INTRODUCTION

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The neuregulins (NRGs)¹ were originally identified in searches for ligands of the receptor tyrosine kinase erbB2 (1-3) and as neuronally produced factors that stimulate the synthesis of muscle acetylcholine receptors (acetylcholine receptor-inducing activity; see Ref. 4) and the proliferation of Schwann cells (glial growth factor; see Refs. 5 and 6). In vitro and in vivo studies now implicate the NRGs in the regulation of a large number of biological processes (7-10). Known functions of NRGs include regulation of synapse formation and maintenance, cell proliferation, apoptosis, differentiation decisions, and neuronal migration. Transgenic knockout mice lacking NRG have nervous system developmental defects and die at mid-embryogenesis (embryonic days 10-11) due to abnormalities in heart development (11, 12).

At least 15 NRG protein isoforms are produced from a single NRG gene (6, 13-16). Most of these NRG isoforms are synthesized as transmembrane proteins (see Fig. 1). Full-length transmembrane (TM)-NRG is found on the cell surface of TM-NRG-expressing cells (17), and TM-NRG can be proteolytically processed to release the NRG extracellular domain into the medium (17, 18). Thus, TM-NRG may act both as a juxtacrine (direct cell-cell contact) signaling protein (19-21) and as the precursor for a diffusible, paracrine signaling molecule.

The extracellular epidermal growth factor-like domain of TM-NRGs activates the receptor tyrosine kinases erbB2, erbB3, and erbB4. Most prior NRG studies have focused on the interaction of the NRG extracellular domain with these receptor tyrosine kinases (RTKs) and the biological consequences of erbB2/erbB3/erbB4 activation by NRG. In contrast, this study focused on the long intracellular region of TM-NRG isoforms (see Fig. 1). The high degree of amino acid sequence conservation of this intracellular region (4) suggests that it has important biological functions. Grimm and Leder (22) recently reported that one form of the NRG cytoplasmic tail (the b-tail) can activate apoptosis in TM-NRG-transfected HEK 293 cells. Two other potential biological functions of the NRG cytoplasmic tail are regulation of NRG protein trafficking and of proteolytic release of the NRG ectodomain into the extracellular space (see Refs. 13, 17, 23, and 24). Another intriguing possibility is that transmembrane NRG may function not only as a receptor ligand but also as a receptor and that the NRG cytoplasmic tail mediates outside-in signal transduction. If NRG acts as a "receptor" and the RTKs erbB2, erbB3, and erbB4 are its "ligand," bi-directional signaling could occur between cells expressing TM-NRG and cells expressing the RTKs erbB2, erbB3, and erbB4. The idea of bi-directional signaling between cells expressing an RTK TM ligand and cells expressing the cognate RTK was first suggested by Pfeffer and Ullrich (25), and recent in vivo and in vitro studies of the interaction between the TM ligand LERK-2 and the RTK Nuk (26-28) have strongly supported this hypothesis. Thus, several potential biological roles for the cytoplasmic tails of NRG and other RTK TM ligands are supported by experimental evidence; however, no proteins interacting with these cytoplasmic tails have been molecularly identified.

As an approach to assessing these potential functions of the NRG cytoplasmic tail and to determine the mechanism by which these functions are carried out, we have used the yeast two-hybrid system to isolate brain proteins that interact with the cytoplasmic tail of NRG. We report evidence that the nonreceptor kinase LIMK1 (29-32) interacts with the NRG cytoplasmic tail. We show that LIMK1 and the NRG cytoplasmic tail physically associate in vitro and in cultured cells. In vivo, NRG and LIMK1 have overlapping expression patterns in the mammalian nervous system, and we show that these proteins are co-localized at the neuromuscular synapse. Although the cellular functions of LIMK1 remain unknown, LIMK1 hemizygosity has been implicated in the pathogenesis of the visuospatial constructive cognitive defect of Williams syndrome (33-35). Our findings suggest the possibility that the interaction of LIMK1 with NRG may play a role in the formation of neuromuscular synapses and of neuronal circuitry that mediates specific cognitive functions.

	EXPERIMENTAL PROCEDURES

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Nomenclature Note-- Throughout this paper, neuregulin and the abbreviation NRG refer only to the proteins encoded by the first discovered NRG gene (1-6). These proteins might now be considered forms of NRG1 in light of the recent discovery of related proteins encoded by two other NRG family genes. These NRG1-related proteins have been dubbed NRG2 (or Don-1) (36-38) and NRG3 (39). All of the protein isoforms that are the subject of this study are produced from transcripts of the NRG1 gene.

Reagents-- The cDNA clones encoding rat NRG isoforms are described in Ref. 13. The cDNA clone that encodes the full-length murine LIMK1 was a gift from E. Robertson (Department of Molecular and Cellular Biology, Harvard University) (31). The yeast two-hybrid bait vector pBTM116 and prey vector pVP16 were provided by S. Hollenberg (Fred Hutchinson Cancer Research Center, Seattle, WA) (40). The mouse brain library in pVP16, the PER bait, and the PER prey were provided by C. Weitz and N. Gekakis (Harvard Medical School). Polyclonal antibody 1310, raised against the common region of the NRG cytoplasmic tail, and the immunizing peptide were a gift from T. Burgess (Amgen, Inc.) (immunizing peptide, CNSFLRHARETPDSYRDS) (17). Antibody sc-537, also recognizing the common region of the NRG cytoplasmic tail (immunizing peptide, FLRHARETPDSYRDSPHSER) and anti-Myc mouse monoclonal antibody 9e10 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibody sc-537 was used for the immunohistochemical experiments because at the time these experiments were conducted, little Ab 1310 remained. Antibodies sc-537 and 1310 have given similar results in our experiments. Mouse monoclonal antibody 7D5, directed against the NRG ectodomain, was purchased from NeoMarkers (Fremont, CA). Anti-SV2 hybridoma supernatant was a gift of Dr. Kathy Buckley (Harvard Medical School) (41). The anti-FLAG mouse monoclonal antibody M2 was purchased from International Biotechnologies, Inc. (New Haven, CT). All secondary antibodies for Western blot and immunofluorescence experiments were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Transfection of COS-7 Cells-- Culture conditions for COS-7 cells were as follows: 37 °C; 8% CO₂; medium consisting of Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Plasmid Constructs-- Details of plasmid construct structure are provided in the legend to Fig. 2. To make yeast bait constructs, the target sequences (NRG and TGF- cytoplasmic tails) were amplified by polymerase chain reaction (PCR). The PCR products were gel purified and subcloned into pBTM116 using the EcoRI and BamHI sites. As illustrated in Fig. 2, the bait proteins are fusions of the LexA DNA binding domain (N-terminally fused) and the bait sequence (C-terminally fused).

Yeast Two-hybrid Screening and Assays-- The yeast two-hybrid screening reported here employed the bait plasmid pBTM116, the prey plasmid pVP16, and the yeast strain L40 (40). The bait construct (NRGc bait) used for screening the library encodes a fusion between the LexA DNA binding domain and the portion of the NRG cytoplasmic tail common to all transmembrane NRGs. The two-hybrid expression library screened was prepared by N. Gekakis and C. Weitz (Harvard University). Each prey plasmid in the library encodes a fusion protein consisting of: 1) a nuclear localization sequence, 2) the VP16 transactivation domain, and 3) the protein encoded by a brain cDNA (see Fig. 2). This library was prepared from mRNA obtained from the brain of a 3-week-old mouse. The first strand cDNA synthesis was random primed to minimize bias toward C-terminal sequences and was size-selected for a length of 300-800 base pairs. This length is sufficient to encompass individual protein domains but, in many cases, may encode only a portion of a protein. The partial-length prey resulting from this size selection may be advantageous in allowing identification of interactions between the bait protein and proteins for which a full-length prey protein would not interact in a two-hybrid assay, either because the full-length protein is membrane-associated or because it contains a regulatory domain that blocks interaction with the bait. The library had 2 × 10⁶ primary recombinants.

Production of GST Fusion Proteins and in Vitro Binding Assay-- The GST expression vector pGEX-4T-1 and GST fusion protein constructs GST-NRGas-tail and GST-NRGc-tail were transformed into the bacterial strain BL21. An overnight culture in 2×YT medium was diluted 1:50 into 50 ml of fresh 2×YT and incubated at 37 °C in a shaking incubator for 90 min. Isopropyl-1-thio--D-galactopyranoside was then added to the culture to a final concentration of 0.1 mM, the culture was incubated for an additional 4 h, and then the bacteria were pelleted at 2500 × g. The pellet was washed once with 7 ml of STE buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The bacteria were resuspended in 5 ml of cold STE containing 100 µg/ml lysozyme and incubated on ice for 15 min. Five hundred microliters of 100 mM dithiothreitol and 1 ml of 10% sarkosyl w/v were added, and the volume was brought to 10 ml with cold STE. The bacteria were lysed by freezing and thawing five times in a dry ice-ethanol bath. The lysate was cleared by centrifugation at 16,000 × g for 20 min at 4 °C. The supernatant was transferred to fresh tubes, and Triton X-100 was added to a final concentration of 2% v/v. The lysate (10 ml) was then incubated with 100 µl of glutathione-agarose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C. The beads were settled by centrifugation at 700 × g, washed four times with PBS and twice with TENT buffer (1% Triton X-100, 5 mM EDTA, 150 mM NaCl, 10 mM Tris-HCl, pH 7.5), and used for in vitro binding assays without further treatment.

Immunoblotting-- Samples were heated to 95 °C for 5 min immediately prior to loading on SDS-polyacrylamide minigels. Resolved proteins were then transferred to polyvinylidene difluoride membranes (Millipore) using a CAPS transfer buffer (10 mM CAPS, 10% methyl alcohol, pH 11) (44). The membranes were blocked with 5% nonfat dry milk in TBS buffer (100 mM Tris, 0.9% NaCl, pH 7.5) for 1 h at room temperature. The membranes were incubated overnight at 4 °C with 0.5 µg/ml Ab 9e10 (for LIMK1), 0.3 µg/ml Ab 1310 (for NRGs), or 0.5 µg/ml 7D5 (for NRGs) in 5 ml of the blocking solution. The membranes were washed four times with TTBS buffer (100 mM Tris, 0.9% NaCl, 0.1% Tween 20, pH 7.5) at room temperature. Bound primary antibodies were visualized with a horseradish peroxidase-conjugated goat anti-mouse IgG (1:50,000 in TTBS) or goat anti-rabbit IgG (1:50,000 in TTBS) and SuperSignal chemiluminescent substrate system (Pierce). For stripping and reprobing, blots were incubated in 2% SDS, 62.5 mM Tris-HCl, pH 6.8, 100 mM -mercaptoethanol for 1 h at 60 °C. The membrane was then washed twice in TTBS buffer and probed as described above.

Immunoprecipitation-- COS-7 cells were co-transfected with expression vectors encoding NRGs (full-length TM-NRGs or cytoplasmic tail only; cf. Figs. 5 and 6) and Myc-LIMK1 or were transfected with Myc-LIMK1 only (as a negative control). Sixty hours after transfection, cells were lysed in 400 µl of NDET buffer (150 mM NaCl, 0.5% sodium deoxycholate, 6 mM EDTA, 10 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1 mM PMSF, 2.5 µg/ml aprotinin, 2 µg/ml antipain, and 2 µg/ml leupeptin) per 100-mm dish. Lysate from two dishes was pooled and centrifuged at 700 × g for 15 min to remove cellular debris and nuclei. To preclear, 1 ml of lysate was incubated with 10 µl protein A-Sepharose (Sigma) for 1 h at 4 °C. All incubations were performed with continuous gentle agitation. The protein A-Sepharose beads were then spun down at 700 × g, and the supernatant was transferred to a fresh tube containing 10 µg of Ab 1310. After incubation for 1 h at 4 °C, 10 µl of protein A-Sepharose was added to the lysate/antibody mixture and incubated overnight at 4 °C. The immune complex was washed four times with NDET buffer and eluted in 40 µl of 2× SDS sample buffer containing dithiothreitol. For analysis of LIMK1 expression levels, an aliquot of the preprecipitation cell lysate was mixed with an equal volume of 2× SDS sample buffer with dithiothreitol. The samples were heated to 95 °C for 5 min immediately prior to loading, and 15 µl of each sample was loaded on an 8% SDS gel (see Figs. 5 and 6A) or a 12% SDS gel (see Fig. 6B). Eluted proteins and preprecipitation lysates were analyzed by Western blot as described above.

Antibody Production-- Anti-LIMK1 antibody was produced by immunizing a rabbit with the synthetic peptide -acetyl-KETYRRGESSLPAHPEVRD. The underlined amino acids correspond to the 18 C-terminal amino acids of the mouse and rat LIMK1 proteins (31, 45). For immunizations, the peptide was conjugated to horseshoe crab hemocyanin (Sigma H1757) using glutaraldehyde. Immunizations and harvesting of serum were performed by Covance Research Products (Denver, PA). Serum from the rabbit was analyzed by Western blot and affinity purified using the immunizing peptide conjugated to Affi-Gel 15 (Bio-Rad) according to the manufacturer's instructions. The affinity-purified antibody preparation had an immunoglobulin concentration of 1.5 mg/ml, as estimated from Coomassie Blue-stained SDS gels.

Immunohistochemistry-- An adult rat was anesthetized with 4% chloral hydrate and perfused through the heart with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Tissues were dissected and immersed in 15% sucrose/0.1 M phosphate until they sank, then transferred to 30% sucrose/0.1 M phosphate for 2-3 days and frozen in OCT compound using liquid N₂. 10-µm frozen sections were cut in a cryostat and dried onto SuperfrostPlus slides (Fisher Scientific). For immunohistochemistry, tissue was processed as follows: 1) washed three times with histology PBS (100 mM sodium phosphate, pH 7.4, 150 mM sodium chloride), 2) blocked for 1 h at room temperature in PBS blocking buffer (0.1 M phosphate buffer, pH 7.4, 150 mM NaCl, 0.2% nonfat dry milk, 1% BSA, 0.3% Triton X-100), 3) incubated overnight at 4 °C in primary antibodies diluted in PBS blocking buffer, 4) washed with histology PBS, 5) incubated for 1 h at room temperature with secondary antibodies in PBS blocking buffer, 6) washed with histology PBS, and 7) coverslipped with Vectashield Mounting Medium (Vector Laboratories). Antibody concentration/dilutions used are as follows: -SV2 hybridoma supernatant, 1:50; -NRG cytoplasmic tail antibody sc-537, 1.0 µg/ml; -LIMK1, 1.5 µg/ml; LRSC-conjugated donkey anti-rabbit, 1:200; and FITC-conjugated donkey anti-mouse, 1:200. The specificity of anti-LIMK1 antibody was confirmed by preabsorbing the antibody to the immunizing peptide (15 µg/ml) for 2 h at room temperature. The specificity of antibody sc-537 labeling was confirmed by preabsorbing the antibody to the Ab 1310 immunizing peptide (10 µg/ml), the sequence of which overlaps the sequence of the sc-537 immunizing peptide (see under "Reagents" above). The specificity of anti-SV2 labeling was assessed by omitting the anti-SV2 antibody. Experiments using each primary antibody alone with both secondary antibodies demonstrated the absence of bleedthrough and cross-labeling. Sections were viewed through a Zeiss Axiophot microscope using a × 100 oil immersion lens. Images were recorded with an MTI 300T-RC CCD camera using the NIH Image software.

	RESULTS

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The LIM Domain Region of LIMK1 Interacts with the Neuregulin Cytoplasmic Tail in the Yeast Two-hybrid System-- The sequence of the NRG cytoplasmic tail is highly conserved between mammals and birds (Fig. 1), suggesting that this region of the NRG protein serves one or more important biological functions. We are particularly interested in the possibility that transmembrane NRGs may serve as cell surface receptorsi.e. that NRGs are receptors in addition to being ligands for the erbB2/erbB3/erbB4 receptor tyrosine kinases. If so, one role of the NRG cytoplasmic tail may be to transduce signals received through the NRG extracellular domain. To test the hypothesis that the NRG cytoplasmic tail is involved in signal transduction, we conducted a yeast two-hybrid screen to identify proteins that interact with the NRG cytoplasmic tail.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Schematic representation of a transmembrane NRG isoform. As a consequence of alternative splicing, multiple NRG isoforms are synthesized. Most are transmembrane proteins. All contain an epidermal growth factor-like domain in the extracellular portion of the protein. This domain is necessary and sufficient for activation of the receptor tyrosine kinases erbB2/erbB3/erbB4. Due to alternative splicing, transmembrane NRGs with three different cytoplasmic tail forms are synthesized. The TM-NRG isoform illustrated has the longest form of the tail (a-tail). This a-tail consists of the 157 amino acids common to all NRG cytoplasmic tails (# to *, solid line) and a 217 amino acid sequence unique to the a-tail (* to C terminus, broken line). The shortest variant of the NRG cytoplasmic tail (c-tail) consists only of the 157-amino acid common region (i.e. ends at the *). The b-tail isoform consists of the 157 amino acid common region plus 39 amino acids unique to the b-tail (not shown here). For each domain of the TM-NRG isoform illustrated, the percentage of amino acids identical between rat and chicken TM-NRG is shown. Also shown is the number of amino acids in each domain. Note the high conservation of amino acid sequence for the cytoplasmic tail. Proteolysis of the extracellular juxtamembrane region (arrow) can release a soluble, bioactive form of NRG. Alternatively, uncleaved TM-NRG at the cell surface may activate cognate RTKs on cells contacting the NRG-expressing cell. This direct cell-cell contact mode of signaling has been called "juxtacrine" signaling (19, 20). This diagram is adapted from Ref. 2. A rat NDF 2a isoform is illustrated (GenBank accession number U02318). Ig-like, immunoglobulin-like domain; EGF-like, epidermal growth factor-like domain; TM, transmembrane domain.

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Summary of plasmid constructs. A, schematic diagrams of the types of recombinant proteins used in this study. Illustrated here are the yeast two-hybrid NRG cytoplasmic tail common region bait used for screening (NRGc-bait), the LIMK1 prey isolated in the two-hybrid screen (LIMK1-ldr), full-length LIMK1 with an N-terminal Myc epitope tag (Myc-LIMK1), a TM-NRG c-tail isoform (NRGc), FLAG-tagged NRG cytoplasmic tail common region (flag-NRGc-tail), and the GST-NRG cytoplasmic tail common region fusion protein (GST-NRGc-tail). In the diagram of Myc-LIMK1, the amino acid sequence encoded by the LIMK1-ldr is shown in black. Full-length LIMK1 consists of two LIM motifs (LIM1 and LIM2), a Dlg-homology region (DHR), a serine/proline rich domain (S/P), and a protein kinase domain. Numbers in the figure refer to amino acid numbers of GenPep sequences. B, tabulation of constructs used in this study. The amino acid sequence encoded by each construct is referenced to the amino acid numbering for a corresponding GenPep entry. Each of the NRG constructs, except the NRGas bait, contains the 157-amino acid cytoplasmic tail common region (amino acids 266-422 of U02323 and U02316; # to * in Fig. 1). The Myc and FLAG epitope tag sequences are underlined. Vectors used for each construct are indicated. LexA, LexA DNA binding domain; NLS, nuclear localization signal; VP16TA, VP16 transactivation domain. See under "Experimental Procedures" for details of construct creation.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   The LIM domain region of LIMK1 interacts specifically with the NRG cytoplasmic tail in yeast two-hybrid assays. A, the NRGc bait interacts with LIMK1-ldr but not with a control prey. A -galactosidase filter assay is shown. Blue color of the yeast colony (seen here as dark gray) indicates interaction of the bait protein and the prey protein. The NRGc bait contains the entire amino acid sequence of the NRG cytoplasmic tail that is common to all TM-NRGs. The LIMK1-ldr contains the LIM domain region of LIMK1 (cf. Fig. 2). This photograph was taken 2 h after addition of substrate. However, blue color of the yeast transformed with both NRGc bait and LIMK1-ldr was already evident at 30 min, which is as early as for the positive control (PER bait/PER prey), a known strong interaction. The NRGc bait/PER prey assay is a negative control. Assay of three separate colonies for each bait/prey combination is shown. B, the LIMK1-ldr interacts with the NRGc bait but not with control baits. Although the LIMK1-ldr interacts strongly with the NRG cytoplasmic tail, it does not interact with the cytoplasmic tail of TGF-, a receptor tyrosine kinase transmembrane ligand structurally related to NRG. It also does not interact with the PAS domain of PER (PER-bait), a protein sequence unrelated to NRG. -galactosidase assay results from panel A are included here for comparison. -Galactosidase activity was assessed by filter and solution assay. For filter assay, -galactosidase activity was determined from the time taken for colonies to turn blue at 30 ° C: +++, <30 min; ++, 30-60 min; +, 60-120 min; , no color after 120 min. For solution assay, -galactosidase activity was calculated as described under "Experimental Procedures."

LIMK1 Interacts with the Cytoplasmic Tail of Neuregulin in Vitro and in Cultured Cells-- The LIMK1-ldr prey used in the two-hybrid assays includes only the LIM domain region of LIMK1. We used an in vitro binding assay and a co-precipitation assay to determine whether full-length LIMK1 binds to the NRG cytoplasmic tail common region. For the in vitro binding assay, COS-7 cells were transfected with an expression construct encoding full-length LIMK1. A lysate prepared from these transfected cells was incubated with GST fusion proteins attached to glutathione-agarose beads (Fig. 4). LIMK1 bound to the GST-NRGc-tail protein, which consists of the entire NRG cytoplasmic tail common region, but not to GST alone. LIMK1 also did not bind to a fusion protein containing the 217-amino acid sequence unique to the longest NRG cytoplasmic tail isoform but lacking the NRG cytoplasmic tail common region (GST-NRGas-tail; cf. Fig. 2B). These results demonstrate that full-length LIMK1 binds specifically to the NRG cytoplasmic tail common region. The GST-NRGas-tail result also indicates that the interaction of the NRG a-tail isoform with LIMK1 (see below) appears to be restricted to the portion of the a-tail common to all TM-NRG isoforms.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   LIMK1 binds to the neuregulin cytoplasmic tail in vitro. Lysates from cells transfected with Myc-LIMK1 or mock-transfected were incubated with the indicated GST fusion proteins. Lane 4 demonstrates that LIMK1 binds to a NRG cytoplasmic tail common region-GST fusion protein (GST-NRGc-tail). However, LIMK1 does not bind to GST alone (lane 1) or to a fusion protein containing the a-tail specific sequence of the NRG cytoplasmic tail (GST-NRGas-tail; lane 2). Lanes 3 and 5 are negative controls. LIMK1 bound to the fusion protein was detected by Western blotting using an anti-Myc antibody. The same lysate preparation was used for the incubations shown in lanes 1, 2, and 4; thus, the concentration of LIMK1 used in each of these incubations is identical. This experiment has been performed three times, each time with similar results.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   LIMK1 is co-immunoprecipitated with neuregulin from lysates of transfected COS-7 cells. Top, lysates of COS-7 cells co-transfected with TM-NRG (NRGc) and LIMK1 or singly transfected with LIMK1 were immunoprecipitated using anti-NRG Ab 1310. Co-precipitated LIMK1 was detected by Western blotting using anti-Myc Ab 9E10. No band is seen in lane 2, demonstrating that the precipitation of LIMK1 from the lysate of co-transfected cells (lane 1) is specifically dependent on the precipitation of NRG. This experiment has been performed three times, each time with similar results. Bottom, Western blots of the preprecipitation lysates demonstrate that similar amounts of LIMK1 were present in the lysates of the doubly and singly transfected cells used for the co-precipitation analysis shown in the top panel.

TM-NRGs with a-, b-, and c-cytoplasmic Tail Forms Interact with LIMK1-- Transmembrane NRGs with three different cytoplasmic tail sequences are known. All share the 157-amino acid "common region" sequence that we used as the bait in our two-hybrid screen. The c-tail isoform is composed exclusively of this 157-amino acid "common region." The b-tail is the common region plus 39 additional amino acids. The a-tail is the common region plus 217 additional amino acids (cf. Figs. 1 and 6C). Because we used the common region of the NRG cytoplasmic tail as the bait for the screen that identified LIMK1 as a NRG-interacting protein, we expected that LIMK1 would also interact with a-tail and b-tail NRGs.

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Binding of LIMK1 to the three known isoforms of the neuregulin cytoplasmic tail. A, assay of LIMK1 co-precipitation with TM-NRGs. LIMK1 co-precipitated with TM-NRGs having each type of cytoplasmic tail. COS-7 cells were co-transfected with expression constructs encoding Myc-LIMK1 and a full-length TM-NRG (NRGa, NRGb, or NRGc), as indicated above blots. TM-NRGs were immunoprecipitated with Ab 1310, and co-precipitated LIMK1 was detected with antibody 9e10 (top panel). The blot was stripped and reprobed with anti-NRG antibody 7D5 to assess the amount of precipitated TM-NRG (middle panel). The multiple bands seen for individual NRG isoforms are likely due to heterogeneous glycosylation. Analysis of cell lysates on a separate blot demonstrated that similar amounts of Myc-LIMK1 were expressed in each transfection (bottom panel). This experiment has been performed twice; both times with similar results. B, assay of LIMK1 co-precipitation with NRG cytoplasmic tail isoforms expressed as cytosolic proteins. Top panel, more LIMK1 co-precipitated with the NRG c-tail isoform than with the b-tail; no specific co-precipitation was detected with the a-tail isoform. COS-7 cells were co-transfected with the Myc-LIMK1 expression vector and with expression constructs encoding FLAG-tagged NRG cytoplasmic tails (flag-NRGa-tail, flag-NRGb-tail, or flag-NRGc-tail), as indicated above blots. The amount of NRG precipitated (middle panel) and the amount of LIMK1 (bottom panel) in the original lysates were assessed on separate blots. In the middle panel, a band corresponding to the heavy chain of Ab 1310 (used for the immunoprecipitation) is seen in each lane (labeled IgG); this band overlaps the flag-NRGa-tail band. Similar amounts of LIMK1 were expressed in each co-transfection. This experiment has been performed twice, both times with similar results. C, yeast two-hybrid assay. Yeast two-hybrid assays of the interaction between the isoforms of the NRG cytoplasmic tail and the LIMK1 LIM domain region parallel the co-precipitation results for the NRG cytoplasmic tail isoforms expressed as cytosolic proteins (B). Filter assays were quantitated as described for Fig. 3. For solution assay, -galactosidase activity was calculated as described under "Experimental Procedures."

LIMK1 and Neuregulin Are Co-localized at the Neuromuscular Synapse-- The results described above demonstrate a physical interaction between recombinantly expressed NRG and LIMK1. These findings suggest the hypothesis that NRG and LIMK1 interact in vivo. An important prediction of this hypothesis is that NRG and LIMK1 will have an overlapping expression pattern. Neuregulin is known to play an important role in the development, maintenance, and regeneration of the neuromuscular synapse (reviewed in Ref. 9) and in the interaction of motor and sensory neurons with Schwann cells (Refs. 52-55 and references therein). Therefore, to test whether NRG and LIMK1 are co-expressed in vivo, we examined the expression of the NRG and LIMK1 genes in the spinal cord and dorsal root ganglia of the adult rat and we determined whether the NRG and LIMK1 proteins are co-localized at neuromuscular synapses.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Anti-LIMK1 antibody specifically labels Myc-LIMK1 in transfected COS-7 cells. The specificity of affinity-purified antibody was tested by Western blot analysis using lysates of Myc-LIMK1 transfected COS-7 cells. Anti-LIMK1 antibody recognized the same 71 kDa band as antibody 9e10. Neither antibody recognized this band in mock-transfected cells (), indicating that the 71-kDa band is Myc-LIMK1. No band was seen when anti-LIMK1 antibody was preincubated with the immunizing peptide.

View larger version (79K): [in this window] [in a new window]   Fig. 8.   Neuregulin and LIMK1 are co-localized at neuromuscular synapses. A section of adult rat muscle was double-labeled for LIMK1 (A) and for a marker of nerve terminals, SV2 (B). An adjacent section was double-labeled for TM-NRG (D) and SV2 (E). Both LIMK1 (A) and TM-NRG (D) co-localized with SV2. Control experiments confirmed specificity of labeling with each antibody (see under "Experimental Procedures" for details). A DIC image for each section is also shown (C and F). Scale bar, 20 µm.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

The cytoplasmic tail of transmembrane NRGs has a highly conserved amino acid sequence, but its functions are unknown. We hypothesized that the NRG cytoplasmic tail plays a role in signal transduction and searched for interacting proteins involved in this process. Here we have reported characterization of the interaction between the NRG cytoplasmic tail and LIMK1, a nonreceptor kinase likely to be involved in intracellular signal transduction. Through a yeast two-hybrid screen, we discovered that the LIM domain region of LIMK1 can physically associate with the NRG cytoplasmic tail. In vitro binding studies showed that full-length LIMK1 binds to the NRG cytoplasmic tail, and co-immunoprecipitation experiments revealed that full-length LIMK1 can associate with transmembrane NRGs in mammalian cells. In contrast, the LIMK1 LIM domain region does not interact with the cytoplasmic tail of TGF-, an RTK transmembrane ligand previously reported to associate with a kinase activity (50, 51) These results demonstrate a specific physical interaction between NRG and LIMK1. In agreement with previously published results (32, 33, 55, 56, 59), we find that both LIMK1 and TM-NRG are expressed in spinal cord and dorsal root ganglia. Now we have found that LIMK1 is also co-localized with TM-NRG at neuromuscular synapses. Taken together, the physical interaction and co-localization of these proteins strongly suggest that NRG and LIMK1 functionally interact in vivo.

LIMK1 was discovered in several screens for novel kinases (29-32). Like NRG, LIMK1 is most highly expressed within the nervous system and appears to be principally expressed by neurons, not glia (29-32, 59). Human genetic studies implicate LIMK1 hemizygosity as the cause of the cognitive defect found in Williams syndrome (33-35). Features of Williams syndrome include a specific cognitive profile characterized by pronounced weakness in visuospatial constructive cognition but relative strength in language and auditory rote memory, mild or moderate mental retardation, congenital heart and vascular disease, dysmorphic facial features, and infantile hypercalcemia. Although it seems likely that LIMK1 hemizygosity results in lower than normal amounts of LIMK1 protein being produced, the cellular mechanisms underlying the nervous system abnormalities of Williams syndrome are unknown. In vitro studies indicate that overexpression of LIMK1 can inhibit proliferation of fibroblasts (62) and differentiation of PC12 cells (63), but the relevance of these findings to the in vivo functions of LIMK1 is unclear. Our finding of an interaction between the NRG cytoplasmic tail and LIMK1 suggests that the pathogenesis of Williams syndrome may involve an abnormality in signaling through NRG-as-receptor, perhaps resulting in abnormalities of axon guidance (see discussion of Lerk-2/Nuk interaction, below).

LIMK1 has four structural domains: a double LIM domain, a Dlg-homology region (DHR/PDZ) domain, a serine/proline-rich region, and a kinase domain (Fig. 2). LIMK1 was the first protein found to combine LIM domains with a kinase domain. Each of the two LIM domains is comprised of two zinc finger structures. Although LIM domains were first described in homeodomain proteins (47-49) that may serve as transcription factors, it is now recognized that the LIM domain is a protein-protein interaction motif found in proteins with a wide variety of functions (46). LIMK1 has been shown to bind several isoforms of protein kinase C; for the protein kinase C  isoform, it has been demonstrated that this binding is through the second (more C-terminal) of the two LIMK1 LIM domains (64). Together with the results presented here, this finding suggests the possibility that the NRG-LIMK1 interaction regulates protein kinase C activity. The LIM domain region of LIMK1 has also been shown to bind the LIMK1 kinase domain (65). Thus, the NRG cytoplasmic tail may compete with the LIMK1 kinase domain for binding to the LIM domains, and this competition may regulate the phosphorylation activity of LIMK1.

The amino acid sequence of the LIMK1 kinase domain is unusual in that it precisely fits neither the signature for serine/threonine nor for tyrosine kinases. Nonetheless, biochemical evidence indicates that this is an active kinase domain with a preference for serine residues (31, 32, 60). In vitro, LIMK1 has been shown to autophosphorylate and to phosphorylate myelin basic protein. Curiously, when assayed using myelin basic protein as a substrate, the specific activity of LIMK1 isolated from A431 cells is 100-fold greater than for recombinant LIMK1 produced in COS-7 cells (60). No natural substrates of LIMK1 have been defined. We considered it possible that the NRG cytoplasmic tail would act as a substrate for LIMK1, but results from preliminary experiments testing this hypothesis have been negative.²

NRG produced by motor neurons is believed to regulate synthesis of the postsynaptic muscle acetylcholine receptors at developing and mature neuromuscular synapses (for review, see Ref.9), to regulate apoptosis and function of Schwann cells capping the nerve terminals (52-54), and to regulate interaction of motor neuron axons with myelinating Schwann cells (see Ref. 8 and references therein). Immunohistochemical studies using antibodies directed against the NRG extracellular domain indicate that NRG is concentrated at neuromuscular synapses in both the motor nerve terminal (58, 66) and in the synaptic cleft (67-69). Our results extend these previous studies by demonstrating that the NRG cytoplasmic tail is also concentrated at the adult neuromuscular synapse (cf. also Ref. 66).

Within what component(s) of the synapse is the NRG tail located? Although the NRG extracellular domain can be released from TM-NRG by proteolytic cleavage, it is unlikely that the NRG cytoplasmic tail (a portion of the NRG protein located in the cytoplasmic compartment of the nerve terminal) is released from the nerve terminal. Thus, it is probable that most of the synaptic labeling by the NRG cytoplasmic tail antibody (Fig. 8) is due to NRG in the nerve terminal. There is, however, evidence that muscle expresses the NRG gene at low levels (66, 68, 70, 71), so the possibility that there is NRG cytoplasmic tail associated with the postsynaptic membrane cannot be excluded.

In contrast to NRG, LIMK1 has not previously been reported to be concentrated at synapses. Although in the early embryo LIMK1 is widely expressed (31, 59), in the adult, LIMK1 is principally expressed in the nervous system (29-32, 45, 60). Our reverse transcription PCR studies of LIMK1 expression in adult rat spinal cord and dorsal root ganglia are consistent with published in situ hybridization data (32, 59) suggesting LIMK1 expression by motor and sensory neurons. In situ hybridization studies have also demonstrated expression of LIMK1 by various neurons of the adult brain (29, 32, 59), and immunohistochemical studies have determined that LIMK1 is present in the cytoplasm and nuclei of hippocampal and cerebellar neurons (29). However, no description of the protein distribution in motor or sensory neurons has previously been reported, nor has there been any published observation of LIMK1 in axons or concentrated at synapses.

It seems most likely that synaptic LIMK1, like synaptic NRG cytoplasmic tail, is principally in the nerve terminal, rather than in the synaptic cleft or muscle components of the synapse. Because LIMK1 is a cytoplasmic protein, it is unlikely to be released from the nerve terminal. Further, Northern blot analysis of adult mouse muscle did not detect LIMK1 expression (Refs. 29 and 32; however, see also Ref. 60), and LIMK1 labeling is not seen in the cytoplasm of rat muscle (Fig. 8). Thus, we conclude that our evidence strongly indicates the co-localization of LIMK1 and the NRG cytoplasmic tail in the nerve terminal. Because NRG has important functions in the regulation of neuromuscular synapse formation and maintenance, the finding that the NRG cytoplasmic tail-interacting protein LIMK1 is also concentrated at this synapse supports the hypothesis that the NRG cytoplasmic tail and LIMK1 play important roles in the regulation of neuromuscular synapses.

Why many NRGs are synthesized as transmembrane proteins rather than soluble proteins is unknown. Part of the answer to this puzzle is likely to reside in the functions of the cytoplasmic tail, because the amino acid sequence of this region is highly conserved. Although we favor the idea that the interaction of LIMK1 with NRG mediates outside-in signaling, the LIMK1-NRG interaction could mediate any of the potential functions of the NRG cytoplasmic tail, including regulation of NRG trafficking and proteolytic processing (see Refs. 13, 17, 23, and 24) or apoptosis (22). Indeed, these are not mutually exclusive possibilities; for example, a consequence of outside-in signaling through NRG might be increased proteolytic processing of TM-NRG.

The cytoplasmic tails of two other RTK TM ligands, TGF- and Lerk-2, are known to associate with kinases. In Chinese hamster ovary cells stably expressing TM TGF-, TM TGF- can be cross-linked to a protein complex that includes serine, threonine, and tyrosine kinase activities (50, 51). These kinase activities have been demonstrated by an in vitro phosphorylation assay of the precipitated complex using myelin basic protein and histone 2B as substrates. The binding of this kinase(s) to TGF- apparently has different structural requirements than the binding of NRG to LIMK1, because the association of kinase activities with the TGF- cytoplasmic tail depends on a pair of cysteines in the tail (51), but the NRG common region lacks cysteines. A protein of 86 kDa that is a component of the cross-linked complex may be the TGF- associated kinase, because its association with the TGF- cytoplasmic tail also depends on the cysteine pair. However, the identity of this protein has not yet been reported.

To date, the strongest evidence for bi-directional signaling through an RTK TM ligand/RTK pair comes from studies of the interaction between the transmembrane RTK ligand Lerk-2 and its RTK receptor Nuk. Genetically altered mice lacking the Nuk RTK have a defect in anterior commissure formation; whereas genetically altered mice that make a mutant form of Nuk with no kinase activity have no defect in anterior commissure formation. Lerk-2 is expressed by the neurons of which the axons are misrouted in the mice lacking the Nuk RTK, and Nuk is expressed by the cells over which these axons migrate (26). One interpretation of these results is that Lerk-2 serves as a receptor guiding these axons. This interpretation is supported by studies in a model system demonstrating that when Lerk-2 expressing cells are co-cultured with Nuk expressing cells, the Lerk-2 becomes tyrosine-phosphorylated on its cytoplasmic tail (27, 28).

LIMK1 is the first molecularly identified protein known to interact with the cytoplasmic tail of an RTK TM ligand. The experiments reported here demonstrate physical interaction between NRG and LIMK1 and a similar cellular and subcellular distribution of these proteins. The fact that both NRG and LIMK1 are localized at neuromuscular synapses supports the hypothesis that this interaction is functionally significant. To elucidate the biological functions of the NRG cytoplasmic tail/LIMK1 interaction, experiments now under way seek to determine how the interaction of NRG and LIMK1 is regulated, the downstream events triggered by this interaction, and the functional consequences of the interaction.