α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)*
-type glutamate receptors mediate most fast excitatory synaptic transmission in the brain. These receptors are heterotetramers containing at least two distinct glutamate receptor (GluR) subunits (GluR1–4). Interestingly, AMPA receptors recycle rapidly at the plasma membrane, and activity-dependent changes in synaptic AMPA receptor number regulate synaptic strength. This critical role for glutamate receptor trafficking in synaptic function and plasticity has motivated intensive study of proteins involved in receptor clustering at the postsynaptic density (PSD).
A major breakthrough in understanding synaptic organization was the discovery that proteins containing postsynaptic density-95, discs large, zonula occludens (PDZ) domains are abundant at the PSD and play key roles in recruiting receptors (Kornau et al., 1997
; Craven and Bredt, 1998
; Garner et al., 2000
; Sheng and Sala, 2001
). PDZ domains are modular protein–protein interaction motifs that bind to short peptide sequences that often occur at the COOH termini of their protein ligands. The prototypical PDZ protein, PSD-95, a membrane-associated guanylate kinase, was first found to interact with the COOH termini of N
-aspartate (NMDA) receptor 2 subunits and certain K+
channels. More recent studies have shown that PICK1 (Xia et al., 1999
) and GRIP/ABP, other synaptic PDZ proteins (Dong et al., 1997
; Srivastava et al., 1998
), bind directly to the tail of AMPA receptor subunit GluR2 and regulate receptor clustering and/or retention at the PSD (Osten et al., 2000
). PDZ domains from SAP-97, a PSD-95–related protein, bind the tail of GluR1, but this appears not to regulate synaptic AMPA receptors (Sans et al., 2001
; Klocker et al., 2002
The first transmembrane protein found to interact with AMPA receptors is the tetraspanning protein stargazin (Chen et al., 2000
), which is mutated in epileptic stargazer mice (Letts et al., 1998
). In addition to absence epilepsy, stargazer mice show cerebellar ataxia (Noebels et al., 1990
). Cellular and physiological studies of these mutant mice show that stargazin is required for surface expression of AMPA receptors in cerebellar granule cells, whereas AMPA receptors in many forebrain neurons are intact (Chen et al., 1999
; Hashimoto et al., 1999
). Expression of AMPA receptor protein subunits GluR2 and GluR4 in stargazer granule cells is largely maintained, but these receptors are not delivered to the cell surface. This AMPA receptor deficiency is preserved in cultured granule cells from stargazer, suggesting it is a cell autonomous defect (Chen et al., 2000
). Transfecting these cultures with stargazin restores the normal distribution of AMPA receptor function.
Stargazin also has a COOH-terminal PDZ-binding site that associates with PSD-95 (Chen et al., 2000
). Importantly, transfecting stargazer granule cells with a stargazin mutant lacking the PDZ-binding site restores extrasynaptic but not synaptic AMPA receptors. This suggests two separable roles for stargazin effects on AMPA receptors. First, stargazin is essential for the delivery and/or maintenance of plasma membrane AMPA receptors. Second, PDZ domain interactions with stargazin mediate synaptic clustering of AMPA receptors. In support of this two-step model, overexpression of stargazin in hippocampal slice cultures selectively augments the number of extrasynaptic AMPA receptors (Schnell et al., 2002
). Clustering of these additional receptors to the synapse requires increased levels of the synaptic stargazin anchor, PSD-95 (Schnell et al., 2002
). Interestingly, the PDZ-binding site of stargazin contains a consensus site for numerous neuronal protein kinases, and phosphorylation of this site disrupts interaction with PSD-95 (Choi et al., 2002
) and prevents synaptic clustering of AMPA receptors (Chetkovich et al., 2002
). Although the PDZ-binding site of stargazin serves as a protein kinase A substrate in vitro, it is not clear which kinases phosphorylate the site in neurons.
Stargazin protein has four transmembrane domains and shares some sequence homology with γ-1, an auxiliary subunit of skeletal muscle calcium channels (Letts et al., 1998
). In cell transfection studies, stargazin modulates the functional properties of neuronal calcium channels (Letts et al., 1998
; Klugbauer et al., 2000
; Green et al., 2001
; Rousset et al., 2001
), and aberrant calcium channel regulation may explain the spike-wave seizure phenotype of stargazer mice (Zhang et al., 2002
). In addition to γ-1 and stargazin/γ-2, six additional γ subunit–related genes have been identified, and some of these can apparently also modulate calcium channel function (Klugbauer et al., 2000
; Burgess et al., 2001
; Chu et al., 2001
; Moss et al., 2002
). The relationship between calcium channel and AMPA receptor regulation by stargazin and other γ subunits is unclear. This dual role for stargazin and relatives is especially intriguing because AMPA receptors function at the PSD, whereas calcium channels are highly expressed at nerve terminals compared with the PSD.
In this study, we define a family of stargazin-related proteins, γ-3, γ-4, and γ-8, that regulate AMPA receptors. This group of four proteins, in contrast to other calcium channel γ subunits and other related four-pass transmembrane proteins, promote surface expression of functional AMPA receptors. Therefore, we refer to this subset as the family of transmembrane AMPA receptor regulatory proteins (TARPs). TARP family members each show specific complementary patterns of expression throughout the brain and collectively appear to occur in all neuronal types in adult brain. A specific TARP isoform also associates with AMPA receptors in developing brain and in glial cells. In brain regions that express multiple isoforms, TARP complexes remain strictly segregated, suggesting differential functions for distinct AMPA receptor/TARP combinations. Immunofluorescent and electron microscopic studies show that TARPs occur in dendrites, but not axons or presynaptic terminals, and specifically cluster at the PSD together with AMPA receptors. Cerebellar granule cells from stargazer mice, which lack any TARP expression, show immature glycosylation and diminished surface expression of AMPA receptors, suggesting that the receptors are retained in an intracellular domain. These studies identify a universal role for the family of TARPs in regulating surface expression of mature AMPA receptors throughout the brain.