SAP97 and CASK mediate sorting of NMDA receptors through a previously unknown secretory pathway Okunola Jeyifous 1 , Clarissa L Waites 1 , Christian G Specht 1 , Sho Fujisawa 2 , Manja Schubert 3 , Eric I Lin 4 , John Marshall 5 , Chiye Aoki 2 , Tharani de Silva 3 , Johanna M Montgomery 3 , Craig C Garner 1,6 & William N Green 4,6 Synaptic plasticity is dependent on the differential sorting, delivery and retention of neurotransmitter receptors, but the mechanisms underlying these processes are poorly understood. We found that differential sorting of glutamate receptor subtypes began in the endoplasmic reticulum of rat hippocampal neurons. As AMPA receptors (AMPARs) were trafficked to the plasma membrane via the conventional somatic Golgi network, NMDA receptors (NMDARs) were diverted from the somatic endoplasmic reticulum into a specialized endoplasmic reticulum subcompartment that bypasses somatic Golgi, merging instead with dendritic Golgi outposts. This endoplasmic reticulum subcompartment was composed of highly mobile vesicles containing the NMDAR subunits NR1 and NR2B, the microtubule-dependent motor protein KIF17, and the postsynaptic adaptor proteins CASK and SAP97. Our data demonstrate that the retention and trafficking of NMDARs in this endoplasmic reticulum subcompartment requires both CASK and SAP97. These findings indicate that NMDARs are sorted away from AMPARs via a non-conventional secretory pathway that utilizes dendritic Golgi outposts. NMDARs regulate synaptic plasticity by functioning as coincidence detectors that integrate synapse-specific information with the over- all excitability of neuronal cells 1 . NMDAR activation is essential for eliciting changes in synaptic strength, primarily by regulating the levels of postsynaptic AMPARs 2,3 . Numerous studies have demon- strated that NMDARs and AMPARs are independently delivered to nascent synapses during synaptogenesis 4–6 and to mature synapses during synaptic plasticity 2,3 . Mechanistically, the sorting of these receptors into distinct vesicles 4,6 and/or their differential retention at the postsynaptic density (PSD) 7 may underlie these differences, although the relative contribution of each mechanism remains unclear. Studies of AMPAR biogenesis, transport and synaptic delivery suggest a three step mechanism, wherein receptor subunits are synthesized in the somatic endoplasmic reticulum and Golgi, transported to the plasma membrane by constitutive membrane flow 8,9 and subsequently internalized into recycling endosomes 10 before their activity-dependent reinsertion at extrasynaptic sites 11,12 and capture by scaffold proteins in the PSD 7,13 . It is unclear whether NMDARs follow a similar biosynthetic pathway and/or at what stage they are sorted from AMPARs. Studies suggest that members of the membrane-associated guanylate kinase (MAGUK) family of synaptic scaffold proteins (for example, SAP97, PSD-95, SAP102 and CASK) 7 , the protein GRIP/ABP, and subclasses of microtubule and actin-dependent motor proteins contribute to the differential sorting and trafficking of NMDA and AMPARs 14–18 . For example, SAP97 associates with AMPAR subunits during their biosynthesis and transport to the plasma membrane 15 and GRIP1 and KIF5 participate in the synaptic delivery of these receptors 19 . Similarly, subunits of the NMDAR form complexes with CASK, Velis/MALS, Mint and KIF17 on vesicles that move rapidly along dendritic microtubules 14,19 at rates that are distinct from vesicles carrying AMPARs 6,20 . Addi- tional reports indicate that NMDAR subunits also form complexes with SAP102, sec8 and mPins 21,22 , as well as SAP97 (refs. 23–25). However, it remains unclear when and where each complex forms or how each contributes to individual steps in the biogenesis, transport and recycling of NMDARs. We explored the mechanisms that underlie sorting of AMPARs versus NMDARs. We found that NMDARs were trafficked via a SAP97- and CASK-dependent pathway from somatic endoplasmic reticulum to a dendritic endoplasmic reticulum subcompartment, and subsequently to Golgi outposts. In contrast, AMPARs followed the conventional route from somatic endoplasmic reticulum and Golgi to reach the plasma membrane. These data not only provide new insights into the cellular mechanisms underlying glutamate receptor sorting, but also indicate that dendritic Golgi outposts 26 are part of a functionally distinct secretory pathway. © 2009 Nature America, Inc. All rights reserved. Received 25 March; accepted 8 June; published online 20 July 2009; doi:10.1038/nn.2362 1 Department of Psychiatry and Behavioral Science, Stanford University, Palo Alto, California, USA. 2 Center for Neural Science and Department of Biology, New York University, New York, New York, USA. 3 Department of Physiology, University of Auckland, Auckland, New Zealand. 4 Department of Neurobiology, University of Chicago, Chicago, Illinois, USA. 5 Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island, USA. 6 These authors contributed equally to this work. Correspondence should be addressed to C.C.G. (cgarner@stanford.edu) or W.N.G. (wgreen@midway.uchicago.edu). NATURE NEUROSCIENCE ADVANCE ONLINE PUBLICATION 1 ARTICLES