Ralf Enz

Expression of GABA Receptor ρ1 and ρ2 Subunits in the Retina and Brain of the Rat

We have investigated the distribution of GABA receptor ρ1 and ρ2 subunits in the rat central nervous system. Cloning of rat ρ1 and ρ2 cDNA fragments revealed similarities to the corresponding human sequences of 99% (ρ1) and 88% (ρ2) at the protein level. Whereas the human ρ2 subunit has no consensus sequence for phosphorylation by protein kinase C, the cytoplasmic loop of the rat sequence contains two such sites. Use of the polymerase chain reaction with reverse‐transcribed total RNA (RT‐PCR) from different brain tissues revealed that transcript for the ρ1 subunit was present in the retina only. The ρ2 mRNA was detected in all brain regions, with the highest level of expression in the retina. In situ hybridization of retinal sections revealed that ρ1 and ρ2 transcripts are present in the inner nuclear layer. RT‐PCR and in situ hybridization of isolated retinal cells showed that both ρ subunits are present in rod bipolar cells. Since these cells express bicuculline‐insensitive GABA receptors, our results further support the idea that ρ subunits are part of the GABAC receptor.

Synaptic clustering of GABAC receptor ρ-subunits in the rat retina

Polyclonal antibodies which recognize the ρ‐subunits of the GABAC receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABAC receptors at synaptic sites. During postnatal development diffuse ρ‐immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the ρ‐immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the ρ‐antiserum were performed. The experiments showed that ρ‐immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABAC receptor ρ‐subunits coassemble with GABAA receptor subunits, double‐labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABAC receptor expressing puncta did not coincide with GABAA receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABAA and GABAC receptor subunits are coassembled. Similar double‐labelling experiments were also performed to find out whether GABAC receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABAC receptors consist of ρ‐subunits and are not coassembled with GABAA‐ or glycine‐receptor subunits.

Expression of the mRNA of Seven Metabotropic Glutamate Receptors (mGluR1 to 7) in the Rat Retina. An In Situ Hybridization Study on Tissue Sections and Isolated Cells

We have studied the expression of mRNAs for seven metabotropic glutamate receptors (mGluR1–7) in the retina of the adult rat by in situ hybridization with tissue sections and isolated cells using [α35S]dATP‐labelled oligonucleotide probes. Hybridization revealed the expression of six of the metabotropic receptor mRNAs, mGluR1, 2 and 4–7, in the retina, while mGluR3 was not detected. Each of the expressed receptor mRNAs showed a distinct pattern of expression. In the outer nuclear layer, corresponding to photoreceptor somata, no labelling was detected. In the outer part of the inner nuclear layer, putative horizontal cells were labelled for mGluR5. More proximal in this layer, corresponding to the position of bipolar cell somata, there was strong labelling for mGluR6. A small number of bipolar cells were also labelled for mGluR5 and mGluR7. In situ hybridization with isolated cells showed that mGluR6 was expressed by rod bipolar cells. Subsets of amacrine cells, with cell bodies along the border between the inner nuclear layer and the inner plexiform layer, were positive for mGluR1, 2, 4 and 7, suggesting considerable heterogeneity of these receptors among amacrine cells. None of the seven metabotropic receptor mRNAs was expressed in isolated Müller glial cells. In the ganglion cell layer, virtually every ganglion cell and displaced amacrine cell was labelled for mGluR1 and mGluR4. Some cells in this layer (˜20% of the total), most likely both ganglion cells and displaced amacrine cells, were also labelled for mGluR2 and mGluR7. These findings suggest that metabotropic glutamate receptors are considerably more widespread among neurons in the retina than indicated by previous physiological and pharmacological investigations.

Immunocytochemical localization of the GABAC receptor ? subunits in the cat, goldfish, and chicken retina

Polyclonal antibodies against the N‐terminus of the rat ρ 1 subunit were used to study the distribution of γ‐aminobutyric acid C (GABAC) receptors in the cat, goldfish, and chicken retina. Strong punctate immunoreactivity was present in the inner plexiform layer (IPL) of all three species. The punctate labelling suggests a clustering of the GABAC receptors at synaptic sites. Weak label was also found in the outer plexiform layer (OPL) and over the cell bodies of bipolar cells. Double immunostaining of vertical sections with an antibody against protein kinase C (PKC) showed the punctate immunofluorescence to colocalize with bipolar cell axon terminals. In the goldfish retina, the axon terminals of Mb1 bipolar cells were enclosed by ρ‐immunoreactive puncta. In the chicken retina, several distinct strata within the IPL showed a high density of ρ‐immunoreactive puncta. The results suggest a high degree of sequence homology between the ρ subunits of different vertebrate species, and they show that the retinal localization of GABAC receptors is similar across different species. J. Comp. Neurol. 380:520–532, 1997.

The actin-binding protein Filamin-A interacts with the metabotropic glutamate receptor type 7

A yeast two‐hybrid screen identified Filamin‐A as a binding partner of the metabotropic glutamate receptor type 7b (mGluR7b) splice variant. In addition, Filamin‐A interacted with mGluR4a, mGluR5a, mGluR5b, mGluR7a and mGluR8a. Domain mapping revealed that alternative splicing of mGluR4, mGluR7 and mGluR8 C‐termini regulated the interaction. A conserved tyrosine within mGluR C‐termini was identified to mediate the binding to Filamin‐A. Protein interactions were verified in biochemical assays using recombinant and native proteins. Finally, co‐expression of Filamin‐A and mGluR7 splice variants was shown in brain regions. These findings suggest that Filamin‐A may physically link metabotropic glutamate receptors to the actin cytoskeleton.

Different binding motifs in metabotropic glutamate receptor type 7b for filamin A, protein phosphatase 1C, protein interacting with protein kinase C (PICK) 1 and syntenin allow the formation of multimeric protein complexes.

Metabotropic glutamate receptor (mGluR) type 7-mediated neurotransmission depends critically on its regulation by associated molecules, such as kinases, phosphatases and structural proteins. The splice variants mGluR7a and mGluR7b are defined by different intracellular C-termini, and simultaneous or exclusive binding of interacting proteins to these domains modulates mGluR7-mediated signalling. However, molecular determinants defining binding regions for associated proteins within mGluR7 C-termini are mostly unknown. In the present study, we have mapped the binding domains of four proteins [filamin A, protein phosphatase (PP) 1C, protein interacting with protein kinase C (PICK) 1 and syntenin] interacting with the mGluR7b variant, and show that the alternatively spliced distal part of the mGluR7b C-terminus was sufficient for the interactions. By individual substitution of all mGluR7b isoform-specific amino acids with alanine and construction of a series of deletion constructs, residues important for the interactions were identified and binding regions could be defined. Interestingly, mGluR7b contains an unusual PP1C-binding motif, located at the N-terminus of the binding domains for PICK1 and syntenin. Consistently, binding of PP1C and PICK1 or PP1C and syntenin to mGluR7b was not competitive. Furthermore, PICK1, but not PP1C, interacted physically with syntenin. Our results represent a molecular description of the binding mechanisms of four mGluR7-associated proteins, and indicate the formation of ternary protein complexes composed of mGluR7b, PP1C, PICK1 and syntenin.

Cereblon is expressed in the retina and binds to voltage-gated chloride channels

MINT‐6823070: CIC‐2 (uniprotkb:O54822) physically interacts (MI:0218) with CRBN (uniprotkb:Q0P564) by two hybrid (MI:0018) MINT‐6823160, MINT‐6823197: CIC‐2 (uniprotkb:O54822) physically interacts (MI:0218) with CRBN (uniprotkb:Q56AP7) by pull down (MI:0096) MINT‐6823105: CIC‐2 (uniprotkb:O54822) physically interacts (MI:0218) with IK (uniprotkb:A4FUY8) by two hybrid (MI:0018)

Group I Metabotropic Glutamate Receptors Bind to Protein Phosphatase 1C MAPPING AND MODELING OF INTERACTING SEQUENCES

The modulation of neurotransmitter receptors by kinases and phosphatases represents a key mechanism in controlling synaptic signal transduction. However, molecular determinants involved in the specific targeting and interactions of these enzymes are largely unknown. Here, we identified both catalytic γ-isoforms of protein phosphatase 1C (PP1γ1 and PP1γ2) as binding partners of the group I metabotropic glutamate receptors type 1a, 5a, and 5b in yeast cells and pull-down assays, using recombinant and native protein preparations. The tissue distribution of interacting proteins was compared, and protein phosphatase 1C was detected in dendrites of retinal bipolar cells expressing the respective interacting glutamate receptors. We mapped interacting domains within binding partners and identified five amino acids in the intracellular C termini of the metabotropic glutamate receptors type 1a, 5a, 5b, and 7b being both necessary and sufficient to bind protein phosphatase 1C. Furthermore, we show a dose-dependent competition of these C termini in binding the enzyme. Based on our data, we investigated the structure of the identified amino acids bound to protein phosphatase 1C by homology-based molecular modeling. In summary, these results provide a molecular description of the interaction between protein phosphatase 1C and metabotropic glutamate receptors and thereby increase our understanding of glutamatergic signal transduction.

RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors.

In the central nervous system, synaptic signal transduction depends on the regulation of neurotransmitter receptors by interacting proteins. Here, we searched for proteins interacting with two metabotropic glutamate receptor type 8 isoforms (mGlu8a and mGlu8b) and identified RanBPM. RanBPM is expressed in several brain regions, including the retina. There, RanBPM is restricted to the inner plexiform layer where it co‐localizes with the mGlu8b isoform and processes of cholinergic amacrine cells expressing mGlu2 receptors. RanBPM interacts with mGlu2 and other group II and group III receptors, except mGlu6. Our data suggest that RanBPM might be associated with mGlu receptors at synaptic sites.

Expression of glycine receptor subunits and gephyrin in single bipolar cells of the rat retina.

We studied the expression of glycine receptor (GlyR) subunits and gephyrin in the adult rat retina. Reverse transcribed RNA was amplified by polymerase chain reaction (RT-PCR) with primers designed to recognize GlyR alpha 1, alpha 2, alpha 3, beta subunits, and gephyrin. Using RNA isolated from the whole retina, signals for all four GlyR subunits and gephyrin could be observed. In rod bipolar cells, in contrast, we detected a subset of GlyR subunits, alpha 1 and beta, and no gephyrin. Patch-clamp recording employing two subtype-specific blockers of the GlyR, picrotoxinin and cyanotriphenylborate (CTB), indicated that the GlyR in rod bipolar cells is a heteromeric protein composed of the alpha 1 and beta subunit. Moreover, the absence of detectable amounts of gephyrin mRNA suggests that the anchor protein is not required for the function of GlyRs in rod bipolar cells.