ARTICLES 520 NATURE CELL BIOLOGY VOLUME 5 | NUMBER 6 | JUNE 2003 NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex Nathalie Sans 1 , Kate Prybylowski 1 , Ronald S. Petralia 1 , Kai Chang 1 , Ya-Xian Wang 1 , Claudia Racca 1 , Stefano Vicini 2 and Robert J. Wenthold 1 NMDA (N-methyl-D-aspartate) receptors (NMDARs) are targeted to dendrites and anchored at the post-synaptic density (PSD) through interactions with PDZ proteins. However, little is known about how these receptors are sorted from the endoplasmic reticulum and Golgi apparatus to the synapse. Here, we find that synapse-associated protein 102 (SAP102) interacts with the PDZ-binding domain of Sec8, a member of the exocyst complex. Our results show that interactions between SAP102 and Sec8 are involved in the delivery of NMDARs to the cell surface in heterologous cells and neurons. Furthermore, they suggest that an exocyst–SAP102–NMDAR complex is an important component of NMDAR trafficking. Ionotropic glutamate receptors (GluRs) mediate most excitatory neu- rotransmission in the central nervous system and are concentrated at the PSDs of excitatory synapses 1,2 . Although significant progress has been made in identifying the mechanisms involved in the organization and the local receptor trafficking at synapses 3–7 , considerably less is known about GluR processing earlier in the secretory pathway. Recent studies have shown that PDZ proteins are involved in GluR processing in the endoplasmic reticulum. NMDAR1 splice variants containing the C2′ cassette interact with a PDZ protein to overcome the ER retention of the RRR motif in the C1 cassette, allowing the subunits to exir from the endoplasmic reticulum 8–10 . The interaction of SAP97 with GluR1 occurs in the endoplasmic reticulum and is most probably critical for delivery of receptor complexes to synapses 11–13 . Therefore, in addition to their well-recognized role in anchoring and regulating receptors at the synapse, PDZ proteins also are involved in the early events of assembly, processing and delivery of receptor proteins. The addition of new or recycled membrane proteins to the plasma membrane is essential for maintaining existing synaptic structures, as well as for generating new membranes during development and synap- tic plasticity. The exocyst, also known as the Sec6/8 complex, has been implicated in the secretory process 14,15 ; it consists of eight proteins (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) that form a complex with a relative molecular mass (M r ) of approximately 750,000. The exocyst is thought to direct intracellular membrane vesi- cles to their sites of fusion with the plasma membrane 16 and exocyst proteins are often concentrated at points of rapid membrane addition, such as the growth cone of a neuron 17 . However, the mechanisms by which this complex moves a general or specific cargo are not known. The exocyst is also associated with intracellular compartments, includ- ing the endoplasmic reticulum, Golgi apparatus and trans-Golgi net- work (TGN), and antibody inhibition affects its targeting to the plasma membrane 16,18–20 . In addition, a number of small GTPases that are known to regulate membrane trafficking have recently been shown to associate with exocyst proteins 21 . SAP102, a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ proteins 22–24 , has a wider distribution and is more abundant in the cytoplasm than PSD-95 (ref. 8), the major MAGUK in the PSD. During early development, SAP102 is the major MAGUK expressed in neurons; its expression parallels that of newly formed synapses and precedes that of synaptic PSD-95 (ref. 25). This suggests that SAP102 may interact with NMDARs during delivery to synapses and function as the preferred partner of NMDARs at imma- ture synapses, whereas PSD-95 may be the preferred partner at mature synapses. To investigate further the functional relationship between SAP102 and NMDARs, we used yeast two-hybrid screening to identify other proteins that interact with SAP102. Here, we report that Sec8 binds directly to the PDZ domain of MAGUK proteins and forms a complex containing NMDARs. RESULTS Identification of Sec8 as a binding partner of SAP102 The three PDZ domains of SAP102 were used as bait to probe a yeast two-hybrid mouse E17 embryonic library. The screen yielded three clones containing a 2.1-kb insert and two clones containing a 1.6-kb insert, encoding a 626-amino acid and a 557-amino acid open reading frame (ORF) of Sec8, respectively. Sequence analysis showed that the clones contained a type-1 PDZ-binding domain (TXV) at their car- boxyl termini (Fig. 1a). This motif is present in the rat sequence, but not in the mouse sequence in the NCBI database (accession numbers U32498 and AF022962, respectively), which is probably the result of a sequencing error in the original submitted mouse sequence. The 1 Laboratory of Neurochemistry, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Building 50, Room 4146, 50 South Drive, Bethesda, MD 20892-8027, USA. 2 Department of Physiology and Biophysics, Georgetown University Medical Center, Room BSB225, 3900 Reservoir Road NW, Washington DC 20057-1421, USA Correspondence should be addressed to N.S. (e-mail: sansn@nidcd.nih.gov) © 2003 Nature Publishing Group