scrutiny due to environmental health concerns particularly its association with neurodevelopmental defects. While the insecticidal actions and acute toxicity of CPF are attributed to its oxon metabolite (CPO) which potently inhibits the cholinergic enzyme acetylcholinesterase (AChE), there is significant evidence that CPF, CPO, and other organophosphates may affect a variety of neuronal targets and processes that are not directly related to AChE. Previously, in adult rat sciatic nerves ex vivo and postnatal neurons from rats in vitro we observed that CPF and CPO impaired the movements of vesicles and mitochondria in axons. Here, in embryonic neurons from rats in culture, we evaluated 24h exposures to CPF and CPO across picomolar to micromolar concentrations for effects on fast axonal transport of membrane bound organelles (MBOs) that contained the amyloid precursor protein (APP) tagged with the fluorescent marker, Dendra2 (APPDendra2). The most notable observations of this study were concentration-dependent decreases in the velocity and percentage of MBOs moving in the anterograde direction, an increase in the number of stationary MBOs, and an increased frequency of pauses associated with both CPF and CPO. These effects occurred at concentrations that did not significantly inhibit AChE activity, they were not blocked by cholinergic receptor antagonists, and they were not associated with compromised cell viability. These effects of CPF and CPO may be significant given the importance of axonal transport to neuronal development as well the function of fully developed neurons.

Background: SAP102 is a scaffolding protein that regulates NMDA receptor function. Results: Specific SAP102 spliced isoform is enriched at synapses to regulate NMDA receptor surface expression. Conclusion: I2 region-containing SAP102 is required for the regulation of subunit-specific NMDA receptor surface expression. Significance: This is the first study showing that SAP102 synaptic targeting is critical for regulating NMDA receptor trafficking.