INTRODUCTION

Top Abstract Introduction Procedures Results Discussion References

Gelsolin is a Ca²⁺- and phosphatidylinositol 4,5-bisphosphate-regulated actin-binding protein (1). It has been implicated in the regulation of the actin cytoskeleton and the modulation of membrane-cytoskeletal cross-talks (2-4). Many gelsolin-like proteins have been identified and they appear to have evolved from an ancestral single segment gene that has duplicated multiple times to form proteins with 3- or 6-fold repeats (5). Recent three-dimensional structure analyses show that the segments within gelsolin (6), as well as segments from different gelsolin family members, have a similar core structure (7-9). Nevertheless, the gelsolin family of proteins have distinct actin binding characteristics and intracellular localizations. For example, unlike most gelsolin members, CapG is a nuclear as well as cytoplasmic protein (10) and it does not sever filaments (11). Therefore, the conserved residues in each protein appear to maintain the basic folds of the repeated segments, while actin binding per se involves residues customized for each segment and for each protein.

Flightless I (fliI)¹ is a recently identified member of the gelsolin family (12). It was discovered as a mutation in Drosphila melanogaster that leads to flightlessness. This phenotype is accompanied by disorganization of the indirect flight muscle myofibrils (13). Other more severe fliI mutations lead to late larval or pupal death. Eggs lacking maternally supplied fliI show incomplete cellularization, abnormal furrow formation, and impaired gastrulation. Defective cellularization of the syncytial blastoderm is associated with a disorganized cortical actin cytoskeleton (14). The flightless and cellularization phenotypes suggest that fliI is required for actin organization during myogenesis and embryogenesis, respectively. The human flightless I (FLI) locus has been mapped to a region deleted in the Smith-Magenis syndrome (15), which is associated with a spectrum of developmental and behavioral abnormalities. The COOH-terminal half of human FLI has 31% identity and 52% similarity to human gelsolin (12), and has the same 6-fold segmental repeat typical of many gelsolin family members (Fig. 1A). Since FLI is more divergent from gelsolin than other gelsolin family members such as CapG, adseverin, and villin (1), it probably arose from the prototypical ancestral protein very early during phylogeny and evolved independently (16). Therefore, it is necessary to determine whether FLI is an actin-binding protein, and how it interacts with actin.

The NH₂-terminal half of FLI is distinct from that of the other previously identified gelsolin members. It contains 16 tandem 23-amino acid leucine-rich repeat motif (LRR) (12) (Fig. 1A) found in an emerging collection of proteins (17). Close to 100 proteins containing this motif have been identified thus far. Proteins in this LRR superfamily have diverse cellular localizations (extracellular, cytoplasmic, transmembrane, and nuclear) and functions (receptor ligand binding, signal transduction, cell adhesion, development, bacterial virulence, DNA repair, and RNA processing). The unifying theme among these diverse functions is molecular recognition. The LRR motif contributes to protein-protein interactions, either directly as the ligand binding module, or as a regulator to enhance affinity and/or specificity of binding to a separate ligand-binding site. In a few cases, the LRR binding partners have been identified. The ligand:LRR protein pairs include glycoprotein hormones:G-protein coupled receptors (18), collagen:matrix proteoglycans (such as decorin) (19), protein phosphatase-1:sds22 (required for the completion of mitosis) (20), neurotrophins:trk receptors (21), Ras:yeast adenylate cyclase (Cyr1) (22, 23), and pancreatic RNases:RNase inhibitor (24).

Among the LRR modules identified thus far, the FLI LRR 23-amino acid repeats fit the consensus for a LRR subgroup consisting of proteins which can potentially interact with Ras-like ligands (17, 25). This group includes Rsp-1 (also known as Rsu-1), which binds Raf-1 (26) and the yeast adenylate cyclase (Cyr1). Rsp-1 has 35% identity and 53% similarity to FLI. It binds Raf-1 (26) and suppresses the transformation activity of v-Ras (27). Cyr1 is regulated by Ras, and its LRR motif is required for membrane association and Ras binding (22). The similarity in LRR motifs raises the intriguing possibility that FLI may mediate interactions with Ras homologs (25). This is plausible because Ras transformation disrupts the actin cytoskeleton, and Rac1, a small G-protein which drives membrane ruffling (28), has been implicated in Ras transformation. Gelsolin severing and capping may be regulated by Rac1 (29), and gelsolin suppresses Ras-induced transformation in foci assays (30). We therefore used a variety of approaches to determine if FLI binds Ras or its downstream effectors. In addition, we tried to identify other FLI-LRR binding partners, to begin a molecular characterization of this novel member of the gelsolin and LRR superfamilies.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Plasmid Constructions-- The human FLI cDNA which was inserted into pBluescript SK() vector (Stratagene) through the EcoRI site, was kindly provided by H. D. Campbell (The Australian National University). This cDNA (12) is missing the AT nucleotides of the ATG initiation codon (GenBank accession U01184). To introduce a translation initiation codon, the cDNA was excised with EcoRV and SpeI in the multiple cloning region upstream and downstream of the EcoRI site, and subcloned into pGEM-5Zf(+) vector (Promega). The resultant clone contains a short fusion sequence 5' of the original fliI cDNA (5'-ccATGgccgcgggatatcgaattccgGAG-3') and was used for construction of all the yeast and mammalian FLI expression vectors described here. The FLI LRR domain construct was generated by SmaI digestion (nucleotide 1399), and encompasses amino acids 1-466. This construct extends past the end of the LRR repeat at amino acid 380 into part of the linker between the LRR- and gelsolin-like domains (Fig. 1A). A gelsolin-like domain construct which contains an initiation codon and spans FLI amino acid 496-1269 was constructed by introducing an initiation codon using polymerase chain reaction primers.

In Vitro Transcription and Translation-- Full-length and truncated FLI cDNA were cloned into the pTM1 vector (31). 1 µg of cDNA was added to the T7 polymerase-coupled reticulocyte lysate system (TNT, Promega) in the presence of Trans³⁵S-label (ICN Biomedical, Inc.), and in vitro transcription and translation were carried out in a 50-µl volume according to the manufacturer's protocol.

Actin-Sepharose Binding-- 2 and 4 µl of the in vitro transcribed and translated product was diluted to 80 µl with a buffer containing 2 mM Tris-HCl, 0.2 mM CaCl₂, and 0.1% gelatin, pH 7.5, and added to 20 µl of packed actin-Sepharose beads (32). The samples were incubated for 30 min at room temperature, and the beads were then washed three times in the same buffer. Proteins bound to the beads were analyzed by SDS-polyacrylamide gel electrophoresis, and radioactive bands were detected by autoradiography. In some samples, 4 µl of 3 mg/ml actin or bovine serum albumin was added to determine if binding to actin-Sepharose was reduced by competition with soluble actin.

Binding to Small GTPases-- GST-Ras, GST-Ras^Val-12, GST-RhoA, and GST-CDC42 (gifts of A. Hall, University College London and M. White, University of Texas Southwestern Medical Center) were expressed in BL21 and purified with glutathione-Sepharose (Pharmacia). They were charged with GTP or GDP. ³⁵S-Labeled FLI and LRR produced by in vitro transcription and translation were added to the beads and binding was determined as described (33).

Yeast Two-hybrid Screening-- The LexA based yeast two-hybrid system (34) was used initially to identify candidate FLI LRR interactive clones. The FLI LRR domain was fused in-frame to the 3'-end of the sequence encoding the LexA DNA-binding domain of the yeast two-hybrid vector pBTM116. The construct (pLEX-LRR) was used as a bait to screen a GAL4 activation domain-based human HeLa matchmaker cDNA library (pGAD-GH vector, CLONTECH). The yeast L40 strain (MATa trp1 leu2 his3 LYS2::lexA-His URA3::lexA-lacZ) was sequentially transformed, and potential interactors were identified as described previously (34). Approximately 1.6 million transformants were screened. Positive plasmids were isolated and tested against pLEX-lamin to rule out nonspecific interaction. Pairwise assays were also used to detect interaction with small GTPases (plasmids provided by M. White and J. Frost, University of Texas Southwestern Medical Center).

Northern Blotting-- ³²P-Labeled probes were synthesized with random primers by the Ready-To-Go DNA labeling kit (Pharmacia). Full-length FLI, H186 FLAP, and -actin cDNAs were used as templates. The premade poly(A) RNA human tissue blot, purchased from OriGene (Rockville, MD) was hybridized in buffer containing 0.2% SDS, 5 × SSPE, 5 × Denhardt's solution, 100 µg/ml denatured salmon sperm DNA, 50% formamide, 10% dextran sulfate at 42 °C for 14-16 h. The membrane was washed with 2 × SSC, 0.1% SDS for 5 min at room temperature 3 times, and with 0.25 × SSC, 0.1% SDS at 65 °C for 30 min 2 times. The membrane was exposed to x-ray film for 1 h to 4 days. Membrane was stripped between probes.

cDNA Cloning of FLI LRR-associated Protein (FLAP)-- A mouse I.M.A.G.E. consortium (LLNL) EST clone (ID 532888) whose 5' sequence (GenBank accession number AA068950) (36) is homologous to the two-hybrid human FLAP cDNA insert, was purchased from the ATCC. It was ³²P-labeled and used to screen a mouse skeletal muscle 5'-Stretch Plus gt11 cDNA library (CLONTECH). Approximately 8 × 10⁵ plaques were screened. The inserts in the positive plaques were amplified with the Expand Long Template polymerase chain reaction system (Boehringer Mannheim), using the gt11 LD-insert screening amplimer set (CLONTECH). The polymerase chain reaction products were cloned into the pGEM-T vector (Promega), and nucleotide sequences were determined by manual and automatic sequencing using external and internal primers.

Expression of Recombinant LRR-binding Partner and Antibody Production-- The H186 FLAP cDNA was released from the HeLa matchmaker vector pGAD GH with SpeI and XhoI. The insert was ligated to the bacterial expression vector pGEX-KG (37) digested with XbaI and XhoI. The resultant plasmid was transformed into DH5 (Life Technologies) or BLR (Novagen). Bacteria were grown to an OD₆₀₀ of 0.5 and GST-H186 FLAP fusion protein (GST-H186 FLAP) synthesis was induced with 0.5 mM isopropyl-1-thio--D-galactopyranoside for 3 h at 37 °C. The bacteria were lysed and fusion protein was purified with glutathione-Sepharose. Eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by staining with 0.3 M cupric chloride without fixation. The GST-H186 FLAP band was excised and used for rabbit immunization. The antibody from an early bleed, which recognizes H186 FLAP but not GST, was used for Western blotting.

Cell Culture and Transfection-- The FLI clone containing the inserted initiation codon (as described above) was further modified by attaching a COOH-terminal HA epitope tag (YPYDVPDYA). The construct was subcloned into pcDNA3 (Invitrogen) via the BamHI and XhoI sites in the multiple cloning region. H186 FLAP was cloned into pCMV5 (38). The expressed protein contains a 12-residue Myc tag (MEQKLISEEDLN) at the NH₂ terminus, 218 amino acids from H186 FLAP, and 31 residues of pCMV5 sequence acquired because H186 does not contain its own stop codon. The FLAP-1 cDNA has its own translation start and stop codons and was cloned into pcDNA3. DNA was transfected singly or in combination (at a 1:1 weight ratio) into human embryonic kidney 293 cells (HEK293) by calcium phosphate precipitation. Cells were cultured for 24-36 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

Immunoprecipitation-- Transfected cells (in 60 mm dishes) were labeled with Trans³⁵S-label (100 µCi/ml) for 2 h in methionine-free medium and lysed in 300 µl of RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 2 mM EGTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM sodium vanadate, 30 mM sodium pyrophosphate, 0.2 units/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 0.025% sodium azide). The lysate was centrifuged at 10,000 × g for 30 min. 30-50 µl of 12CA5 anti-HA hybridoma culture medium (gift of R. Gaynor, University of Texas Southwestern Medical Center) was added to 150 µl of the lysis supernatant. After incubation for 2 h at 4 °C, 20 µl of packed Protein A-Sepharose (Pharmacia) was added and incubation continued for 2 h at 4 °C with continuous rocking. Beads were washed twice with RIPA buffer, once with a buffer without detergent (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.025% sodium azide), and finally with 50 mM Tris-HCl, pH 6.8. They were boiled in SDS gel sample buffer with 4 M urea and analyzed by SDS-polyacrylamide gel electrophoresis, 7.5% acrylamide or 5-15% gradient acrylamide gels were used. Immunoprecipitated proteins were detected by Western blotting using the ECL system (Amersham) or by autoradiography.

	RESULTS

Top Abstract Introduction Procedures Results Discussion References

FLI Binding to Actin-- To determine if FLI is an actin-binding protein, FLI and the FLI LRR domain (Fig. 1A) were expressed by in vitro transcription and translation (Fig. 1B). In each case, a single in vitro transcribed product which migrated on a SDS-polyacrylamide gel with mobility consistent with the calculated molecular mass was generated (145 and 68 kDa, respectively, for FLI and LRR). The in vitro transcription products were incubated with actin-Sepharose in the presence and absence of excess soluble actin. FLI bound actin-Sepharose in a dose-dependent manner (Fig. 1C, left panel, lanes 1 and 2). Addition of actin monomers inhibited FLI binding to actin-Sepharose, as evidenced by the lack of FLI in the pellet, and its retention in the supernatant (compare lanes 3 with lanes 1). Densitometry scanning shows that doubling the amount of in vitro transcription product results in a 145% increase in FLI associated with actin-Sepharose (lanes 1 and 2) and addition of excess actin reduced FLI binding to actin-Sepharose to 16% (lanes 1 and 3). In contrast, bovine serum albumin did not decrease binding (data not shown). Thus, FLI bound actin specifically. This is not surprising, but until now, FLI has not been formally established as an actin-binding protein. In contrast to full-length FLI, in vitro transcribed and translated FLI LRR (Fig. 1B, right panel) did not bind actin-Sepharose (data not shown), suggesting that actin binding was mediated through the gelsolin-like domain.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   In vitro synthesis of FLI and LRR and binding to actin-Sepharose. A, the FLI domains. The NH2-terminal half has sixteen 23-amino acid leucine-rich repeats. The COOH-terminal half has six segmental repeats resembling that of the gelsolin family. GLD, gelsolin-like domains. a.a.#, amino acid residue number; nt#, nucleotide number. The LRR-HA construct used in this paper extends past the true LRR domain to residues 466. B, in vitro transcription and translation. 35S-Labeled FLI and LRR were synthesized in vitro, and the reaction products were analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. 2, 4, and 6 µl of FLI or LRR reaction products were loaded on the left and right panels, respectively. C, actin-Sepharose binding. Lanes 1 and 3, 2 µl of in vitro synthesized FLI, with and without soluble actin, respectively. Lane 2, 4 µl in vitro synthesized FLI. P, pelleted beads; S, supernatant. The entire pellet and one-fourth of the supernatant were analyzed. Densitometry scanning of the supernatants and pellets show that the combined amount of radioactivity recovered was 84, 204, and 92 units for lanes 1-3, respectively. The amounts of FLI bound to actin-Sepharose were 33, 20, and 5%, respectively, when expressed as percent of total input.

Lack of Interaction between FLI-LRR and Ras and Other Small GTPases-- The close resemblance between FLI-LRR with those of Cyr1 and Rsp-1 raises the possibility that FLI may also interact with Ras or its downstream effectors. This possibility was examined in two ways. First, two-hybrid pairwise assays were used to identify interactions in vivo. Second, recombinant Ras and Ras^Val-12, the consitutitvely active form of Ras, were expressed as fusion proteins with GST, and their binding to in vitro transcribed and translated LRR was determined by sedimenting GST fusion proteins with glutathione beads. Neither assay was able to detect evidence for Ras interaction with LRR. In the two-hybrid assay, LRR did not interact with Ras^Val-12 or Ras (data not shown), although Ras^Val-12 interacted with its known effectors, Ral GDS (39), Raf (34), and Cyr1 (34), under identical conditions. In addition, there was no evidence for LRR binding to other small G proteins, such as Rac2, RhoA, or CDC42 (data not shown). Likewise, we did not detect specific binding of in vitro synthesized LRR to GST-Ras^Val-12, GST-Ras, GST-CDC42, or GST-RhoA, either in the presence of GTP or GDP with the GST bead pull down assay (data not shown).

Identification of a Novel FLI LRR-binding Partner-- Two-hybrid screens using LRR fused with the LexA DNA-binding domain as bait yielded four interactive clones that did not bind the negative control, lamin. All of these clones have similar DNA sequences, and one (H186) was selected for further analysis. H186 survived additional stringent tests for specific interactions (Table I). H186 interacted with LRR regardless of whether it was fused to the LexA DNA-binding domain or the transcriptional activation domain. It did not bind lamin, and it still bound LRR when fused with the GAL4 DNA-binding domain instead of the LexA domain (Table I). In contrast, H186 did not bind the FLI gelsolin-like domain. We will call this protein H186 FLI LRR-associated protein (H186 FLAP).

View this table: [in this window] [in a new window]   Table I Two hybrid pairwise assays for FLI-LRR binding to FLAP Assays were performed using the LexA DNA-binding domain and GAL 4 activation domain, except in the bottom lane, where LRR fused to the GAL4 DNA-binding domain was assayed against the H186 FLAP-activation domain. FLAP-1 (R) indicates cloning in the antisense direction. Interaction was detected by the -galactosidase assay.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Co-immunoprecipitation of FLAP with FLI LRR. HEK293 cells were transfected with expression vectors for HA epitope tagged-LRR (LRR-HA), FLI-HA, H186 FLAP, or FLAP-1, either singly or in combination. P, immunoprecipitate; S, supernatant after immunoprecipitation. A, FLAP binding to LRR-HA. Transfected cells were 35S-labeled, lysed, and HA-tagged proteins were immunoprecipitated with anti-HA, and analyzed on a 5-15% gradient gel. Labeled proteins were detected by autoradiography. One-tenth of the amount of cell lysates used for immunoprecipitation was loaded in the supernatant lanes. Only H186 FLAP was expressed at sufficiently high level to be detected by the eye in the supernatants (right panel, lanes 4 and 5). B, FLAP-1 binding to LRR-HA. LRR-HA was immunoprecipitated with anti-HA, and Western blotting with anti-HA and anti-FLAP was used to detect LRR-HA and FLAP-1, respectively. Lane 1, cells transfected with FLAP-1; lane 2, LRR-HA and FLAP-1; lane 3, LRR-HA. LC, IgG light chain. The secondary antibody used to detect -HA also cross-reacted with the mouse IgG HC in the immunoprecipitate and LRR-HA comigrated with the IgG HC. The staining of IgG HC accounts for the appearance of a faint band in the lane 1 pellet, at a position overlapping with that of LRR-HA in lanes 2 and 3. C, Western blotting to show that FLAP-1 co-immunoprecipitated with FLI. Overexpressed proteins were immunoprecipitated with anti-HA or anti-FLAP, and the proteins in the immunoprecipitates were analyzed by gel electrophoresis in a 7.5% acrylamide gel. Immunoprecipitated proteins were identified by Western blotting. Lane 1, cells transfected with FLAP-1; lane 2, FLAP-1 and FLI-HA; lane 3, FLI-HA; lane 4, FLAP-1 and FLI-HA; lane 5, FLI-HA; lanes 6 and 7, supernatants of lanes 4 and 5, respectively.

Tissue Distribution of FLAP-- H186 FLAP is 0.65 kb and contains an open reading frame which contains a potential translation initiation codon but no termination codon. The translated amino acid sequence in this frame, or in the other two frames (which are interrupted by multiple stop codons), has no strong homology to known proteins in the GenBank data base. It matches mouse EST clone sequences AA068950 and AA106954 by BLAST search (40), except for the existence of two inserts in the latter sequence (Fig. 4C). These results raise the possibility that H186 FLAP is not full-length, and that there may be multiple splice variants or multiple FLAP genes.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Northern blot analyses of FLAP and FLI distributions in human tissues. A human tissue poly(A) RNA blot was probed with FLI (A), H186 FLAP (B), and -actin (C) cDNAs sequentially. The blot was stripped between hybridization with each probe. Two exposures of the actin blot were shown. The size of RNA standards are indicated in kb.

cDNA Cloning of Mouse FLAP-- To determine the sequence of full-length FLAP, a mouse skeletal muscle cDNA library was screened with a mouse embryonic carcinoma EST clone (GenBank sequence number AA068950) which has extensive homology to the human H186 FLAP clone (Fig. 4C). Phage clones with inserts ranging from 0.9 to 3 kb were identified. The 2.7-kb clone (called FLAP-1) contains an open reading frame which encodes for a protein of 628 amino acids (Fig. 4A). The shorter clone (FLAP-1a, 1.6 kb) overlaps with the first clone and extends the 5'-untranslated sequence by 11 base pairs (Fig. 4C). The 5'-untranslated sequence of the mouse cDNA and mouse embryonic carcinoma EST clones are identical to each other, but not to human H186 FLAP. In contrast, the coding sequences of the mouse skeletal muscle and human HeLa H186 FLAP clones are highly homologous (95% identity, 100% similarity at the amino acid level), except for the existence of a 148-amino acid insert (residues 52-199) and a 24-amino acid insert (residues 253-276) in the former. Since these inserts are found in two independently isolated muscle cDNA clones, they are unlikely to result from cloning artifacts. Furthermore, parts of the first insert and the entire second insert are found in another mouse embryonic carcinoma EST clone (sequence AA106954) and in a human skeletal muscle EST clone (sequence AA180174), respectively. The existence of several cDNA inserts and multiple mRNA strongly indicate that there are tissue and/or species-specific alternative splice variants.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Sequence analyses of mouse skeletal muscle FLAP-1. A, predicted amino acid sequence. The predicted coiled coil regions are shown in bold. Inserts found in the mouse skeletal muscle cDNA clones but not in human H186 FLAP are underlined. The nucleotide sequence is deposited in GenBank (accession number AF045573). B, predicted location of the coiled coil regions, with the PAIRCOIL program (43). A score of above 0.5 indicates high probability of coiled coil structure. C, alignment of mouse skeletal muscle FLAP-1 cDNA clones with H186-FLAP and EST clones. FLAP-1 and FLAP-1a were isolated from a mouse skeletal muscle cDNA library. H186-FLAP is the human HeLa LRR-interactive plasmid identified by two-hybrid screens. Sequences AA06895 and AA106954 are derived from mouse embryonic carcinoma EST clones. Sequence AA180174 is derived from a human skeletal muscle EST clone. Solid bars, sequenced regions. *, initiation codon; ×, termination codon. Lines linking solid bars denote deleted regions compared with FLAP-1. Shaded bar, 5'-untranslated region of the human H186 FLAP which diverges from the mouse sequences.

Evidence for the Binding of Full-length FLAP-1 to FLI-- FLAP-1 fused to the LexA DNA-binding domain interacted with FLI-LRR in the two-hybrid assay to an extent comparable to that of H186 FLAP (Fig. 5) (Table II). In contrast, FLAP-1 cloned in the antisense orientation was not positive.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Interaction of FLAP-1 with FLI LRR in the two-hybrid system. The LexA based yeast two-hybrid system was used. DBD, DNA-binding domain; AD, activation domain. FLAP-1(R), cloned into the vector in the reverse (antisense) orientation and lamin are used as negative controls. Four randomly selected clones from each transformation were shown.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

FLI is unique among the members of the LRR and gelsolin superfamilies. Although many LRR proteins contain additional modules implicated in molecular recognition (17), such as epidermal growth factor repeats, immunoglobulin domains, G-protein coupled receptors, and leucine zippers, FLI is the first actin-binding LRR protein to be identified. FLI is also unusual in the gelsolin family, because it is the first example of the bridging of a motif with no known actin binding function to gelsolin proper. Villin is the only other gelsolin family protein with an extension identified thus far, but its extension binds actin and is much shorter (46). The novel juxtaposition of the LRR tandem repeats with the gelsolin 6-fold repeats suggests that FLI may link the actin cytoskeleton to other structures in the cell in a far more direct manner than has been envisioned for the more traditional gelsolin-like proteins. The membrane signaling systems are favored candidates for interaction with FLI, because many LRR proteins and actin regulatory proteins are involved in signal transduction and adhesion, and the FLI LRR repeats best fit the consensus found in several Ras interactive proteins (17, 25).

In this paper, we demonstrate that FLI binds actin, establishing it as a bona fide member of the gelsolin family. This is expected because of its homology to gelsolin, and is consistent with the effects of fliI mutations on actin organization in Drosophila embryos and flight muscles. Nevertheless, interaction with actin cannot be assumed a priori because FLI is far more divergent than the other traditional members of the gelsolin family. Although a comparison of FLI and gelsolin suggests that residues essential for the structural integrity the S1 core are preserved in FLI S1 (7, 47), some of the other FLI segments have unique insertions (5, 12). FLI binding to actin was demonstrated by using in vitro synthesized FLI. This assay has been used to identify proteins which bind monomeric actin with high affinity. Most gelsolin family members bind actin-Sepharose (48), although CapG does not (11). Therefore, FLI is likely to be bind actin with high affinity. It will be important to determine whether FLI caps and severs actin filaments like other gelsolin family proteins. These issues cannot be addressed at present because we cannot isolate soluble recombinant FLI.² Preliminary results suggest that endogenous FLI is a low abundance protein and we have not purified enough to allow functional characterizations.

Despite the resemblance of the FLI LRR to the LRR of known Ras effectors such as Cyr1 and Rsp-1, we were unable to demonstrate FLI LRR binding to Ras. FLI LRR also did not bind several Ras downstream effectors, and other Ras-related small G proteins. These proteins are, therefore, unlikely to be high affinity FLI interactive partners, although we cannot rule out low affinity binding or linkage through other interactive proteins. The negative result is not surprising, because the LRR motif is probably used to present a platform for interaction with other proteins, while binding specificity is dictated by the nonconsensus amino acids in the repeats. Furthermore, FLI does not have the same numbers of LRR repeats as Cyr1 and Rsp1 (16, 24, and 7 repeats, respectively) and it is flanked by unique sequences as well.

We identified a novel FLI LRR interactive partner, FLAP, by two-hybrid screens, and confirmed its specific binding to FLI by immunoprecipitation. Northern blotting and cDNA analyses indicate that there are multiple FLAP messages, which are most likely generated through differential splicing. Skeletal and cardiac muscles have high level expression of the 3.3-kb FLAP mRNA and the FLI mRNA as well, lending further support to the possibility that FLAP and FLI are in vivo partners. FLAP is predicted to form coiled coils at its middle and COOH-terminal regions. -Helical coiled coils are the most common assembly motif in proteins and provide the potential for homotypic or heterotypic interactions (44, 49). The FLAP coiled coils are predicted to have a high tendency for dimerization, in a manner similar to that of well characterized cytoskeletal proteins like tropomyosins, myosins, and kinesins. However, FLI has fewer heptads and may not assemble into a fibrous structure. On the other hand, short coiled coils have been identified in many other proteins and they participate in important heterotypic interactions. These include the cyclic GMP-dependent protein kinase, the dimer of heterotrimeric G proteins, and the basic leucine zippers of certain transcription factors (reviewed in Ref. 49).

In analogy, we hypothesize that the FLAP coiled coils bind FLI LRR. While this has not been established directly, it is supported by the finding that both H186 FLAP and FLAP-1 bind LRR, even though the former has a large deletion in the NH₂-terminal region upstream of the coiled coil domain. In this context, it should be noted that there is precedent for LRR protein interaction with -helical structures. The best example is the binding of decorin, a small proteoglycan with eleven 24-amino acid LRR repeats, to the triple helix of collagen (50). How decorin affects collagen fibrillogenesis is predicted by molecular modeling based on the crystal structure of the RNase inhibitor (51, 52). The entire RNase inhibitor molecule is composed of 15 tandem alternating 28- and 29-residue LRR repeats with alternating short -strands and -helices parallel to a common axis. This symmetrical arrangement folds into an open, nonglobular protein with a horseshoe shape, which is lined by -strands in its inner surface and -helices on its outer surface. RNase inhibitor inhibits RNase, and the crystal structure of the complex shows that RNase binds to a broad region in the concave face (52). Decorin, which has fewer and shorter repeats than RNase inhibitor, is predicted to fold into an arch shape with an internal cavity just large enough to accommodate a single triple helical collagen molecule (50). Decorin therefore acts a spacer to prevent lateral fusion of collagen fibrils and to guide collagen fibril assembly (50). In analogy, the FLAP coiled coil may insert into the concave FLI LRR binding surface.

The identification of FLI as an actin-binding protein and the discovery of a novel coiled coil ligand for the its LRR domain suggests that FLI is a linkage protein between the cytoskeleton and an as yet unidentified structure in the cell. Based on the intimate connections between the cytoskeleton and the plasma membrane, we favor the possibility that FLAP is part of the membrane cytoskeleton.