The GABA ρ1 subunit interacts with a cellular retinoic acid binding protein in mammalian retina

Neuroscience 136 (2005) 467– 475

THE GABA ␳1 SUBUNIT INTERACTS WITH A CELLULAR RETINOIC ACID BINDING PROTEIN IN MAMMALIAN RETINA X.-Q. SONG,a F. MENG,a D. J. RAMSEY,b H. RIPPSa,b AND H. QIANa,b*

GABAA0r), that are pharmacologically distinct from classic GABAA receptors (Barnard et al., 1998; Bormann, 2000; Zhang et al., 2001; Johnston et al., 2003). Three ␳ subunits (␳1, ␳2, ␳3) have been cloned from mammalian retinal cDNA libraries (Cutting et al., 1991; Zhang et al., 1995; Ogurusu and Shingai, 1996); they are distributed in many parts of the brain, but are expressed predominantly on retinal neurons (Wegelius et al., 1998; Enz and Cutting, 1999; Qian and Ripps, 2001; Zhang et al., 2001; Rozzo et al., 2002). Although the various subunits form GABA-sensitive homologous receptors when expressed in oocytes, the precise subunit stoichiometry of GABAC receptors in retinal neurons has not been established. Nevertheless, it is likely that this class of receptor is composed of heteromers of ␳ subunits (Zhang et al., 1995), possibly in combination with the ␥2 subunit of the GABAA receptor (Qian and Ripps, 1999; Milligan et al., 2004; Pan and Qian, 2005). Particularly relevant for the present study is the fact that the majority of the intracellular exposure for each subunit consists of a large cytoplasmic loop that is thought to play a crucial role in interactions with cellular proteins. Interactions of neurotransmitter receptors with cytoplasmic proteins have been implicated in many functions, such as receptor clustering and anchoring (Moss and Smart, 2001). Using a traditional yeast two-hybrid system, three proteins were identified as interacting with the large intracellular loop between the third and the fourth transmembrane domains of GABA ␳ subunits. A splice variant of the glycine transporter GLYT-1 interacts with the ␳1 subunit (Hanley et al., 2000), the microtubule-associated protein 1B (MAP-1B) binds with the ␳1 subunit (Hanley et al., 1999), and a novel splice variant of PKC-␨ interacts with all three ␳ subunits (Croci et al., 2003). However, evidence of such interactions does not a priori indicate functional significance. Thus, although MAP-1B binds microtubules and is co-localized with GABAC receptors at the presynaptic terminals of retinal bipolar cells and cone photoreceptors (Billups et al., 2000; Pattnaik et al., 2000), the expression pattern of GABAC receptors in MAP-1B deficient mice was shown to be indistinguishable from that of wild-type animals (Meixner et al., 2000). Despite the fact that the conventional yeast two-hybrid system is a powerful and versatile technique widely used to identify binding partners, the method has limitations. Most notable is the fact that the detection of protein–protein interactions relies on transcriptional activation of nuclear reporter genes. Therefore, the fusion proteins used in the conventional yeast two-hybrid system have to be transported to the nucleus where post-translational modifications may differ from those that take place in the cyto-

a Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 West Taylor Street, Chicago, IL 60612, USA b Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA

Abstract—Interactions between the intracellular domain of ligand-gated membrane receptors and cytoplasmic proteins play important roles in their assembly, clustering, and function. In addition, protein–protein interactions may provide an alternative mechanism by which neurotransmitters activate intracellular pathways. In this study, we report a novel interaction between the GABA ␳1 subunit and cellular retinoic acid binding protein in mammalian retina that could serve as a link between the GABA signaling pathway and the control of gene expression in neurons. The interaction between the intracellular loop of the human GABA ␳ subunit and cellular retinoic acid binding protein was identified using a CytoTrap XR yeast two-hybrid system, and was further confirmed by co-precipitation of the human GABA ␳ subunit and cellular retinoic acid binding protein from baboon retinal samples. The cellular retinoic acid binding protein binding domain on the human ␳1 subunit was located to the C-terminal region of human GABA ␳ subunit, and the interaction of the human GABA ␳ subunit with cellular retinoic acid binding protein could be antagonized by a peptide derived from within the binding domain of the ␳1 subunit. Since cellular retinoic acid binding protein is a carrier protein for retinoic acid, we investigated the effect of GABA on retinoic acid activity in neuroblastoma cells containing endogenously expressed cellular retinoic acid binding protein. In the absence of the ␳1 receptor, these cells showed enhanced neurite outgrowth when exposed to retinoic acid and GABA had no effect on their response to retinoic acid. In contrast, cells stably transfected with the human ␳1 subunit showed a significantly reduced sensitivity to retinoic acid when exposed to GABA. These results suggest that the GABA receptor subunit effectively altered gene expression through its interaction with the cellular retinoic acid binding protein pathway. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: GABA␳1 subunit, GABAC receptor, protein–protein interaction, CytoTrap yeast two-hybrid system, baboon retina, neurite outgrowth.

GABA ␳ subunits are believed to form a subclass of GABA receptors, referred to as GABAC receptors (also called *Correspondence to: H. Qian, Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 West Taylor Street, Chicago, IL 60612, USA. Tel: ⫹1-312-413-7347; fax: ⫹1-312-996-7773. E-mail address: [email protected] (H. Qian). Abbreviations: CRABP, cellular retinoic acid-binding protein; hSOS, human SOS (“Son Of Sevenless”); MAP-1B, microtubule-associated protein 1B; RA, retinoic acid; SD/galactose-Leu-Ura, galactose based synthetic dropout with no leucine and uracil.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.08.018

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plasm. To overcome this problem, several strategies have been designed to study protein interactions outside the yeast nucleus (Thaminy and Stagljar, 2002). One of these, the CytoTrap two hybrid system, commercially available from Stratagene (La Jolla, CA, USA), was used in the present study to detect proteins that interact with the GABAC receptor. Using the intracellular loop of the human GABA ␳1 subunit as bait, we identified cellular retinoic acid-binding protein (CRABP) as a potential binding partner with the GABAC receptor in primate retina. Retinoic acid (RA) is a potent signaling molecule which most notably controls nuclear gene activation, and CRABP has been implicated in regulating the availability of the hydrophobic RA molecule to its site of action in the nucleus (Donovan et al., 1995; Li and Norris, 1996). Thus, the interaction between the ␳1 subunit and CRABP could alter the efficacy of the RA signal, thereby providing a novel mechanism by which the GABAC receptor modulates gene expression in neurons.

EXPERIMENTAL PROCEDURES Yeast two-hybrid screening A commercially available CytoTrap two-hybrid system (Stratagene) was used to identify GABAC receptor binding partners. The system is based on the translocation of active human SOS (hSOS, the human equivalent of Drosophila “Son Of Sevenless” protein) to its site of action at the inner leaflet of the plasma membrane. The bait protein is expressed as a fusion protein with hSOS, and the prey is targeted to the membrane via a myristoylation signal sequence. Interacting fusion proteins recruit SOS to the membrane where it stimulates guanyl nucleotide exchange on yeast Ras, thus rescuing a temperature-sensitive mutant (cdc25H␣) to grow at 37 °C. The large intracellular loop between the third and the fourth transmembrane domains (amino acids 359 – 454) of the human GABA ␳1 subunit was PCR amplified and subcloned in frame into NcoI/SalI sites of the bait vector pSOS. The construct was co-transfected into a mutant yeast strain cdc25H␣ with a bovine retina cDNA library cloned in pMyr vector (Stratagene). The yeast cells were cultured in a galactose based synthetic dropout with no leucine and uracil (SD/galactose–Leu–Ura) medium for 4 days at 37 °C.

Co-precipitation The large intracellular domain of the human ␳1 subunit as well as three fragments (amino acids 359 –377, 376 – 428, and 435– 454) were cloned in frame into the GST-His fusion vector, pET42 (Novagen, Madison, WI, USA). The constructs were transformed in BL21-CodonPlus (DE3)-RP bacteria (Stratagene). Protein expression was induced with 1 mM IPTG for 3 h at 37 °C. The fusion proteins were lysed in 50 mM NaPO4 (pH8.0), 150 mM NaCl, 1 mM imidazole, 1% Triton X-100, and protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The lysate was incubated with a homogenate of baboon retina, and pulled down with Ni-NTA agarose beads (Qiagen, Valencia, CA, USA) that bind with the histidine tag on the fusion protein. After rinses with 10 mM imidazole wash buffer, the precipitated proteins were eluted and denatured in sample buffer at 95 °C for 5 min, separated on 10% SDS/PAGE gel, and analyzed by Western blot with anti-CRABP antibody (ABR, Golden, CO, USA). The baboon retinas were obtained through the shared tissue program at the Biological Resources Laboratory, University of Illinois at Chicago, and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Neurite measurements SHSY5Y neuroblastoma cells (ATCC, Manassas, VA, USA), stably transfected with the human GABA ␳1 subunit (kindly provided by Dr. David S. Weiss, University of Alabama at Birmingham, and referred to as SHp5-␳1) were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS and 100 U/ml penicillin and streptomycin. Cells were treated with various concentrations of RA and/or GABA for 48 h. Neurite length was determined from DIC images of cultured cells taken with an Axiovert 100 M inverted microscope (Zeiss, Germany). Because many cells extended processes that formed contacts with the neurites of neighboring cells, only those neurites that projected from the cell body to the distal end of the process were measured. For each experimental condition, 10 microscopic fields of 0.03 mm2 were randomly selected, and neurite length measured from the cell body to the tip of the neurite. Averaged data were compared using a two-tail Student’s t-test and plotted as means⫾S.D. To determine whether chloride currents induced by activation of the GABA receptor were responsible for any observed effects on neurite extension, neuroblastoma cells were transiently transfected with a glycine receptor subunit. The cells were transfected by nucleofection (Amaxa, Gaithersberg, MD, USA) with a 4:1 mixture of rat glycine ␣2 subunit cloned in pcDNA vector and an EGFP plasmid. The cDNA for the rat glycine ␣2 subunit was obtained by RT-PCR from rat retina. The clone was confirmed by nucleotide sequencing (Akagi et al., 1991), and its ability to form a functional glycine receptor was confirmed by current recordings from Xenopus oocytes (data not shown). EGFP was used as a fluorescent marker of successful transfection. Observations and data analysis were similar to those described above.

Immunocytochemistry Cells were fixed with 3% formalin for 15 min, stained with primary antibodies (anti-human ␳1 subunit, 1:100, Santa Cruz (Santa Cruz, CA, USA) and anti-CRABP, 1:250, ABR) at room temperature for 2 h, and followed by one hour incubation with the secondary antibodies (FITC tagged donkey anti-goat, 1:200 and TRITC tagged donkey anti-mouse, 1:200; Jackson ImmunoResearch Laboratory, West Grove, PA, USA). Fluorescent cell images were obtained with a Leica confocal microscope (Leica Microsystems Inc.).

RESULTS Interaction of human ␳1 subunit with CRABP in mammalian retina The CytoTrap yeast two-hybrid system was used to screen a bovine retinal cDNA library (Stratagene) for new partners interacting with the large intracellular loop of the human GABAC receptor ␳1 subunit (␳1). Approximately 1.3 million recombinants were screened with a pSOS-␳1 construct. Two hundred yeast colonies, grown at 37 °C, were isolated from the selection plate. Among them, 23 colonies contained an insert sequence code for bovine CRABP type I (CRABP I, CRABP for short) (Shubeita et al., 1987). To confirm the interaction between CRABP and the GABAC receptor ␳1 subunit, yeast were co-transformed with pSOS-␳1 and pMyr-CRABP, pSOS-␳1 and pMyr, or pSOS and pMyr-CRABP. All the transformants grew on selection medium (SD/galactose–Leu–Ura) at permissive temperature (25 °C), but only those containing both the human GABAC receptor ␳1 subunit and CRABP could grow when the temperature was increased to 37 °C. In addition, growth at 37 °C was only observed on plates with galac-

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Fig. 1. Interaction of GABA ␳1 subunit and CRABP in mammalian retina. (A) Western blot analysis detects a 16 kDa CRABP-specific band from a baboon retinal sample. (B) Co-precipitation of the human ␳1 subunit intracellular domain (␳1-IL) and CRABP. Upper panel: Coomassie-stained SDS-PAGE illustrates the concentration of GST-His and GST-His fusion protein. Note that approximately equivalent amounts of the different size proteins were used for the experiment. Lower panel: Western blot of CRABP co-precipitated from a baboon retinal sample. CRABP co-precipitated only with the human ␳1 fusion protein (GST-His-␳1-IL).

tose, but not with glucose, which does not induce the expression of pMyr fusion protein. Since GABA ␳ subunits are expressed predominantly in retina, it is important to recall that CRABP is also expressed in various cell types in the vertebrate retina (Saari et al., 1978; Milam et al., 1990; Fischer et al., 1999). Fig. 1A shows that a Western blot stained with a monoclonal

antibody to CRABP detects a single band at about 16 kDa from samples prepared from baboon retina. To investigate the interaction of the GABAC receptor and CRABP present in primate retina, we made a GST-His fusion protein with the large intracellular domain of the human GABAC receptor ␳1 subunit (GST-His-␳1-IL). As a control, GST-His vector protein was also made. These two proteins were dis-

Fig. 2. The CRABP binding site is located in the C-terminal region of ␳1-IL. (A) Schematic diagrams of the fusion proteins used in the experiment. (B) The ability of each construct to co-precipitate CRABP from baboon retina. Numbers above each lane correspond to each of the constructs shown in A. Upper panel: SDS-PAGE of the fusion proteins illustrates the different molecular weights of the various peptides. Lower panel: Western blot of CRABP in the precipitate. Strong signals were detected when the fusion protein contained the full length human ␳1 intracellular loop (lane 1) or the C-terminal region (lane 4). On the other hand, only weak signals appeared when fusion proteins with the N-terminal region (lane 2) or the middle region (lane 3) of the intracellular loop were used, despite the addition of much higher amounts of protein (upper panel). With only the GST-His protein, no CRABP signal was detected (lane 5).

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tinguishable by their respective molecular sizes (45 kDa vs 26 kDa for GST-His-␳1-IL and GST-His, respectively) as noted on the SDS gel (Fig. 1B, upper panel). These fusion proteins were incubated with samples of baboon retina, and pulled down by the Ni-NTA beads for His tagged proteins. When the precipitates were probed by Western blot with the CRABP antibody (lower panel, Fig. 1B), a 16 kDa band was detected only in the GST-His-␳1-IL pull down sample, but not in the control (GST-His). These results indicate that the large intracellular domain of the human GABAC receptor ␳1 subunit can interact with CRABP expressed in the primate retina. The C-terminal region of the ␳1 intracellular loop mediates CRABP interaction To localize the CRABP binding site on the large intracellular loop of the human GABAC receptor ␳1 subunit, we constructed three GST-His fusion proteins containing amino acids 359 –377, 376 – 429, or 435– 454 of the human GABAC receptor ␳1 subunit (Fig. 2A). Similar coprecipitation experiments were performed with these fusion proteins, and the results indicate that only the C-terminal fragment (435– 454) of the human ␳1 intracellular loop exhibited significant binding with CRABP (Fig. 2B). To further delineate the CRABP binding site

on the human ␳1 subunit, we synthesized a 12-amino acid peptide (RIDTHAIDKYSR) that corresponded to a portion of the C-terminal end of the ␳1-IL (443– 454 of human ␳1 subunit) (Fig. 3A). The interaction between CRABP and the human ␳1 intracellular loop could be inhibited by this competitive peptide. When the retinal extracts were first incubated with 20 pM of the peptide for one hour at room temperature, and then mixed with GSTHis-␳1 fusion proteins, co-precipitation of CRABP was blocked when the fusion proteins were pulled down by Ni-NTA beads (Fig. 3B). Similarly, the peptide also blocked interaction of CRABP with the C-terminal fragment of the human ␳1 intracellular loop. GABA modulates RA-induced cell growth and neurite elongations in neuroblastoma cells RA is an important signaling molecule that participates in many cellular processes, such as development and differentiation (Napoli, 1996). The most notable action of RA is the control of nuclear gene activation. However, RA is a hydrophobic molecule, and it needs a carrier molecule for delivery to its site of action in the nucleus. CRABP binds RA with high affinity and could serve as an intracellular retinoid transporter (Saari et al., 1978; Donovan et al., 1995). The interaction between the GABAC receptor ␳1

Fig. 3. Interaction between ␳1-IL and CRABP was antagonized by a competing peptide. (A) The schematic diagram of the GST fusion protein and the competitive peptide sequence. (B) Co-precipitation of CRABP from a baboon retinal sample. Upper panel: SDS-PAGE of the fusion proteins. Lanes 1 and 2: ␳1-IL (359 – 454aa). Lanes 3 and 4: ␳1-IL (435– 454 aa). Lane 5: GST-His. Lower panel: Western blot of CRABP signal precipitated from a baboon retinal sample. CRABP signal was observed with fusion proteins of full length (lane 1), or the C-terminal region (lane 3) of the intracellular loop. The interacting CRABP signal was blocked by the addition of the peptide (lane 2 and lane 4). Again, the GST-His protein did not interact with CRABP (lane 5).

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Fig. 4. Expression of the human ␳1 subunit and CRABP in neuroblastoma cells. Immunocytochemical staining for the ␳1 subunit (A), CRABP (B), and a merged image (C) from SHp5-␳1 cells stably transfected with the human ␳1 subunit. Strong immunofluorescence was observed in these cells with prominent expression of the GABA ␳ subunit at the cell membrane and of CRABP in the cytoplasm. (D) An example of the GABA (10 ␮M)-elicited chloride current from a neuroblastoma cell stably transfected with human ␳1 subunit. (E) Western blot analysis reveals the expression of CRABP in these cells. (F) Immunocytochemical staining for the GABA ␳1 subunit (left) and a matched DIC image (right) of wild type neuroblastoma cells (SHSY5Y). Endogenous expression of the GABAC receptor was not observed in these cells.

subunit with CRABP could therefore link the GABA signal to gene regulation. To investigate the functional significance of the interaction between CRABP and the GABAC receptor ␳1 subunit, we used a neuroblastoma cell line stably transfected with the human ␳1 subunit (SHp5-␳1). Fig. 4A–C shows immunocytochemical results for the GABA receptor and endogenously expressed CRABP in this cell line. The majority of the expressed GABA receptor is localized on the plasma membrane (Fig. 4A), and CRABP is mainly distributed in the cytoplasm (Fig. 4B). A merged image illustrating the distribution of the GABA receptor and CRABP is shown in Fig. 4C. The expressed GABA receptors were functional, and the cell responded to 10 ␮M GABA with slow, sustained chloride currents that characterize the GABAC receptor (Fig. 4D). The endogenously expressed CRABP could also be detected on Western blot (Fig. 4E). On the other hand, non-transfected cells (SHSY5Y) lacking the GABAC receptor showed no immunostaining with antibody to the human ␳1 subunit (Fig. 4F), and GABA failed to elicit any membrane currents (data not shown). It is noteworthy that the neuroblastoma cell line is sensitive to RA, and application of RA elicits a series of cellular responses including enhanced neurite elongation (Kim et al., 2000; Simpson et al., 2001). To examine the effect of the interaction between the GABAC receptor and CRABP on the RA signal pathway, we measured neurite extension under various culture conditions. Examples of cell morphology in response to RA and RA⫹GABA are

shown in Fig. 5A–C for cells expressing the GABAC ␳1 receptor. Note that RA alone elicited an elongation of neurite length as compared with cells in normal culture medium (control). However, the RA-induced neurite extension was largely inhibited when the GABAC receptors on these cells were activated by 10 ␮M GABA (Fig. 5C). We performed similar experiments to those shown in Fig. 5A–C on non-transfected (wild type) neuroblastoma cells, on cells expressing the GABA ␳1 subunit, and on cells expressing the rat glycine ␣2 subunit (as a control for gated chloride channels). Quantitative analysis of averaged data obtained under the various culture conditions is shown as bar graphs in Fig. 5D–F. For each experiment, neurite length was normalized to that measured in control, and it is evident that in each case RA (100 nM) significantly enhanced (25–50%) neurite outgrowth. The lower response to RA observed in cells transiently transfected with the glycine receptor subunit (25% enhancement) compared with those of stably expressing cells (⬃50% enhancement) may be due to the nature of the transfection procedure. That aside, it is apparent that irrespective of which cell line was tested, neither the application of (GABA 10 ␮M) nor glycine (10 ␮M) exerted an effect on neurite extension. Moreover, when GABA⫹RA was applied to non-transfected (SHSY5Y) cells (Fig. 5D), or when glycine⫹RA was applied to cells expressing the glycine receptor (Fig. 5F), the RA-induced neurite elongation was unaffected by these receptor agonists. However, for cells expressing the ␳1 receptor subunit (SHp5-␳1), the

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Fig. 5. Activation of the GABAC receptor inhibited RA-induced neurite growth. Examples of DIC images of SHp5-␳1 cells under normal culture conditions (A), after 48 h exposure to 100 nM RA (B), and following incubation in 100 nM RA⫹10 ␮M GABA (C). Neurite length was measured from the cell body to the distal tip of the process (white bars in each image). The bar graphs show the averaged neurite lengths measured under various culture conditions for (D) SHSY5Y cells (wild type neuroblastoma cells), (E) SHp5-␳1 cells (neuroblastoma cells expressing the GABAC ␳1 subunit), and (F) SHSY5Y-Gly-␣2 cells (neuroblastoma cells transiently transfected with the glycine ␣2 subunit). For transient expression, EGFP was co-transfected as a marker and only fluorescent cells were counted. The data were averaged over three experiments, each consisting of 25 independent neurite measurements. Note that RA significantly enhanced neurite outgrowth for all cell types (*** P⬍0.001; * P⬍0.05). The RA-induced outgrowth was not affected by the application of 10 ␮M GABA on wild type neuroblastoma cells, or by 10 ␮M glycine on cells transfected with the glycine ␣2 subunit. However, application of GABA (10 ␮M) to cells stably expressing ␳1 subunit significantly diminished their responsiveness to RA.

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RA-induced increase in neurite length was greatly reduced when GABA was coapplied with RA (Fig. 5E), i.e. there was no significant difference between the results with GABA⫹RA and GABA alone. Since the rat glycine ␣2 subunit is able to form a homomeric receptor that gates a chloride channel (Akagi et al., 1991), these results indicate that chloride current activation is not the principal mechanism by which GABA suppresses the RA sensitivity of the neuroblastoma cells. It is likely, therefore, that interaction of the ␳1 subunit with CRABP modulates the RA sensitivity of neuroblastoma cells expressing the GABAC receptor.

DISCUSSION Ionotropic neurotransmitter receptors are traditionally thought of as mediating fast synaptic transmission by gating ion channels. Although controlling ion flux is considered the main function of these receptors, the transmembrane structure of the receptor subunits may also serve as an alternative signal transduction mechanism. Indeed, recent studies have demonstrated some “metabotropic” functions for certain types of ionotropic glutamate receptors (Schenk and Matteoli, 2004). For example, AMPA receptors have been reported to suppress adenylyl cyclase by activation of a G-protein (Wang et al., 1997). These receptors also interact with protein tyrosine kinase Lyn (Hayashi et al., 1999) and mitogen-activated protein kinase (MAPK) (Schenk et al., 2005), and they inhibit a cGMP-gated current via a G-protein pathway (Kawai and Sterling, 1999). The GABAC receptor is also a ligand-gated ion channel (Qian and Ripps, 2001; Zhang et al., 2001), and the properties of its channel gating mechanism have been well described (Chang and Weiss, 1999; Bormann, 2000; Sedelnikova et al., 2005). However, whereas the opening of ion channels results from ligand binding on the extracellular surface of the membrane, the receptor undergoes a conformational change which may alter the structure of its intracellular domain and trigger a signal cascade within the cell. Thus, the transmembrane structure of the protein could subserve auxiliary functions, and it is likely that such a signal transduction mechanism would work on a relatively slow time scale to regulate, in concert with the ionic flux, neuronal activity. The results of the present study reveal a novel interaction between the GABAC receptor ␳1 subunit and CRABP that may provide a link between the GABA signal and the control of neuronal gene expression in the mammalian retina. Using the Cyto-trap yeast two-hybrid system, we identified CRABP as a binding partner with the intracellular domain of the ␳1 subunit of the GABAC receptor. Although the specific amino acids within the C-terminus that mediate the interaction were not determined in this study, it is interesting to note that the intracellular binding domain for CRABP on the human GABA ␳1 subunit is shared with the region that interacts with the MAP1B protein (Hanley et al., 1999). Thus, it is highly likely that this region of the receptor plays an important role in communicating with still other cytoplasmic pro-

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teins. However, in our screen of 1.3 million recombinants with the CytoTrap yeast two-hybrid system, we did not detect the various GABAC receptor-interacting proteins found in earlier studies (Hanley et al., 1999, 2000; Croci et al., 2003). The reason for this discrepancy is not clear. One possibility arises from the fundamental differences in the yeast systems used in these studies. While the GABAC receptor was found to interact with several proteins in yeast nuclei to initiate the transcription of reporter genes in the conventional yeast two-hybrid system, the cytoplasmic environment used by the CytoTrap yeast two-hybrid system may not favor these interactions. It is also possible that the cDNA library we obtained from Stratagene was unequally amplified, and produced a distorted representation of cDNA clones. We sought to investigate some of the functional consequences of the interaction between the GABAC receptor and CRABP using a neuroblastoma cell line stably transfected with the human ␳1 subunit. These cells endogenously express CRABP, and thus provide a convenient system with which to study changes in both structure and function resulting from protein–protein interactions. However, application of RA appeared to have little effect on the electrophysiological properties of these cells; patch clamp recordings showed that neither the amplitude nor the kinetics of the GABA-elicited currents were significantly altered (data not shown). In addition, we tested the sensitivity to GABA in Xenopus oocytes before and after coexpressing the human ␳1 subunit with CRABP. No significant change was observed as a result of the presence of CRABP, nor did the sensitivity of the oocyte to GABA change in the presence of RA (results not shown). On the other hand, RA did induce readily detectable changes in the morphology of the neuroblastoma cells, most notably, enhanced neurite outgrowth (Fig. 5). When neurite length was measured, there was a pronounced outgrowth in the presence of RA after 2 days in culture. In contrast, the sensitivity to RA was greatly decreased by activating the GABA receptor in cells expressing the ␳1 subunit, i.e. neurite extension was suppressed (Fig. 5E). However, GABA had no effect on the RA sensitivity of control cells that had not been transfected with the ␳1 subunit (Fig. 5D). In addition, although neurite outgrowth was enhanced by RA in neuroblastoma cells expressing the glycine receptor, activation of the chloride channel gated by the glycine receptor had no significant effect on the sensitivity to RA (Fig. 5F). Thus, our results are consistent with the hypothesis that activation of the GABAC receptor present on the plasma membrane alters the RA signal pathway through its interaction with CRABP in the cytoplasm (Fig. 4). Interestingly, the relation between the GABAC receptor and the activity of RA in retinal neurons may provide a key to understanding the role of GABA in myopia. Indeed, it has been suggested that RA participates in a signaling pathway that mediates form-deprivation-induced myopia (Wallman and Winawer, 2004), and the interaction between GABAC receptors and CRABP could serve as a link between GABA activity and the development of myopia. It

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well known that, in addition to other factors such as genetics, blur of the retinal image triggers a signal transduction cascade that leads to abnormal growth of the sclera, elongation of the eye, and myopia (Wallman and Winawer, 2004). Moreover, recent evidence has shown that intravitreal injection of GABAC receptor antagonists can prevent the induction of form deprivation myopia (Stone et al., 2003). The neurochemical signal that links the activity of retinal neurons to the elastic fibers and collagenous connective tissue of the sclera is largely unknown, but RA has emerged as the most likely candidate from among the several molecules that have been considered as contributing to the ocular deformation of myopia (Wallman and Winawer, 2004). Consistent with this view is evidence that form deprivation in chick is accompanied by an increase in retinal RA (Seko et al., 1998), and that the level of RA can be bidirectionally modulated by making the eyes of chick and guinea-pig either hyperopic or myopic (Seko et al., 1998; McFadden et al., 2004). Although highly speculative, it is apparent that interaction between the ␳1 subunit and CRABP could provide the link between neuronal activity and altered gene expression in the sclera through a RA-mediated pathway. It is not immediately evident where in the retina the interaction between the GABAC receptor and CRABP occurs. In mammalian retina, GABAC receptor ␳1 and ␳2 subunits are expressed predominantly on bipolar cells (Enz et al., 1995, 1996), whereas the ␳3 subunit appears to be expressed in cells located more proximally in the retina (Ogurusu et al., 1997). Immunostaining for CRABP has been seen mainly in the inner plexiform layer and within a few cell bodies of the inner nuclear and ganglion cell layers (Milam et al., 1990). We observed a similar CRABP expression pattern in rat and baboon retina (data not shown). Although mammalian bipolar cell bodies were not labeled by the CRABP antibody, it is possible that, for certain types of bipolar cell, CRABP is expressed in the axon terminal region of the inner plexiform layer. Thus, the interaction between the GABAC receptor and CRABP may occur in a subtype of bipolar cell or in the ␳3-expessing cells. Although CRABP was identified many years ago, and its structure has been well characterized (Donovan et al., 1995; Li and Norris, 1996), the cellular function of this protein is less well understood. It has been proposed that CRABP, with its high affinity for RA, could regulate the effects of RA by controlling its access to nuclear receptors (Li and Norris, 1996). However, the mechanism through which CRABP and the GABAC receptor interact has yet to be explored. It would be of particular interest to know the effects of this interaction on the affinity of CRABP for RA, as well as whether it affects the efficacy of RA in regulating gene transcription in retinal neurons. Acknowledgments—We are grateful to Ruth Zelka for her assistance in neurite measurement, and Dr. David S. Weiss for providing the SH5p-␳1 neuroblastoma cell line. This work was supported by grants from the National Eye Institute (EY-12028, EY-06516, EY 014557, and core grant EY-01792), and a Senior Scientific

Investigator Award (H.R.) from Research to Prevent Blindness, Inc.

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(Accepted 4 August 2005) (Available online 28 September 2005)