Subunit-specific surface mobility of differentially labeled AMPA receptor subunits

Lateral mobility of AMPA-type glutamate receptors as well as their trafficking between plasma membrane and intracellular compartments are major mechanisms for the regulation of synaptic plasticity. Here we applied a recently established labeling technique in combination with lentiviral expression in hippocampal neurons to label individual ACP-tagged AMPA receptor subunits specifically at the surface of neurons. We show that this technique allows the differential labeling of two receptor subunits on the same cell. Moreover, these subunits are integrated into heteromeric receptors together with endogenous subunits, and these labeled receptors are targeted to active synapses. Sequential labeling experiments indicate that there is basal surface insertion of GluR1, GluR2 and GluR3, and that this insertion is strongly increased following potassium depolarization. Moreover, we found that ACP-labeled GluR3 shows the highest surface mobility among GluR1, GluR2, and GluR3. In double-infected neurons the diffusion coefficient of labeled GluR2 at the surface of living neurons is significantly higher in GluR2/GluR3-infected neurons compared to GluR1/GluR2-infected neurons suggesting a higher mobility of GluR2/3 receptors compared to GluR1/2 receptors. These results indicate that surface mobility is regulated by different subunit compositions of AMPA receptors.     

AMPA receptor subunits expressed by single Purkinje cells.

Several subunits of the glutamate receptor of the AMPA subtype have been cloned recently. These subunits, named GluR1, GluR2, GluR3, and GluR4, exist as two splicing variants (flip and flop). We have determined the subset of AMPA receptor subunits expressed by single cerebellar Purkinje cells in culture. This was achieved by combining whole-cell patch-clamp recordings and a molecular analysis, based on the polymerase chain reaction, of the messenger RNAs harvested into the patch pipette at the end of each recording. We found that each single cell expresses the messenger RNAs encoding the following five subunits: the flip and flop versions of GluR1 and GluR2 as well as GluR3flip, GluR2 being the most abundant. In addition, GluR3flop and GluR4flip were scarcely expressed in half of these neurons, and GluR4flop was never detected.     

Immunocytochemical localization of the AMPA receptor subunits in the mesencephalic trigeminal nucleus of the rat

The mesencephalic trigeminal nucleus is composed of large (35-50 microns) pseudo-unipolar neurons. Closely associated with them are small (< 20 microns) multipolar neurons. An unique peculiarity of the pseudo-unipolar perikarya is that they receive synaptic input from various sources, which sets them apart from the dorsal root and cranial nerves sensory ganglia neurons. Whereas glutamate is the best neurotransmitter candidate in pseudo-unipolar neurons, glutamatergic input into them has not yet been reported. AMPA glutamate receptors are implicated in fast excitatory glutamatergic synaptic transmission. They have been localized ultrastructurally at postsynaptic sites. This study demonstrates that the pseudo-unipolar neurons of the mesencephalic trigeminal nucleus express AMPA glutamate receptor subunits, which indicates that these neurons receive glutamatergic input. Serial sections from the rostral pons and midbrain of Sprague-Dawley rats were immunostained with antibodies against C-terminus of AMPA receptor subunits: GluR1, GluR2/3, and GluR4. The immunoreaction was visualized with avidin-biotin-peroxidase/DAB for light and electron microscopy. With GluR1 antibody only the smallest multipolar neurons were recognized as immunopositive within the mesencephalic trigeminal nucleus. GluR2/3 stained the pseudo-unipolar neurons intensely within the entire rostro-caudal extent of the nucleus. In addition the former antibody stained small multipolar neurons within the mesencephalic trigeminal nucleus, though with somewhat larger dimensions than those immunoreactive for GluR1. Whereas the overall staining with GluR4 antibody was scant, those pseudo-unipolar neurons that were stained, were strongly stained. Furthermore, a considerable number of microglial cells within and surrounding the mesencephalic trigeminal nucleus displayed very intense immunoreactivity for GluR4. These results are discussed in the light of the glutamate receptor subunit composition.     

Pharmacological heterogeneity of release-regulating presynaptic AMPA/kainate receptors in the rat brain: study with receptor antagonists

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [(3)H]noradrenaline or [(3)H]serotonin release, striatal [(3)H]dopamine release and cortical [(3)H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [(3)H]noradrenaline, [(3)H]dopamine or [(3)H]serotonin, while it weakly affected the AMPA-mediated release of [(3)H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [(3)H]dopamine and [(3)H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [(3)H]noradrenaline, while the AMPA-mediated [(3)H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]serotonin, but not that of [(3)H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [(3)H]noradrenaline, [(3)H]dopamine, [(3)H]acetylcholine or [(3)H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [(3)H]noradrenaline and [(3)H]acetylcholine, but it potently blocked the AMPA-induced release of [(3)H]serotonin and, less so, of [(3)H]dopamine. AMOA inhibited the AMPA-induced release of [(3)H]serotonin or [(3)H]acetylcholine, but not that of [(3)H]noradrenaline or [(3)H]dopamine. GAMS prevented the AMPA-mediated release of [(3)H]acetylcholine and, more weakly, that of [(3)H]dopamine, but it failed to inhibit the release of [(3)H]noradrenaline or [(3)H]serotonin elicited by AMPA. gamma-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes.     

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate–type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.     

Chronic hyperammonemia alters in opposite ways membrane expression of GluA1 and GluA2 AMPA receptor subunits in cerebellum. Molecular mechanisms involved

Hyperammonemia contributes to altered neurotransmission and cognition in patients with hepatic encephalopathy. Hyperammonemia in rats affects differently high- and low-affinity AMPA receptors (AMPARs) in cerebellum. We hypothesized that hyperammonemia would alter differently membrane expression of AMPARs GluA1 and GluA2 subunits by altering its phosphorylation. This work aims were: 1) assess if hyperammonemia alters GluA1 and GluA2 subunits membrane expression in cerebellum and 2) analyze the underlying mechanisms.Hyperammonemia reduces membrane expression of GluA2 and enhances membrane expression of GluA1 in vivo. We show that changes in GluA2 and GluA1 membrane expression in hyperammonemia would be due to enhanced NMDA receptors activation which reduces cGMP levels and phosphodiesterase 2 (PDE2) activity, resulting in increased cAMP levels. This leads to increased protein kinase A (PKA) activity which activates phospholipase C (PLC) and protein kinase C (PKC) thus increasing phosphorylation of GluA2 in Ser880, which reduces GluA2 membrane expression, and phosphorylation of GluA1 in Ser831, which increases GluA1 membrane expression. Blocking NMDA receptors or inhibiting PKA, PLC or PKC normalizes GluA2 and GluA1 phosphorylation and membrane expression in hyperammonemic rats.Altered GluA2 and GluA1 membrane expression would alter signal transduction which may contribute to cognitive and motor alterations in hyperammonemia and hepatic encephalopathy.     

Orexin decreases mRNA expressions of NMDA and AMPA receptor subunits in rat primary neuron cultures

Orexin is one of the orexigenic neuropeptides in the hypothalamus. Orexin neurons in the lateral hypothalamus (LH) project into the cerebral cortex and hippocampus in which the receptors are distributed in high concentrations. Therefore, to elucidate the actions of orexin in the cerebral cortex, we examined its effects on the mRNA expressions of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A, NR2B) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunits (GluR1, GluR2) following 6-day application of orexin-A or orexin-B to rat primary cortical neuron cultures. The mRNAs of NR1 and NR2A subunits were significantly decreased by orexin-A and orexin-B at concentrations over 0.1 μM and 0.01 μM, respectively. The mRNA expression of NR2B subunit was also significantly decreased by orexin-A and orexin-B only at the concentration of 1 μM. Moreover, orexin-A and orexin-B at concentrations over 0.01 μM significantly decreased the mRNA expressions of AMPA receptor subunits, GluR1 and GluR2. The present study demonstrated that orexins significantly suppressed RNA expressions of NMDA and AMPA receptor subunits in cortical neuron cultures, suggesting that orexin may regulate the higher functions of the cerebral cortex as well as be involved in energy regulation in the hypothalamus.     

Facilitation of AMPA receptor synaptic delivery as a molecular mechanism for cognitive enhancement

Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.     

The AMPA receptor subunits GluR-A and GluR-B reciprocally modulate spinal synaptic plasticity and inflammatory pain

Ca(2+)-permeable AMPA receptors are densely expressed in the spinal dorsal horn, but their functional significance in pain processing is not understood. By disrupting the genes encoding GluR-A or GluR-B, we generated mice exhibiting increased or decreased numbers of Ca(2+)-permeable AMPA receptors, respectively. Here, we demonstrate that AMPA receptors are critical determinants of nociceptive plasticity and inflammatory pain. A reduction in the number of Ca(2+)-permeable AMPA receptors and density of AMPA channel currents in spinal neurons of GluR-A-deficient mice is accompanied by a loss of nociceptive plasticity in vitro and a reduction in acute inflammatory hyperalgesia in vivo. In contrast, an increase in spinal Ca(2+)-permeable AMPA receptors in GluR-B-deficient mice facilitated nociceptive plasticity and enhanced long-lasting inflammatory hyperalgesia. Thus, AMPA receptors are not mere determinants of fast synaptic transmission underlying basal pain sensitivity as previously thought, but are critically involved in activity-dependent changes in synaptic processing of nociceptive inputs.     

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Intracellular membrane trafficking of glutamate receptors at excitatory synapses is critical for synaptic function. However, little is known about the specialized trafficking events occurring at the postsynaptic membrane. We have found that two components of the exocyst complex, Sec8 and Exo70, separately control synaptic targeting and insertion of AMPA-type glutamate receptors. Sec8 controls the directional movement of receptors towards synapses through PDZ-dependent interactions. In contrast, Exo70 mediates receptor insertion at the postsynaptic membrane, but it does not participate in receptor targeting. Thus, interference with Exo70 function accumulates AMPA receptors inside the spine, forming a complex physically associated, but not yet fused with the postsynaptic membrane. Electron microscopic analysis of these complexes indicates that Exo70 mediates AMPA receptor insertion directly within the postsynaptic density, rather than at extrasynaptic membranes. Therefore, we propose a molecular and anatomical model that dissects AMPA receptor sorting and synaptic delivery within the spine, and uncovers new functions of the exocyst at the postsynaptic membrane.     

Kainate binding to the AMPA receptor in rat brain

Displacement of [³H]AMPA and [³H]CNQX by kainate was measured in membranes and solubilized fractions from rat brain. In soluble fractions, plots of [³H]AMPA and [³H]CNQX binding displaced by kainate resulted in one-site fits with K_i values in the range of 1–3 M. In membranes, plots of [³H]AMPA binding displaced by kainate resulted in graphs which were better fit by twosite regression analysis than by a one-site fit. The K_i value for the high-affinity component of these two-site fits was 3–9 M and the low-affinity component K_i was in the range of 70–120 M; similar values were determined for kainate displacement of [³H]CNQX. The presence of thiocyanate ions had no effect on kainate displacement of [³H]CNQX. Since the affinity for kainate of the presumed synaptic AMPA receptor is in the range of EC₅₀ values for kainate determined from physiological studies, these data contribute further evidence for the idea that kainate binding to synaptic AMPA receptors may be responsible for many of kainate's physiological effects.     

Desensitization dynamics of the AMPA receptor

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Serotonin 5-HT receptor blockade enhances Ca(2+)/calmodulin-dependent protein kinase II function and membrane expression of AMPA receptor subunits in the rat hippocampus: implications for memory formation

Stimulation of hippocampal 5-HT(1A) receptors impairs memory retention. The highly selective 5-HT(1A) antagonist, WAY-100635, prevents the cognitive deficits induced not only by 5-HT(1A) stimulation but also by cholinergic or NMDA receptor blockade. On this basis, the effects of WAY-100635 on molecular events associated with memory storage were explored. In rat hippocampus, WAY-100635 produced a rapid increase in phosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and in Ca(2+)-independent CaMKII and protein kinase A (PKA) enzyme activity. This increase was followed a few hours later by an enhanced membrane expression of AMPA receptor subunits, especially of the GluR1 subunit phosphorylated at the CaMKII site, pGluR1(Ser831). The same qualitative effects were found with the weaker 5-HT(1A) antagonist NAN-190. The effects of both antagonists were no longer apparent in rats with a previous 5-HT depletion induced by the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA), suggesting that 5-HT(1A) receptor blockade removes the tonic inhibition of 5-HT through 5-HT(1A) receptor stimulation on excitatory hippocampal neurons, with the consequent increase in PKA activity. In addition, administration of WAY-100635 potentiated the learning-specific increase in the hippocampus of phospho-CaMKII, Ca(2+)-independent CaMKII activity, as well as the phosphorylation of either the CaMKII or the PKA site on the AMPA receptor GluR1 subunit. This study suggests that blockade of hippocampal 5-HT(1A) receptors favours molecular events critically involved in memory formation, and provides an in vivo molecular basis for the proposed utility of 5-HT(1A) receptor antagonists in the treatment of cognitive disorders.     

Modeling the role of lateral membrane diffusion in AMPA receptor trafficking along a spiny dendrite

AMPA receptor trafficking in dendritic spines is emerging as a major postsynaptic mechanism for the expression of plasticity at glutamatergic synapses. AMPA receptors within a spine are in a continuous state of flux, being exchanged with local intracellular pools via exo/endocytosis and with the surrounding dendrite via lateral membrane diffusion. This suggests that one cannot treat a single spine in isolation. Here we present a model of AMPA receptor trafficking between multiple dendritic spines distributed along the surface of a dendrite. Receptors undergo lateral diffusion within the dendritic membrane, with each spine acting as a spatially localized trap where receptors can bind to scaffolding proteins or be internalized through endocytosis. Exocytosis of receptors occurs either at the soma or at sites local to dendritic spines via constitutive recycling from intracellular pools. We derive a reaction–diffusion equation for receptor trafficking that takes into account these various processes. Solutions of this equation allow us to calculate the distribution of synaptic receptor numbers across the population of spines, and hence determine how lateral diffusion contributes to the strength of a synapse. A number of specific results follow from our modeling and analysis. (1) Lateral membrane diffusion alone is insufficient as a mechanism for delivering AMPA receptors from the soma to distal dendrites. (2) A source of surface receptors at the soma tends to generate an exponential-like distribution of receptors along the dendrite, which has implications for synaptic democracy. (3) Diffusion mediates a heterosynaptic interaction between spines so that local changes in the constitutive recycling of AMPA receptors induce nonlocal changes in synaptic strength. On the other hand, structural changes in a spine following long term potentiation or depression have a purely local effect on synaptic strength. (4) A global change in the rates of AMPA receptor exo/endocytosis is unlikely to be the sole mechanism for homeostatic synaptic scaling. (5) The dynamics of AMPA receptor trafficking occurs on multiple timescales and varies according to spatial location along the dendrite. Understanding such dynamics is important when interpreting data from inactivation experiments that are used to infer the rate of relaxation to steady-state.     

Differential regulation of dendrite complexity by AMPA receptor subunits GluR1 and GluR2 in motor neurons

Activity-dependent developmental mechanisms in many regions of the central nervous system are thought to be responsible for shaping dendritic architecture and connectivity, although the molecular mechanisms underlying these events remain obscure. Since AMPA glutamate receptors are developmentally regulated in spinal motor neurons, we have investigated the role of activation of AMPA receptors in dendritic outgrowth of spinal motor neurons by overexpression of two subunits, GluR1 and GluR2, and find that dendrite outgrowth is differentially controlled by expression of these subunits. Overexpression of GluR1 was associated with greater numbers of filopodia, and an increase in the length and complexity of dendritic arbor. In contrast, GluR2 expression did not alter dendritic complexity, but was associated with a moderate increase in length of arbor, and decreased numbers of filopodia. Neither GluR1 nor GluR2 had any effect on the motility of filopodia. In addition, GluR1 but not GluR2 expression increased the density of dendritic puncta incorporating a GFP-labeled PSD95, suggesting that GluR1 may mediate its effect in part by augmenting the number of excitatory synapses within motor neuron dendrites. Together these results suggest that in spinal motor neurons, AMPA receptors composed of GluR1 subunits may facilitate neurotrophic mechanisms in these neurons, permitting sustained dendrite outgrowth and synaptogenesis, whereas expression of AMPA receptors containing GluR2 acts to preserve existing dendritic arbor. Thus, the observed downregulation of GluR1 in motor neurons during postnatal development may limit the formation of new dendrite segments and synapses, promoting stabilized synaptic connectivity.     

KIF13A drives AMPA receptor synaptic delivery for long-term potentiation via endosomal remodeling

The regulated trafficking of AMPA-type glutamate receptors (AMPARs) from dendritic compartments to the synaptic membrane in response to neuronal activity is a core mechanism for long-term potentiation (LTP). However, the contribution of the microtubule cytoskeleton to this synaptic transport is still unknown. In this work, using electrophysiological, biochemical, and imaging techniques, we have found that one member of the kinesin-3 family of motor proteins, KIF13A, is specifically required for the delivery of AMPARs to the spine surface during LTP induction. Accordingly, KIF13A depletion from hippocampal slices abolishes LTP expression. We also identify the vesicular protein centaurin-α1 as part of a motor transport machinery that is engaged with KIF13A and AMPARs upon LTP induction. Finally, we determine that KIF13A is responsible for the remodeling of Rab11-FIP2 endosomal structures in the dendritic shaft during LTP. Overall, these results identify specific kinesin molecular motors and endosomal transport machinery that catalyzes the dendrite-to-synapse translocation of AMPA receptors during synaptic plasticity.     

Cellular distribution of AMPA receptor subunits and mGlu5 following acute and repeated administration of morphine or methamphetamine

Ionotropic AMPA receptors (AMPAR) and metabotropic glutamate group I subtype 5 receptors (mGlu5) mediate neuronal and behavioral effects of abused drugs. mGlu5 stimulation increases expression of striatal‐enriched tyrosine phosphatase isoform 61 (STEP₆₁) which internalizes AMPARs. We determined the rat brain profile of these proteins using two different classes of abused drugs, opiates, and stimulants. STEP₆₁ levels, and cellular distribution/expression of AMPAR subunits (GluA1, GluA2) and mGlu5, were evaluated via a protein cross‐linking assay in medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral pallidum (VP) harvested 1 day after acute, or fourteen days after repeated morphine (8 mg/kg) or methamphetamine (1 mg/kg) (treatments producing behavioral sensitization). Acute morphine decreased GluA1 and GluA2 surface expression in mPFC and GluA1 in NAc. Fourteen days after repeated morphine or methamphetamine, mGlu5 surface expression increased in VP. In mPFC, mGlu5 were unaltered; however, after methamphetamine, STEP₆₁ levels decreased and GluA2 surface expression increased. Pre‐treatment with a mGlu5‐selective negative allosteric modulator, blocked methamphetamine‐induced behavioral sensitization and changes in mPFC GluA2 and STEP₆₁. These data reveal (i) region‐specific distinctions in glutamate receptor trafficking between acute and repeated treatments of morphine and methamphetamine, and (ii) that mGlu5 is necessary for methamphetamine‐induced alterations in mPFC GluA2 and STEP₆₁.     

The phosphorylation state of GluR1 subunits determines the susceptibility of AMPA receptors to calpain cleavage

The alpha-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor (AMPAR) is an ionotropic glutamate receptor that governs most of excitatory synaptic transmission in neurons. In vitro biochemical assay has shown that calpain, a Ca2+-activated protease, can cleave AMPAR GluR1 subunits. Our physiological study found that calpain, which was activated by prolonged stimulation of the N-methyl-D-aspartate receptor (100 microM, 10 min), caused a substantial suppression of AMPAR currents in cortical neurons. Since the phosphorylation sites of GluR1 by several protein kinases are located in close proximity to the calpain cleavage sites, we investigated the effect of phosphorylation on the susceptibility of GluR1 to calpain cleavage. Interestingly, we found that the calpain regulation of AMPAR currents was diminished by inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) but was augmented by inhibition of protein phosphatase 1/2A (PP1/2A). In agreement with this, in vitro assay showed that the calpain-induced proteolytic cleavage of GluR1 C-terminal fusion protein was strongly potentiated by adding the purified active CaMKII, and GluR1 phosphorylated at Ser831 by CaMKII is much more sensitive to calpain cleavage. Taken together, our data suggest that calpain activation suppresses AMPA receptor currents via proteolytic cleavage of GluR1 subunits, and the susceptibility of AMPARs to calpain cleavage is determined by the phosphorylation state of GluR1 subunits, which is mediated by CaMKII-PP1/2A activity.     

Rapsyn-mediated clustering of acetylcholine receptor subunits requires the major cytoplasmic loop of the receptor subunits

During synaptogenesis at the neuromuscular junction, nicotinic acetylcholine receptors (AChRs) are organized into high-density postsynaptic clusters that are critical for efficient synaptic transmission. Rapsyn, an AChR associated cytoplasmic protein, is essential for the aggregation and immobilization of AChRs at the neuromuscular junction. Previous studies have shown that when expressed in nonmuscle cells, both assembled and unassembled AChR subunits are clustered by rapsyn, and the clustering of the alpha subunit is dependent on its major cytoplasmic loop. In the present study, we investigated the mechanism of rapsyn-induced clustering of the AChR beta, gamma, and delta subunits by testing mutant subunits for the ability to cocluster with rapsyn in transfected QT6 cells. For each subunit, deletion of the major cytoplasmic loop, between the third and fourth transmembrane domains, dramatically reduced coclustering with rapsyn. Furthermore, each major cytoplasmic loop was sufficient to mediate clustering of an unrelated transmembrane protein. The AChR subunit mutants lacking the major cytoplasmic loops could assemble into alphadelta dimers, but these were poorly clustered by rapsyn unless at least one mutant was replaced with its wild-type counterpart. These results demonstrate that the major cytoplasmic loop of each AChR subunit is both necessary and sufficient for mediating efficient clustering by rapsyn, and that only one such domain is required for rapsyn-mediated clustering of an assembly intermediate, the alphadelta dimer.