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Comparative Study
. 2008 Jun 1;105(6):2300-14.
doi: 10.1111/j.1471-4159.2008.05311.x.

Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses

Wendou Yu  1 , Erik I Charych, David R Serwanski, Rong-wen Li, Rashid Ali, Ben A Bahr, Angel L De Blas
Affiliations
Comparative Study

Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses

Wendou Yu et al. J Neurochem. .

Abstract

We have previously shown that the glutamate receptor interacting protein 1 (GRIP1) splice forms GRIP1a/b and GRIP1c4-7 are present at the GABAergic post-synaptic complex. Nevertheless, the role that these GRIP1 protein isoforms play at the GABAergic post-synaptic complex is not known. We are now showing that GRIP1c4-7 and GRIP1a/b interact with gephyrin, the main post-synaptic scaffold protein of GABAergic and glycinergic synapses. Gephyrin coprecipitates with GRIP1c4-7 or GRIP1a/b from rat brain extracts and from extracts of human embryonic kidney 293 cells that have been cotransfected with gephyrin and one of the GRIP1 protein isoforms. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b, indicating that gephyrin directly interacts with the common region of these GRIP1 proteins, which includes PDZ domains 4-7. An engineered deletion construct of GRIP1a/b (GRIP1a4-7), which both contains the aforementioned common region and binds to gephyrin, targets to the post-synaptic GABAergic complex of transfected cultured hippocampal neurons. In these hippocampal cultures, endogenous gephyrin colocalizes with endogenous GRIP1c4-7 and GRIP1a/b in over 90% of the GABAergic synapses. Double-labeling electron microscopy immunogold reveals that in the rat brain GRIP1c4-7 and GRIP1a/b colocalize with gephyrin at the post-synaptic complex of individual synapses. These results indicate that GRIP1c4-7 and GRIP1a/b colocalize and interact with gephyrin at the GABAergic post-synaptic complex and suggest that this interaction plays a role in GABAergic synaptic function.

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Figures

Fig. 1
Fig. 1. GRIP1c4-7 associates with gephyrin in transfected HEK293 cells and in brain extracts
A–C, HEK293 cells were cotransfected with GRIP1c4-7 and gephyrin. Double immunofluorescence labeling was performed with the guinea pig anti-GRIP1c4-7 (green) and the mouse anti-gephyrin antibody (red). Some of the GRIP1c4-7 (A) is recruited at intracellular compartments (white arrowheads) where gephyrin accumulates (B). The overlay (C) also shows cell nuclei that were counter-stained with DAPI. The arrow shows the nucleus of a non-transfected cell. Note that these cells show no immunofluorescence of either GRIP1c4-7 or gephyrin. D, When HEK293 cells were transfected with GRIP1c4-7 only, GRIP1c4-7 showed a diffuse distribution in the cytoplasm and did not form intracellular aggregates. GRIP1c4-7 also associated with plasma membrane (black arrowheads). When HEK293 cells were transfected only with gephyrin, the latter formed aggregates (Fig 2L) similar to the ones shown in panel B. The illustrated expression patterns in A–C are representative of 39 out of 42 cells and in D of 66 out of 73 cells (from 3 and 4 transfection experiments respectively). E, The guinea pig anti-GRIP1c4-7 antibody, but not the corresponding PIS, coprecipitated associated gephyrin (arrowhead, 93 and 96 kD doublet) from extracts of HEK293 cotransfected with gephyrin and GRIP1c4-7, as shown in the immunoblot with the rabbit anti-gephyrin antibody. F, The rabbit anti-gephyrin antibody, but not the corresponding PIS, coprecipitated GRIP1c4-7 (arrowhead, 75 kD) from extracts of the cotransfected HEK293 cells, as shown in immunoblots with a mouse mAb that recognizes GRIP1c4-7. G, The guinea pig anti-GRIP1c4-7 antibody, but not the corresponding PIS, coprecipitated the gephyrin doublet (arrowhead) from a rat brain extract, as shown with the rabbit anti-gephyrin antibody. H, Beads coated with the GST-GRIP1c4-7 fusion protein pulled down gephyrin (arrowhead) from a rat brain extract, but beads coated with GST did not, as shown with the rabbit anti-gephyrin antibody. I, Schematic representation and alignment of various GRIP1 constructs used in this study. GRIP1a is a 135 kD protein with 7 PDZ domains. GRIP1a4-7 is an engineered truncated form of GRIP1a that is identical to amino acids 417–1112 of GRIP1a. GRIP1a4-7 has the same length as GRIP1c4-7. GRIP1c4-7 is a naturally existing short splice variant of GRIP1. Amino acids 36–684 of GRIP1c4-7 are identical to amino acids 452–1100 of GRIP1a. Broken lines at the N and C termini of GRIP1c4-7 represent the 35 and 12 amino acid peptides respectively, which are not present in GRIP1a, GRIP1b or GRIP1a4-7. GRIP1b is identical to GRIP1a except for the N-terminal 18 and 19 amino acid peptides of GRIP1a and GRIP1b respectively. All immunoprecipitation and pull-down experiments were done three times with similar results. Scale bar, 5 μm.
Fig 2
Fig 2. GRIP1a/b and GRIP1a4-7 associate with gephyrin in transfected HEK293 cells and in brain extracts
A–C, HEK293 cells were cotransfected with gephyrin and full-length GRIP1a. Double immunofluorescence labeling was performed with the guinea pig anti-GRIP1a/b (green) and the mouse mAb to gephyrin (red). Full-length GRIP1a (white arrowheads) and gephyrin (black arrowheads) formed cytoplasmic aggregates in HEK293. The overlay (C) also shows cell nuclei that were counter-stained with DAPI. The arrow in C shows the nucleus of a non-transfected cell. The illustrated expression patterns in A–C are representative of 36 out of 49 cells from 3 transfection experiments. D, In HEK293 cells cotransfected with GRIP1a and gephyrin, the guinea pig anti-GRIP1a/b antibody, but not the corresponding PIS, coprecipitated gephyrin (93–96 kD, arrowhead) as revealed in the immunoblot with the rabbit anti-gephyrin antibody. E, The rabbit anti-gephyrin antibody, but not the corresponding PIS, coimmunoprecipitated a very small amount of the full-length GRIP1a (130 kD, arrowhead, faint protein band, first lane from the right) when compared with the input (arrowhead, second lane from the left) from extracts of HEK293 cells cotransfected with gephyrin and GRIP1a, as shown with a mAb to GRIP1a/b, which also recognizes GRIP1a4-7 (see panel K). F, The guinea pig anti-GRIP1a/b antibody but not the corresponding PIS, coprecipitated gephyrin (93–96 kD, arrowhead) from a rat brain extract. G, The rabbit anti-gephyrin antibody but not the corresponding PIS, coprecipitated GRIP1a/b (130 kD, arrowhead) from a rat brain extract. Note that the protein band corresponding to GRIP1a/b is very faint (first lane from the right). H–J, HEK293 cells were cotransfected with engineered GRIP1a4-7 and gephyrin. Double immunofluorescence labeling reveals that GRIP1a4-7 (green) shows both diffuse distribution in the cytoplasm and concentration in cytoplasmic compartments. Some GRIP1a4-7 was recruited to the cytoplasmic compartments where gephyrin (red) accumulates (white arrowhead). The overlay (J) also shows cell nuclei that were counter-stained with DAPI. The arrow in J shows the nucleus of a non-transfected cell. The antibodies used were the same as in panels A–C. The illustrated expression patterns in H–J are representative of 38 out of 47 cells from 3 transfection experiments. K, The anti-gephyrin antibody coprecipitated GRIP1a4-7 (75 kD, arrowhead, first lane from the right) when compared with the input (arrowhead, second lane from the left) from extracts of HEK293 cells cotransfected with gephyrin and GRIP1a 4-7. Note that the density of the band corresponding to the coprecipitated truncated GRIP1a4-7 form (75 kD, arrowhead, first lane from the right) is considerably stronger than that of the co-precipitated full-length GRIP1a protein in panel E (130 kD, arrowhead, first lane from the right). The same mAb was used in panels E and K to identify GRIP1a and GRIP1a4-7 in the immunoblots. L–N, HEK293 cells were single-transfected with gephyrin (L, red), GRIP1a (M, green), or GRIP1a4-7 (N, green). Cell nuclei were counter-stained with DAPI. The illustrated expression patterns in L–N are representative of 66 out of 72 (L), 83 out of 99 (M) and 85 out of 101 (N) cells from 5 transfection experiments. All immunoprecipitation experiments were done three times with similar results. Scale bar, 5 μm.
Fig 3
Fig 3. HA-GRIP1a4-7 colocalizes with postsynaptic GABAARs and extrasynaptic GABAAR clusters in transfected hippocampal pyramidal neurons
A–C, Triple-label immunofluorescence of hippocampal neurons transfected with HA-GRIP1a4-7 cDNA using a combination of mouse mAb to the HA epitope (green, A), rabbit anti-γ2 GABAAR subunit (red, B), and sheep anti-GAD (blue C). Clusters of HA-GRIP1a4-7, in a transfected pyramidal cell, colocalize with γ2-GABAAR clusters at GABAergic synapses (arrows) as shown by their apposition to presynaptic GAD+ terminals. The interneuron, from which the axon containing GAD+ presynaptic terminals originates, has no HA immunoreactivity. Thus the localization of the HA-GRIP1a4-7 in these GABAergic synapses is postsynaptic. Clusters of HA-GRIP1a 4-7 immunoreactivity also colocalize with some non-synaptic γ2-GABAAR clusters (arrowheads), since they are not apposed to presynaptic GAD+ terminals. D, Overlay of A, B and C. Scale bar, 20 μm.
Fig 4
Fig 4. GRIP1c4-7 can associate with GRIP1a in transfected HEK293 cells and in brain extracts
A–C, HEK293 cells were cotransfected with GRIP1c4-7 and GRIP1a. Double immunofluorescence labeling was performed with the guinea pig anti-GRIP1c4-7 antibody (green), which does not recognize GRIP1a/b, and the rabbit anti-GRIP1a/b antibody (red), which does not recognize GRIP1c4-7. The GRIP1c4-7 and GRIP1a formed large aggregates that frequently colocalized (A–C, arrowheads). GRIP1c4-7 does not form aggregates when HEK293 cells are transfected only with GRIP1c4-7 (Fig 1D). The overlay (C) also shows cell nuclei that were counter-stained with DAPI. The arrow shows the nucleus of a non-transfected cell. The illustrated expression patterns in A–C are representative of 45 out of 49 cells from 3 transfection experiments. D, The guinea pig anti-GRIP1a/b antibody but not the corresponding PIS, coprecipitated GRIP1c4-7 (75 kD, arrowhead) from detergent extracts of HEK293 cells cotransfected with GRIP1c4-7 and GRIP1a, as shown by immunoblotting with the rabbit anti-GRIP1c4-7 antibody. E, The guinea pig anti-GRIP1c4-7 antibody, but not the corresponding PIS, coprecipitated GRIP1a (130 kD, arrowhead) from a detergent extract of HEK293 cells cotransfected with GRIP1c4-7 and GRIP1a, as shown by immunoblotting with a rabbit antibody to GRIP1a/b. F, The guinea pig anti-GRIP1c4-7 antibody, but not the corresponding PIS, coprecipitated GRIP1a/b (arrowhead) from the rat brain extract. G, Beads coated with the GST-GRIP1c4-7 fusion protein pulled down GRIP1a/b (arrowhead) from a rat brain extract, while beads coated with GST did not. All the antibodies used in the immunoblots (D–G) were made in rabbit. All immunoprecipitation and pull-down experiments were done three times with similar results. Scale bar, 5 μm.
Fig 5
Fig 5. Purified GRIP1c4-7 and GRIP1a bind to purified gephyrin
A, Purified and solubilized His-gephyrin (113 kD, arrowheads) bound to GST-GRIP1c4-7 but not GST immobilized on glutathione beads. B, Purified His-gephyrin (arrowhead) bound to GST-GRIP1a4-7 and GST-GRIP1a but not GST immobilized on glutathione beads. In A and B His-gephyrin was detected in immunoblots with the rabbit anti-gephyrin antibody. C, Purified His-BIG2CT (31kD, arrow head) bound to GST-β3IL, but not to GST, GST-GRIP1c4-7, GST-GRIP1a4-7, or GST-GRIP1a immobilized on glutathione beads as revealed with an anti-His mAb. The experiments were done three times with similar results. Scale bar, 5 μm.
Fig 6
Fig 6. Gephyrin frequently colocalizes with GRIP1c4-7 and GRIP1a/b at GABAergic synapses in cultured hippocampal neurons
A–D, Triple-label immunofluorescence with mouse mAb to gephyrin (green), rabbit anti-GRIP1c4-7 (red) and sheep anti-GAD (blue) antibodies. E–H, Triple-label immunofluorescence with mouse mAb to gephyrin (green), rabbit anti-GRIP1a/b (red) and sheep anti-GAD (blue) antibodies. Arrows show the colocalization of gephyrin clusters with GRIP1c4-7 or GRIP1a/b clusters at GABAergic synapses. Synaptic clusters are identified by their apposition to GAD+ presynaptic terminals. White arrowheads show colocalization of some gephyrin clusters with GRIP1c4-7 or GRIP1a/b clusters outside GABAergic synapses (not apposed to GAD+ terminals). Crossed arrows show gephyrin clusters that are not colocalized with GRIP1c4-7 or GRIP1a/b clusters and are not apposed to GAD+ terminals. Black arrowheads show GRIP1c4-7 or GRIP1a/b clusters that are not colocalized with gephyrin clusters and are not apposed to GAD+ terminals. Scale bar, 5 μm.
Fig 7
Fig 7. Gephyrin colocalizes with GRIP1c4-7 and GRIP1a/b at the postsynaptic complex of individual synapses in the intact brain
Postembedding EM immunogold labeling of the cerebellum. In all panels, the presynaptic terminal containing synaptic vesicles is located above the synaptic cleft. A, B, Double-labeling with the mouse mAb to gephyrin (small particles, arrowheads) and the rabbit anti-GRIP1c4-7 (large particles, arrows) shows that the two types of gold particles, corresponding to gephyrin and GRIP1c4-7, colocalize postsynaptically at the same synapse. C, D, Double-labeling with the mouse mAb to gephyrin (small particles, arrowheads) and the rabbit anti-GRIP1a/b antibody (large particles, arrows) shows that gold particles corresponding to gephyrin and GRIP1a/b colocalize postsynaptically in the same synapse. Crossed arrows in C, D show gold particles corresponding to presynaptically localized GRIP1a/b. The images correspond to synapses of stellate cells onto Purkinje cell dendrites. Scale bar, 75 nm.
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References

    1. Ataman B, Ashley J, Gorczyca D, Gorczyca M, Mathew D, Wichmann C, Sigrist SJ, Budnik V. Nuclear trafficking of Drosophila Frizzled-2 during synapse development requires the PDZ protein dGRIP. Proc Natl Acad Sci USA. 2006;103:7841–7846. - PMC - PubMed
    1. Bladt F, Tafuri A, Gelkop S, Langille L, Pawson T. Epidermolysis bullosa and embryonic lethality in mice lacking the multi-PDZ domain protein GRIP1. Proc Natl Acad Sci USA. 2002;99:6816–6821. - PMC - PubMed
    1. Bohlhalter S, Mohler H, Fritschy JM. Inhibitory neurotransmission in rat spinal cord: co-localization of glycine- and GABAA-receptors at GABAergic synaptic contacts demonstrated by triple immunofluorescence staining. Brain Res. 1994;642:59–69. - PubMed
    1. Bruckner K, Pablo Labrador J, Scheiffele P, Herb A, Seeburg PH, Klein R. EphrinB ligands recruit GRIP family PDZ adaptor proteins into raft membrane microdomains. Neuron. 1999;22:511–524. - PubMed
    1. Charych EI, Yu W, Li R, Serwanski DR, Miralles CP, Li X, Yang BY, Pinal N, Walikonis R, De Blas AL. A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain. J Biol Chem. 2004a;279:38978–38990. - PubMed

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