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First published online 19 April 2005
doi: 10.1242/jcs.02320

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Cytosolic tail sequences and subunit interactions are critical for synaptic localization of glutamate receptors

The Waksman Institute, Department of Genetics, Rutgers University, Piscataway, NJ 08854, USA

View larger version (28K): [in a new window]   Fig. 1. GLR-1 tail sequences are important for heteromeric AMPA receptor synaptic localization. (Top) The sequences of GLR-1 included in the GLR-1::GFP and GLR-1(tailless)::GFP transgenes. The arrowhead indicates the position of the fluorescent tag (between a.a. 947-948 for full length, and after the final transmembrane domain for the tailless form) used to monitor the protein. (A,C,E,F) GLR-1(tailless)::GFP and (B,D) GLR-1::GFP expressed in different genetic backgrounds. Pictures are of the ventral cord 100 µm anterior of the vulva. (A,B) Wild type, (C,D) glr-1 and glr-2 double deletion, (E) glr-1 deletion, and (F) glr-2 deletion. In (E) and (F), endogenous full-length GLR-2 and GLR-1, respectively, can bring the tailless GLR-1::GFP to the synapse. By contrast, there is no synaptic localization along the ventral nerve cord for (C) GLR-1(tailless)::GFP alone without the endogenous GLR-1 and GLR-2. Bar, is 5 µm. (G) The mean number of clusters per 10 µm of ventral cord length, and (H) the mean cluster size is plotted for the given genotype. `GLR-1' indicates the presence of the glr-1::gfp transgene, whereas `GLR-1T' indicates the presence of the glr-1(tailless)::gfp transgene. Error bars are s.e.m. for all graphs. **P<0.01 compared with GLR-1::GFP in wild type by ANOVA/Dunnett's multiple comparison test. n=20 animals for each genotype.

View larger version (44K): [in a new window]   Fig. 2. GLR-2 tail sequences are required for heteromeric receptor synaptic localization and stability. (Top) The sequences of GLR-2 included in the GLR-2::YFP and GLR-2(tailless)::GFP transgenes. The arrowhead indicates the position of the fluorescent tag (between a.a. 946-947 for full-length, and after the final transmembrane domain for the tailless form) used to monitor the protein. (A,C) GLR-2(tailless)::GFP expressed in wild-type, and glr-1 and glr-2 double deletion, respectively. (B,D) Full-length GLR-2::YFP expressed in wild-type, and glr-1 and glr-2 double deletion, respectively. (A) GLR-2(tailless)::GFP can exit the neuron cell body and populate the synapses of the nerve ring (bracket) and proximal dendrites (arrows) as long as there is endogenous full-length receptor present. (B) However, GLR-2(tailless)::GFP remains in the cell body (arrowhead) when there are no endogenous subunits present. All the images were captured using either YFP or FITC filters, and were taken from the head region of the animal (anterior oriented diagonally down and to the left, dorsal up and to the left). The image in C was taken at a higher exposure to reveal the faint GFP fluorescence of the cell body; consequently, the background autofluorescence from the underlying pharyngeal tissue is also revealed. The neuron shown in all four panels is AVD. Bars, 10 µm.

View larger version (32K): [in a new window]   Fig. 3. Tailless subunits can form complexes with other subunits and conduct GluR function. (A) HA::GLR-1 and FLAG::GLR-2 were contransfected into COS7 cells, which were solubilized in RIPA. Immunoprecipitations (IP) were performed with the indicated antisera, and co-immunoprecipitated proteins were detected by immunoblotting (IB) with the indicated antisera. Arrows indicate the protein pulled-down. (B) HA::GLR-1 and FLAG::GLR-2(tailless), indicated as `FLAG::GLR-2T', were contransfected and analyzed as above. (C) FLAG::GLR-2 and HA::GLR-1(tailless), indicated as `HA::GLR-1T', were contransfected and analyzed as above. For (A-C), `load' indicates 10% of the original lysate. (D) The mean nose-touch sensitivity (percentage of ten trials per animal in which the animal reversed direction upon forward collision with an eyelash) is plotted for the given genotype. `GLR-1 in glr-1' indicates the presence of the glr-1::gfp transgene in a glr-1 mutant, whereas `GLR-1T in glr-1' indicates the presence of the glr-1(tailless)::gfp transgene in a glr-1 mutant. (E) The mean nose-touch sensitivity is plotted for the given genotype. `GLR-2 in glr-2' indicates the presence of the glr-2::gfp transgene in a glr-2 mutant, whereas `GLR-2T in glr-2' indicates the presence of the glr-2(tailless)::gfp transgene in a glr-2 mutant. For (D) and (E), n=13-40 animals for each genotype. ***P<0.001 by ANOVA with the indicated Bonferoni multiple comparison.

View larger version (48K): [in a new window]   Fig. 4. GLR-2 cytosolic tail sequences are sufficient to localize a heterologous membrane protein. (Top) The sequences of PES-10, GLR-1 and GLR-2 included in the TMGFP transgenes. (A) Full-length GLR-1::GFP is localized along the ventral nerve cord in a punctate pattern. (B) TMGFP alone and (C) TMGFP::GLR-1(tail) show no localization. (D) TMGFP::GLR-2(tail) has a similar clustered localization pattern (arrows) to full-length GLR-1::GFP. (A-D) Bars, 5 µm. (E,H) GLR-1::CFP. (F,I) GLR-2::YFP. (G) GLR-1::CFP and GLR-2::YFP colocalize at clusters (arrowheads) in neuron cell bodies (PVC is shown) and along proximal dendrites. (J) GLR-1::CFP and GLR-2::YFP colocalization also occurs at clusters (arrowheads) in the nerve ring (bracket) and along proximal dendrites of neurons in the head. (E-J) Bars, 10 µm. (K) GLR-1::YFP. (N,Q) TMYFP::GLR-2(tail). (L,O,R) SNB-1::CFP from PVD is localized to a small number of presynaptic boutons along the ventral cord neurites. (M) GLR-1::YFP colocalizes near SNB-1-labeled boutons (arrowheads). (P,S) Examples of two animals where TMYFP::GLR-2(tail) colocalizes with SNB-1-labeled boutons (arrowheads). (M,P,S) Merges from K and L, N and O, and Q and R, respectively. (K-S) Bars, 5 µm. All the transgenic animals shown here are of wild-type genetic backgrounds. The images were captured using FITC, CFP and YFP filters, and were taken from the ventral nerve cord region of the animal. (T) The mean number of clusters per 10 µm of ventral cord length, and (U) the mean cluster size is plotted for the given genotype. Error bars are s.e.m. for both graphs. **P<0.01 compared with GLR-1::GFP by ANOVA/Dunnett's multiple comparisons test. n=10-20 animals for each genotype.

View larger version (38K): [in a new window]   Fig. 5. GLR-2 cytosolic tail fusion protein requires the PDZ-binding motif and LIN-10 protein to be localized. We substituted the last three amino acids of the GLR-2 PDZ-binding motif (TLFALE) in TMGFP::GLR-2(tail) to generate TMGFP::GLR-2(TLFALE). (A,B) In wild-type animals, we expressed TMGFP::GLR-2(tail) (A) and TMGFP::GLR-2(TLFALE) (B). (C,D) In lin-10 animals, we expressed TMGFP::GLR-2(tail) (C) and TMGFP::GLR-2(TLFALE) (D). Both B and C show a reduced size of synaptic clusters compared with A, suggesting that both LIN-10 and the PDZ-binding motif of GLR-2 are important for synaptic localization of a GLR-2 tail chimeric protein. (D) These two pathways may compensate for each other as removal of both pathways by expressing a chimeric GLR-2 protein with a mutated PDZ-binding motif in a lin-10 background abolishes the synaptic localization pattern. Bar, 5 µm. (E) The mean number of clusters per 10 µm of ventral cord length, and (F) the mean cluster size is plotted for the given genotype. Error bars are s.e.m. for both graphs. **P<0.01 compared with TLF in wild-type by ANOVA/Dunnett's multiple comparisons test. n=15-20 animals for each genotype.

View larger version (70K): [in a new window]   Fig. 6. Sequence motifs conserved among known GluR tail sequences. (A) The predicted amino acid sequences of five nematode GLR-1 tail sequences (C. elegans (C.e.), C. briggsae (C.b.), C. remanei (C.r.), A. suum (A.s.) and S. stercoralis (S.s.)), aligned by CLUSTALW. This sequence corresponds to amino acids 876-962 of C.e. GLR-1. Black boxes indicate identities present in the majority of sequences. Gray boxes indicate similarities present in the majority of sequences. Lines above the sequence demarcate the conserved sequence motifs identified in D and E. The S.s. GLR-1 sequence is derived from a partial EST, and therefore lacks the final 3' residues. (B) The predicted amino acids of three nematode GLR-2 tail sequences, aligned by CLUSTALW. These sequences correspond to amino acids 919-977 of C.e. GLR-2, and are labeled as in A. (C) The predicted amino acid sequence of three rat long tail AMPAR subunits (R1, R2L, R4), three rat short tail AMPARs (R4S, R3, R2), and GLR-1 and GLR-2, aligned by CLUSTALW and JalView v1.8 Multiple Alignment Editor. Gray boxes indicate identities and similarities present in three or more sequences. (D) The predicted sequence of rat and C.e. GluRs, emphasizing the indicated sequence motif. For the PKA motif, the underlined residue in GluR1 indicates the phosphorylated serine. For the regulated endocytosis motif, the underlined residues in GluR2 indicate the phosphorylated serine and tyrosines. (E) The predicted PDZ ligands for various rat GluRs aligned with GLR-1 and GLR-2. The type of PDZ ligand (I versus II) and its corresponding consensus is shown. `' indicates any hydrophobic residue.

View larger version (112K): [in a new window]   Fig. 7. Sequence motifs conserved among known vertebrate GluR tail sequences. (A) The predicted amino acid sequences of six vertebrate GluR1, five vertebrate GluR2 (long tail isoform) and five vertebrate GluR4 tail sequences, aligned by CLUSTALW. (B) The predicted amino acid sequences of five vertebrate GluR2, eight vertebrate GluR3 and a rat GluR4 (short tail isoform) tail sequences, aligned by CLUSTALW. (C) The predicted amino acid sequences of rat GluR5-1, GluR6 and GluR7 kainate receptors, aligned by CLUSTALW. (D) The predicted amino acid sequences of rat, mouse, and human KA-1 and KA-2 kainate receptors, aligned by CLUSTALW. For all alignments, the majority consensus sequence is highlighted in black. Similarities are highlighted with gray. Lines above a sequence demarcate the conserved sequence motifs identified in Fig. 6D,E.

View larger version (32K): [in a new window]   Fig. 8. A model for GLR-1/GLR-2 subunit association and localization. GLR-1 and GLR-2 subunits are represented by black and gray half-pentagons, respectively. Their carboxy-terminal tail sequences are shown as rectangles projecting into the cytosol. An as yet unknown PDZ-domain protein and LIN-10, which facilitate channel localization, are shown as half-circles and trapezoids, respectively. GLR-1 and GLR-2 can form homomeric channels or heteromeric channels, and the presence one or more carboxy-terminal tail sequences per channel can allow for channel localization. Both the PDZ-binding motif and sequences outside of this motif are required for efficient localization, suggesting that an unknown PDZ domain protein and perhaps LIN-10 might cooperate to localize these channels. Channels that lack the tail sequences cannot interact with these proteins, and therefore fail to become localized.

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