Journal: Neuroscience

Article Title: Group II Metabotropic Glutamate Receptor Interactions with NHERF Scaffold Proteins: Implications for Receptor Localization in Brain

doi: 10.1016/j.neuroscience.2017.03.060

Figure Lengend Snippet: A–B) The C-termini (CT) of mGluR2 (A) and mGluR3 (B) were screened for binding to an array of 96 distinct PDZ domains. The PDZ domains of NHERF-1 and NHERF-2 were the strongest hits for mGluR2 (A) and mGluR3 (B). A complete list of the PDZ proteins on this array has been described previously (He et al., 2006). The data shown here are representative of three independent experiments. C) A summary of the PDZ domains found to interact with either mGluR2/3-CT (black), mGluR2-CT alone (red), or mGluR3-CT alone (blue).

Article Snippet: Primaries used for immunoprecipitation and/or expression studies were: rabbit polyclonal anti-mGluR2/3 (Chemicon, catalog # 06-676), mouse monoclonal anti-mGluR3 (MAb Technologies, catalog# GRM3-2), rabbit polyclonal anti-NHERF-1 (Ab 5199) from ( Yun et al., 2002 ) or rabbit polyclonal anti-NHERF-2 (Ab 2570) from ( Yun et al., 1998 ), and rabbit monoclonal anti-actin (Sigma, catalog # A2066).

Techniques: Binding Assay

Journal: Neuroscience

Article Title: Group II Metabotropic Glutamate Receptor Interactions with NHERF Scaffold Proteins: Implications for Receptor Localization in Brain

doi: 10.1016/j.neuroscience.2017.03.060

Figure Lengend Snippet: HEK293T cells were transiently transfected with mock pcDNA3.1, mGluR2, mGluR2 L872A, mGluR3, mGluR3 L879A, or FLAG NHERF-1 or FLAG-NHERF-2 cDNAs. Robust immunoprecipitation of FLAG-tagged NHERF-1 (left panel, A and B) or NHERF-2 (right panel, A and B) was achieved in all experiments and subsequently examined for co-immunoprecipitation of mGluR2 or mGluR2 L872A (A) and mGluR3 or mGluR3 L879A (B) relative to background co-precipitation signal alone. A) Mutation of the last amino acid of mGluR2 was sufficient to disrupt NHERF-1 association (left, top panel, lanes 2 vs. 4). This lack of association could not be explained by differences in mGluR2 and mutant mGluR2 L872A expression, as similar total levels of protein were used in the study as revealed by comparison to actin labeling (left, bottom panel). It should be noted that the mGluR2/3 antibody detects the C-terminus of mGluR2 and mGluR3; thus, the mutation reduces detection of mGluR2 or mGluR3. Likewise, mutation of the last amino acid of mGluR2 was also sufficient to disrupt NHERF-2 association (right, top panel, lane 2 vs. 4). Similar results were obtained in 2–5 independent experiments. B) Mutation of the last amino acid of mGluR3 was sufficient to disrupt NHERF-1 association (left, top panels, lanes 2 vs. 4, detecting mGluR3 with both a C-terminal (IB: mGluR2/3) and N-terminal (IB: mGluR3-NT) antibody). This lack of association could not be explained by differences in mGluR3 and mutant mGluR3 L879A expression, as similar total levels of protein were expressed in the study as revealed by comparison to actin labeling (left, bottom panel) and comparable detection of mGluR3 input with the N-terminal antibody. Likewise, mutation of the last amino acid of mGluR3 was also sufficient to disrupt NHERF-2 association (right, top 2 panels, lane 3 vs. 4). Similar results were obtained in 2–5 independent experiments.

Article Snippet: Primaries used for immunoprecipitation and/or expression studies were: rabbit polyclonal anti-mGluR2/3 (Chemicon, catalog # 06-676), mouse monoclonal anti-mGluR3 (MAb Technologies, catalog# GRM3-2), rabbit polyclonal anti-NHERF-1 (Ab 5199) from ( Yun et al., 2002 ) or rabbit polyclonal anti-NHERF-2 (Ab 2570) from ( Yun et al., 1998 ), and rabbit monoclonal anti-actin (Sigma, catalog # A2066).

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Expressing, Labeling

Journal: Neuroscience

Article Title: Group II Metabotropic Glutamate Receptor Interactions with NHERF Scaffold Proteins: Implications for Receptor Localization in Brain

doi: 10.1016/j.neuroscience.2017.03.060

Figure Lengend Snippet: Mouse cortical astrocyte cultures expressing mGluR2 or mGluR2 L872A (A and B) and mGluR3 or mGluR3 L879A (C and D) were stimulated with either vehicle (media) or 1 μM LY354740 for designated periods of time. Astrocyte lysates were simultaneously probed for pAKT (Ser473) and total AKT. Graphs depict average fold change ± S.E.M. of normalized pAKT/AKT integrated densities over vehicle treatment. A) Analysis of mGluR2- and L872A-mediated AKT signaling via two-way ANOVA revealed an overall significant effect of receptor on signaling (n = 8, p = 0.0298, *). Enhanced mGluR2 L872A signaling was observed in 6 out of 8 individual experiments. B) Representative immunoblot showing the agonist-dependent activation of pAKT. C) A student’s t-test revealed there was no significant difference between mGluR3- and L879A-mediated AKT signaling (n = 8). D) Representative immunoblot showing the comparably small agonist-dependent activation of pAKT in mGluR3-expressing astrocytes.

Article Snippet: Primaries used for immunoprecipitation and/or expression studies were: rabbit polyclonal anti-mGluR2/3 (Chemicon, catalog # 06-676), mouse monoclonal anti-mGluR3 (MAb Technologies, catalog# GRM3-2), rabbit polyclonal anti-NHERF-1 (Ab 5199) from ( Yun et al., 2002 ) or rabbit polyclonal anti-NHERF-2 (Ab 2570) from ( Yun et al., 1998 ), and rabbit monoclonal anti-actin (Sigma, catalog # A2066).

Techniques: Expressing, Western Blot, Activation Assay

Journal: Neuroscience

Article Title: Group II Metabotropic Glutamate Receptor Interactions with NHERF Scaffold Proteins: Implications for Receptor Localization in Brain

doi: 10.1016/j.neuroscience.2017.03.060

Figure Lengend Snippet: A) Full length NHERF-1 is absent in brain lysates from N1 KO animals and its levels are unchanged in WT and N2 KO mice. Similarly, full-length NHERF-2 is also absent in brain lysates from N2 KO mice and its levels are unchanged in WT and N1 KO mice (B). However, a presumed NHERF-2 splice variant (asterisk) that is predicted to be 24.5 kDa, and runs at approximately 27 kDa, appears upregulated in N2 KO mice and its levels are slightly decreased in N1 KO mice, relative to WT mouse brain samples. The other band detected with the NHERF-2 antibody is unknown; it may reflect a non-specific band as this is a polyclonal antibody that is not affinity purified, or alternatively it may indicate another NHERF-2 splice variant. C-D) Cortical astrocytes were prepared from WT, N1 KO, N2 KO, or N2 KO cultures treated with 250 nM control siRNA or NHERF-1 siRNA. Cultures were then transfected with mock (pcDNA3.1), mGluR2, or mGluR3 cDNAs and after 24 hours of expression and 3 hour serum starvation, were stimulated with either vehicle or 1 μM LY354740 for 10 min. Graphs depict normalized pAKT/AKT integrated densities ± S.E.M. Analysis of the activation of Ser473 AKT (C). Analysis with Two-Way ANOVA revealed no significant effect of genotype, p = 0.86, n.s. However, a significant effect of receptor was observed, in accordance with the differential ability of mock-, mGluR2-, or mGluR3-transfected astrocytes to activate AKT, p < 0.0001, ***. These data are representative of 3–5 independent experiments per condition. D) Western blots characterizing the model system used in this study. Cultured astrocytes were treated with control siRNA, or 100 nM or 500 nM of NHERF-1 siRNA and were probed for NHERF-1 three days following transfection, revealing a dose-dependent knock-down of NHERF-1 with siRNA treatment (top panel). Demonstration of successful transfection of mGluR2 and mGluR3 of cultured astrocytes, as measured by Western blot analysis with an mGluR2/3 C-terminal antibody (bottom panels; lane 1, molecular weight ladder; lanes 2 and 3, lysates from astrocyte cultures from either mock-transfected, mGluR2-transfected, or mGluR3-transfected conditions). Note that the signal for mGluR3 is relatively weak in comparison to mGluR2, despite equal transfection of mGluR2 and mGluR3 plasmids.

Article Snippet: Primaries used for immunoprecipitation and/or expression studies were: rabbit polyclonal anti-mGluR2/3 (Chemicon, catalog # 06-676), mouse monoclonal anti-mGluR3 (MAb Technologies, catalog# GRM3-2), rabbit polyclonal anti-NHERF-1 (Ab 5199) from ( Yun et al., 2002 ) or rabbit polyclonal anti-NHERF-2 (Ab 2570) from ( Yun et al., 1998 ), and rabbit monoclonal anti-actin (Sigma, catalog # A2066).

Techniques: Variant Assay, Affinity Purification, Transfection, Expressing, Activation Assay, Western Blot, Cell Culture, Molecular Weight