Characterization of EHD4, an EH Domain-containing Protein Expressed in the Extracellular Matrix*

To identify proteins that promote assembly of type VI collagen tetramers or stabilize type VI collagen filaments, a two-hybrid screen of a human placenta library was used and a new extracellular protein discovered. The cDNA sequence of the new protein encodes 541 amino acid residues. This cDNA sequence is identical to EHD4, a recently described member of the EH domain family of proteins. Two mRNAs of 4.4 and 3.0 kilobases were present in human skin fibroblasts and most tissues tested but were most prevalent in the heart. The chromosomal localization of the gene for this new protein was determined to be at 15q14-q15. Three polyclonal peptide antibodies were made against synthetic EHD4 peptides. The affinity-purified antibodies were used in immunofluorescent staining of developing limbs and matrices produced by human skin fibroblasts and mouse NIH3T3 fibroblasts in culture. Embryonic rat limb cartilage was strongly stained throughout development, and cultured fibroblasts deposited an extracellular filamentous network containing EHD4. In non-denaturing extracts of fetal bovine cartilage and in human skin fibroblast culture media, two components of ∼220 and 158 kDa were observed, which, after reduction, migrated as a 56-kDa component on SDS-polyacrylamide gel electrophoresis. EHD4 is the first extracellular matrix protein described that contains an EH domain.

The EH domain (Eps15 Homology) 1 was first defined by Wong et al. (1) when characterizing Eps15, a substrate for the epidermal growth factor receptor tyrosine kinase. Subsequently, screening a human fibroblast expression library with EH domains identified several proteins that contained one or more copies of this motif (2). The EH domain contains 80 -100 amino acid residues with 60 residues of consensus sequence and over 50% identity to one another. This domain usually contains a predicted calcium binding EF hand motif. A combinatorial phage display library was used to determine the target sequences that bind to the EH domains of Eps15 and Eps15R, which led to the identification of the tripeptide Asn-Pro-Phe as the optimal ligand (2,3). Some EH domains in other proteins recognized other motifs containing aromatic and hydrophobic amino acids such as Phe-Trp and Trp-Trp (3,4). They are responsible for protein-protein interactions (1,5) and participate in receptor-mediated endocytosis (6) and signaling pathways (7). To date, more than 40 EH domain-containing proteins have been reported (8). They are intracellular proteins found in organisms ranging from yeast to mammals, most of which have multiple EH domains located in their amino termini, adjacent to a coiled-coil domain.
Recently, an EH protein family member called EHD1 (Eps15 Homology Domain 1) was described (9). This protein was originally called Hpast (GenBank™ accession number AF001434), because it was the human analogue of a Drosophila protein called PAST (putative achaete scute target, GenBank™ accession number U70135). EHD1 is a slightly atypical family member, because it contains a single EH domain at the carboxylterminal end of a coiled-coil domain and is the only coiled-coilcontaining member with a single EH domain. Other than a calcium binding EF hand within the EH domain, no other recognizable domains were found in this 62-kDa protein. From Northern blot analysis it was highly expressed in testis, intestine, spleen, and kidney. Immunochemical analysis of mouse embryos showed that EHD1 was expressed in cartilage of the ribs and spinal column at day 15.5 postcoitus. Whole mount in situ hybridization showed that EDH1 expression could be detected in limb buds and pharyngeal arches by day 9.5, in limb buds, sclerotomes, branchial arches, and occipital regions at day 10.5, but at day 17.5 there was no expression in the bones. Cellular localization experiments found EHD1 in several cytoplasmic vesicular structures, including the Golgi apparatus and endocytic vesicles. It is expressed by mesenchymal-derived cells or germ cells that are known to be induced by IGF1. Because of the structural similarities of EHD1 and Eps15, it was speculated that EHD1 is an insulin-like growth factor receptor substrate that mediates the endocytosis of the IGF1 receptor (9).
Exon-trapping experiments identified three new human genes designated EHD2, EHD3, and EHD4 because of their strong similarity to EHD1 (10). The sequence of EHD3 was subsequently corrected in an EMBL/GenBank™ submission (accession number NM014600). The levels of amino acid identity between EHD1 and EHD2, EHD1 and EHD3, and EHD1 and EHD4 are 71%, 86%, and 76%, respectively. They show distinct expression patterns on multiple-tissue Northern blot analysis. Comparison of the amino acid sequences of EHD1-4 revealed almost identical single EH domains containing a predicted calcium binding EF hand. In addition, a bipartite nuclear localization signal and an ATP/GTP binding motif were identified. Surprisingly, no mention was made of the putative coiled-coil region previously identified in EHD1.
We identified EHD4 during a search for proteins potentially involved in type VI collagen filament formation. Type VI collagen filaments are formed by end-to-end association of tetra-mers (11,12). Attempts to reconstitute filaments in vitro from purified type VI collagen tetramers failed, so we proposed that other ligands might be required for type VI collagen fibrillogenesis (12). To find proteins that are potentially involved in type VI collagen filament assembly, the yeast two-hybrid system was used with selected amino-and carboxyl-terminal domains of each ␣(VI) chain to screen a human placenta library (13). EHD4 was found using a bait peptide composed of most of C1 and C2-C5 domains of the ␣3(VI) carboxyl terminus (14). Here we report the complete cDNA sequence of EHD4 and its characterization as an extracellular protein.

EXPERIMENTAL PROCEDURES
Materials-Bovine fetuses were obtained from the local slaughterhouse. A Superscript preamplification system for first-strand cDNA synthesis and the polymerase chain reaction (PCR) kits were from Life Technologies. The Micropoly(A) Pure kit and the Northern Max-Gly kits were from Ambion. The Multiple tissue Northern blot and Protein Medleys (human heart, skeletal muscle, liver, lung, and placenta) were purchased from CLONTECH Laboratories, Inc. The QIAprep spin kit and Wizard PCR preparation kit were from Qiagen and Promega, respectively. Type II collagen monoclonal antibody II6B3 developed by T. F. Linsenmayer was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health and maintained by the Department of Biological Sciences (University of Iowa, Iowa City, IA). Type X (15) and type VI collagen antibodies have been described elsewhere (16). Secondary antibodies used, conjugated with fluorescein or Texas red were from (Sigma Chemical Co.) and Alexa 546 and Alexa 488 from Molecular Probes. The synthetic adjuvant Titermax was from Sigma. The Supersignal substrate for Western blot, N-hydroxysuccinimide-long chainbiotin, Extravidin, the Imject activated immunogen conjugation kits, and the SulfoLink kit were purchased from Pierce Co. The human chondrocyte library was a gift from Dr. K. Doege (Shriners Hospital, Tampa, Fl). The human heart library was purchased from Origene Co. The primer sets and polypeptides for antibody preparations were synthesize by the core facility of Shriners Hospital in Portland. Normal human skin fibroblasts and mouse NIH3T3 cells were from the American Type Culture Collection.
cDNA Sequencing-The isolation of an EHD4 clone (3C-110), containing a 5Ј-ORF of 294 bp and 3Ј-UTR without the poly(A) tail, from a two-hybrid screen of a human placenta library has been previously described (13). The 5Ј-end of the sequence was extended by an additional 877 bp by screening a human heart library using the 3Ј-UTR as a probe and a human chondrocyte library using PCR amplification (94°C, 10 min 3 40 cycles (94°C, 30 s; 55°C, 30 s; 72°C, 60 s) 3 72°C, 7 min 3 4°C) with the primer set designed from gt11 phage (sense: AAGGCACATGGCTGAATATCGACG) and the known sequence (antisense: TCTAGTTTCCAGTGTCCACCCTCCCCAT; Fig. 1, position 1753-1780). The 459 bp of cDNA at the 5Ј-end, which included the initiation codon, was retrieved from the High Throughput Gene Sequences data bank by using the Advanced BLAST Search (www.ncbi.nlm.nih.gov/BLAST). This sequence was checked by sequencing the product from a PCR amplification of human fibroblast cDNA using the primer set as follows: sense: ATGTTCAGCTGGATGGGGCG, position 1-20; and antisense: CCAGTGTCCACCCTCCCCATTCTACAG, position 1755-1772 (Fig. 1).
Radiation Hybrid Mapping-Chromosomal localization of EHD4 was performed using the GeneBridge 4 radiation hybrid panel (Research Genetics, Inc.) according to the protocol provided with the panel. The PCR primer sets used in the screening were designed from the open reading frame (sense: GGCCAAGCACCTCATCAAGATCAAGCTC, position 1518 -1545) and the 3Ј-UTR (antisense: ATCGGGGCACTGG-GATGGAGGCACGC, position 2261-2286). This pair of primers generated a cDNA fragment of 767 bp. The results from screening all 93 templates in the hybrid panel were analyzed by the White Head Institute (www.genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl).
Polyclonal Antibody Preparation-Three peptides for antibody preparation, selected from the translated amino acid sequence of EHD4 were synthesized with an addition cysteine residue at the carboxyl terminus. The polyclonal antibodies were prepared in New Zealand White rabbits. The peptide sequences are QDLFRDIQSLPQ-KAAVRKLNDC (Fig. 1, 279 -299 for pAb 1050), GTTEGPFNQGYGE-GAKEGADEEEWVVAKDKPVYDEC (Fig. 1, 418 -453 for pAb 1051), and GKISGVNAKKEMVTSKLC (Fig. 1, 463-479 for pAb 1306). All the peptides were purified by high performance liquid chromatography on a C18 reverse phase support in 0.01% trifluoroacetic acid using an acetonitrile gradient, and their sequences and purity were checked by direct amino acid sequencing (Applied Biosystems 492) before use for antibody preparations. The peptides were linked to keyhole limpet hemocyanin via their carboxyl-terminal cysteine using the Imject activated immunogen kit. Each injection contained 100 -200 g of purified peptide mixed with an equal volume of Titemax classic adjuvant according to the manufacturer's instruction. A test bleed from each rabbit was collected after the initial injection and two consecutive boosts at 2-week intervals. The sera were purified over an antigen-linked agarose column, which was prepared according to the SulfoLink kit description. The titer of each antibody was checked using peptide-coated enzymelinked immunosorbent assay plates, and their ability to recognize native protein by indirect immunofluorescent staining of human or mouse frozen skin sections.
Protein Preparation-Bovine fetal femur cartilage pieces were collected, frozen in liquid nitrogen, and ground to powder at Ϫ20°C. They were extracted overnight with 3 volumes (v/w) of ice-cold 20 mM Tris-HCl buffer, pH 7.5, containing 0.2 M NaCl and the protease inhibitors 10 mM N-ethylmaleimide, 5 mM benzamidine, 10 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin, and 0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride. The tissue residue was collected by low speed centrifugation and then washed twice with the same buffer containing inhibitors and 1.0 M NaCl, followed by overnight extraction with the same buffer containing inhibitors, 1.0 M NaCl, and 20 mM EDTA. The clarified EDTA extract was dialyzed against 50 mM Tris-HCl buffer, pH 8.3, containing 0.05 M NaCl and 2 M urea. The proteoglycan in the EDTA extract was removed using DEAE anion-exchange column chromatography. The column was equilibrated in the dialysis buffer and eluted with a 1-liter NaCl gradient from 0.1 to 0.3 M at room temperature. The EHD4-containing eluate from the anion-exchange column was further purified over a Sephacryl S-400 column (2.6 ϫ 110 cm) equilibrated in 40 mM Tris-HCl, pH 6.8, buffer containing 2 M urea and 0.2 M sodium sulfate.
Cell Culture-Normal human skin fibroblasts and mouse NIH3T3 fibroblasts were grown in eight-well chamber slides (10 3 cells/chamber) with Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 50 g of ascorbate for 3-5 days.
Indirect Immunofluorescent Staining-The chamber slides with confluent human skin fibroblasts or mouse NIH3T3 cells were treated with the ice-cold acetone for 2 min, air dried, and washed with phosphatebuffered saline before immunofluorescent staining with antibodies using standard procedures. The frozen tissue sections (5 m) from different stages of rat embryo forelimb development were treated with acetone for 10 min and washed with phosphate-buffered saline before staining. Primary antibodies used were polyclonal antibodies against EHD4, type VI collagen, and type X collagen and monoclonal antibody against type II collagen. Negative controls used were the IgG fraction isolated from pre-immune rabbit sera and immune sera pre-treated with the peptide (1 mM) used for immunizing the rabbit.
Northern Blots and Affinity Blot-Multiple tissue Northern blots were done as described in the protocol from CLONTECH using 32 Plabeled 3C-110 as a probe, which consisted of 294 bp of EHD4 ORF and the entire 3Ј-UTR without the poly(A) tail. The mRNA of human skin fibroblasts and the fibroblast Northern blot were prepared according to protocols from the Micropoly(A) Pure kit and the Northern Max-Gly kit, respectively. The affinity blot procedure, using biotinylated type VI collagen as a soluble ligand, has been previously described (17).

Sequence Determination and Domain Analysis-
The entire assembled cDNA sequence of EHD4 and peptide translation, including the complete 3Ј-UTR is illustrated in Fig. 1A. Sequence analysis of clone 3C-110 revealed a short coding region of 294 bp which encodes 98 amino acid residues containing an EF hand calcium binding motif, followed by a 3Ј-untranslated region (Fig. 1, 1339 -2872). Most of the 5Ј-ORF sequence was found in the human cartilage library (644 -1338), and the sequence from positions 322 to 643 was from the human heart library. The 5Ј-UTR, including the first 321 bases of coding sequence, was first obtained from a genomic clone in the High Throughput Gene Sequences data bank (accession number AC039056); however, upon checking, the sequence was found to have one codon missing and two incorrect bases. The poly(A)tail was from the Expressed Sequence Tag data bank (AI341440, reverse). During the course of these investigations, a new group of EH domain-containing protein sequences were entered into the EMBL/GenBank™ data base that were highly similar to EHD1 and were consequently named EHD2, EHD3, and EHD4 (lacking part of the ORF and UTR) (10). EHD4 was identical to the protein described here; therefore, the name EHD4 was adopted. The completed EHD4 cDNA sequence reported here consists of an open reading frame of 1620 bp with the proposed translation-initiating methionine in the strong Kozak consensus sequence context RNNatgY (18). Chromosomal localization of the EDH4 gene on the physical map of chromosome 15 is illustrated in Fig. 2. Nearby markers WI-13757 and WI-11636 have been positioned on the radiation hybrid map enabling EDH4 to be assigned to 15q14-q15.
The domain analysis of the EHD4 protein amino acid sequence using the Simple Modular Architecture Research Tool (SMART; smart.embl-heidelberg.de) revealed a unique aminoterminal domain, a potential coiled-coil domain, and an EH domain with an embedded EF hand calcium binding motif (Fig.  1B). EHD4 contains four cysteine residues, two in the aminoterminal region and two in the calcium binding EF hand motif. Compared with the other EHD proteins, EDH4 has a low score for coiled-coil domain formation using the programs COILS (www.ch.embnet.org/software/COILS_form.html) and Multicoil (nightingale.lcs.mit.edu/cgi-bin/multicoil). There is a predicted ATP/GTP binding site at the amino terminus and a possible bipartite nuclear recognition site in the center of a coiled-coil domain (PSORT program analysis psort.nibb.ac.jp). No signal peptide at the amino terminus was detected (SignalP program, www.cbs.dtu.dk/services/SignalP).
To determine the tissue distribution of the gene, a multiple tissue Northern blot and human skin fibroblast mRNA were probed using the cDNA insert of clone 3C-110. Two messages of 4.4 and 3.0 kb were detected, the smaller of the two being more abundant in all tested tissues and fibroblasts (Fig. 3). Heart had the highest message level followed by pancreas, kidney, placenta, and lung. Fibroblasts also expressed high levels of message.
Immunolocalization of EHD4 -To identify and characterize EDH4 protein in tissues, three polyclonal peptide antibodies were made against peptides selected from the coiled-coil do- FIG. 1. A, the complete assembled cDNA and translated amino acid sequences of EHD4. Nucleotide sequence is above the corresponding amino acid sequence, and the 5Ј sequence obtained from GenBank™ is in lowercase letters. Numbering for the nucleotide sequence is shown to the left and for the amino acid sequence to the right. The EH domain sequence is shaded, and the embedded EF hand motif is in parenthesis.  Fig. 1A. All antibodies recognize the same bands in Western blots of bovine cartilage extracts and fibroblast culture medium (see below). Immunofluorescent staining of fibroblasts culture matrix and tissues was also identical with each antibody (not shown).
In 18-day-old rat embryo limb sections, EHD4 is confined to the cartilage matrix and co-distributed with type II collagen in the extracellular matrix except at the articular surface. Fig. 4  (A and D) shows the similar distributions of EHD4 and type II, respectively, in the elbow. Fig. 4B shows an enlargement of the ulna/humerus stained with EHD4, and Fig. 4C shows the same view stained with EHD4 antibodies preincubated with the peptide used to make the antibody. This negative control demonstrates the specificity of the observed staining. At the articular surface, there is a narrow zone that contains type II collagen, but EHD4 is absent. Enlargements of the ulner/humerus interface are shown in Fig. 4, E and F. In Fig. 4E, EHD4 is stained green and the cell nuclei red, and at the surface of the cartilage there is a region delineated by the presence of cell nuclei that does not contain EHD4. However, this same region is stained for type II collagen (Fig. 4F). Earlier in development at day 14, when EHD4 first appears, EHD4 and type II collagen do not co-distribute. In the clavicle, type II staining delineates the whole bone (Fig. 4H), whereas EHD4 is restricted more to the mid-diaphysis where chondrocytes differentiation is more advanced and where primary endochondral ossification will ultimately initiate (Fig. 4G). It appears while type II collagen is produced early in the differentiation of the condensing mesenchyme to chondroblasts, and EHD4 is produced slightly later in development by more differentiated chondrocytes. The next change in distribution observed occurs just prior to the formation of the secondary centers of ossification. Fig. 4I shows a bone epiphysis 5 days postpartum in which EHD4 labeling is diminished in the center but type II collagen staining is still uniform (Fig. 4L). Type X collagen, which is a marker for hypertrophic chondrocytes and endochondral ossification, is not expressed in this region (Fig. 4J), only in the metaphyseal growth plates. At this stage type VI collagen is concentrated at the articular surface of the epiphysial cartilage (Fig. 4K). Two days later, type X collagen appears in the secondary center of ossification (Fig. 4N). A large area devoid of EHD4 (Fig. 4M) still labels for type II collagen (Fig. 4P). It has a course appearance indicating a physical change in the structure of the cartilage matrix caused by the hypertrophy of chondrocytes and degradation of cartilage prior to calcification. Type VI collagen remains concentrated at the epiphysial surface (Fig. 4O) while EHD4 continues to be excluded from the developing articular cartilage.
Characterization of EDH4 Protein-Because the Northern blot analysis indicated that EHD4 was expressed by fibroblasts, cultures of normal human skin fibroblasts and mouse NIH3T3 fibroblasts were grown to confluence and immunofluorescently labeled to better determine whether EHD4 is an extracellular or an intracellular protein. The proteins in the fibroblast culture medium and cell/matrix extracts were analyzed by Western blot analysis, and the distribution of EHD4 was examined by indirect immunofluorescent staining of fibroblasts grown on chamber slides. The results demonstrated that fibroblasts secreted EHD4 into the media (Fig. 5A). Western blot analysis with an affinity-purified antibody showed disulfide-linked EHD4 oligomers of 158 and 220 kDa were present in unreduced media and a monomer of 56 kDa after reduction. From the apparent molecular masses it would appear that the oligomers are trimers and tetramers, but, owing to the anomalous behavior of these proteins in SDS-PAGE, this is not certain. Two-dimensional gel electrophoresis revealed that both high molecular mass bands are reduced to a 56-kDa band recognized by an EHD4 antibody (data not shown). Therefore, it was concluded that both components are oligomers of EHD4. Furthermore, both mouse and human fibroblast cell cultures deposited an extracellular fibrillar network containing EHD4 (Fig. 5B).
To chemically confirm the presence of EHD4 in cartilage, fetal bovine femoral cartilage was extracted with a series of buffers with increasing matrix-solubilizing properties. It was found that most EHD4 was extracted in 20 mM Tris-HCl, pH 7.5, containing 1 M NaCl and 20 mM EDTA (Fig. 6). The bands recognized by EHD4 antibodies in low salt and high salt-containing Tris buffer without EDTA were thought to be degradation products of EHD4, because they varied in pattern and intensity from one preparation to the next (Fig. 6B). EHD4 protein was purified from the EDTA extract by separation on DEAE-cellulose. Bound material was further chromatographed over a Sephacryl S-400 column (Fig. 7). The apparent sizes of EHD4 protein isolated from culture media and cartilage are identical.
Interactions with Collagens-The affinity of EHD4 for type VI collagen was confirmed by affinity blots of fetal bovine cartilage extract with biotinylated type VI collagen tetramers (Fig. 8). The results showed that the type VI collagen tetramers and EHD4 polyclonal antibody (pAb 1050) recognized the same 158-and 220-kDa components in the gel. Further binding studies (not shown) using EHD4 immobilized on enzyme-linked immunosorbent assay plates and fragments of type VI collagen demonstrated that the binding site was localized in the globular domain of type VI collagen and that the native domain is capable of binding EHD4. Similar assays showed that collagens I, II, III, and V had no affinity for EHD4. Thus, type VI collagen and EHD4 have an affinity for each other in vitro, which confirms the interaction detected in the two-hybrid screen.
As type VI collagen filaments formed in cultures of WI38, HT1080, and MG63 cells in the absence of EHD4 (not shown), EHD4 is not required for type VI collagen formation. Furthermore, the distributions of EHD4 and type VI collagen in cartilage are quite distinct, type VI collagen being concentrated at the periphery of epiphysial cartilage where EHD4 is at its weakest or absent. However, there is a low concentration of type VI collagen in the inter-territorial cartilage matrix (19) where EHD4 is also found, and so an interaction is possible there.

DISCUSSION
EHD4 is a unique extracellular matrix protein in that it contains an EH domain with an embedded calcium binding EF hand. These are characteristics of a family of EH domaincontaining proteins related to Eps15 that were previously thought to be exclusively intracellular. The four EHD proteins are a subset of this family with high sequence similarity. EHD4 was previously assumed to be an intracellular protein because of its structural similarity to EHD1. However, EHD4 has some unique characteristics that make this comparison unreliable. EHD1-3 have a predicted coiled-coil region, presumably used to assemble multimers in a similar manner to Eps15 (20). EHD4 has a very low score for a predicted coiled-coil region but, as we have shown, does assemble into disulfide-bonded multimers in tissues. Because the cysteine residues are not in, or immediately adjacent to, the predicted coiled-coil domain the interaction surfaces for association must involve other regions of the molecule. In addition, PSORT analyses reveal two conserved nuclear targeting sequences in the amino and carboxyl termini of EHD1-3, which are not present in EHD4.
All EHD proteins contain one conserved cysteine residue in the amino-terminal region of the molecules corresponding to C141 of EHD4 (Fig. 1B). EHD2 has a second cysteine located in the coiled-coil domain. EHD4 contains four cysteine residues, two in the calcium binding EF hand, which is rare in this motif (21), and two in the amino-terminal region (Fig. 1B). The third EH domain of Eps15 also contains an EF hand with two cysteine residues, one replacing a Ca 2ϩ -ligating Asp residue, which consequently does not bind calcium (22). The location of the two cysteine residues in the EF hand of EHD4, adjacent to the Asp residues involved in the ligation of calcium, is unique. Because they are in the sequence -Asp-Cys-Asp-Cys-Asp-, they cannot form an intramolecular disulfide cross-link. They are probably involved in the intermolecular disulfide bond forma-tion of polymers by interacting with adjacent EH domains. The calcium binding properties of this domain are unknown, but extraction of EHD4 from cartilage under non-denaturing conditions requires EDTA, indicating a metal ion-dependent interaction with the matrix.
Other notable structural features of the EHD proteins are that they contain a predicted bipartite nuclear localization signal, an ATP/GTP binding domain, and no signal sequence, which are characteristics of intracellular proteins (10). Although unusual, there are several secreted proteins without signal peptides, including fibroblast growth factor (23), interleukin-1 (24), galectins (25), thirodoxin (26), and annexin I (27). ATP/GTP binding domains are also present in extracellular bone and dentin proteins (28) and tubulointerstitial nephritis antigen (22), so the presence of these sequence motifs cannot be used to infer intracellular localization.
The genes for human EHD1-4 were localized to chromosomes 11q13, 19q13.3, 2q21, and 15q11.1, respectively (10). Here we report the localization of EHD4 to be 15q14-q15. This discrepancy may result from using different radiation hybrid panels in the assays. However, the recent deposition of chromosome 15q15 genomic sequences into the data bank, which includes the entire sequence of EHD4, supports the latter localization (GenBank™ accession number AC039056).
Because of the high similarity of the EHD1-4 protein sequences, distribution data reported for EHD1 probably represents the sum of the distributions of EHD1-4, because the antigen used to make the antibody was full-length recombinant EHD1 protein. The regions used as epitopes to make the polyclonal antibodies described here are also similar in EHD1-4. Although the EHD2 and EHD3 proteins have not been characterized, their message levels were high in skeletal muscle and brain (10). The fact that the antibodies against EHD4 are negative on both of these tissues suggests that there is no antibody cross-reactivity with EHD2 and EHD3. In addition, recombinant EHD4 expressed in a cell-free rabbit reticulocyte system was recognized in Western blots by the three EHD4 antibodies, whereas EHD3 was not (not shown). It was also reported from in situ hybridization results that EHD1 expression in developing mouse limbs peaked at day 15.5 in cartilage, preceding hypertrophy and ossification. However, EHD4 was found in the cartilage matrix of developing mouse limbs from day 14 up to postnatal day 7 indicating the antibodies were not recognizing EHD1. Furthermore, because EHD1 contains only one cysteine, it can only be monomeric or form covalently linked dimers. Neither of these was observed in Western blots of the EDTA extracts of bovine cartilage.
The EH domain is a protein-protein interaction motif and binds primarily to Asn-Pro-Phe (type I) sequences in target proteins (3,8,29). Other target sites, including FW, WW, SWG (type II), and H(S/T)F (type III) (30). The 3C-110 clone found in the two-hybrid screen contains the entire EH domain sequence lacking only the first two amino acid residues and should therefore recognize one of these sites. A search for the possible binding site on type VI collagen revealed only one FW type II target site, located in the Kunitz type inhibitor C5 domain of ␣3(VI). Based upon the Kunitz domain crystal structure, the FW residues are outside of the protease binding pocket and are accessible to ligand binding (31). In cell culture type VI collagen, filaments were observed prior to the appearance of EHD4 indicating that EHD4 is not required for the formation of type VI collagen filaments. Furthermore, the filaments of EHD4 do not co-distribute with type VI collagen suggesting that EHD4 is not required for the stabilization of type VI filaments but could still mediate important interactions of type VI with other matrix components in some tissue locations. Other interaction partners for EHD4 that must be present in the extracellular matrix remain to be identified. All four EHD proteins also contain a conserved FW binding site (Fig. 1, position 260 -261) that could be involved in the self-association of EHD proteins. Because cartilage apparently contains EHD1 and EHD4, it also gives rise to the possible existence of heteropolymers, although this would severely limit the extent of disulfide bond formation because EHD1 contains only one cysteine.
The distribution of EHD4 is complex. We have demonstrated that fibroblasts in culture elaborate extracellular matrix filaments containing EHD4. It is, however, not known whether these filaments represent EHD4 deposited on pre-existing fibrils formed by other proteins such as fibronectin or collagens or whether EHD4 can independently assemble into filaments. In developing cartilage, EHD4 is expressed late in the differentiation of chondrocytes and is excluded from the developing articular cartilage similar to cartilage matrix protein (32,33). EHD4 is present in the hypertrophic cartilage of the growth plate, but not of the secondary ossification center, similar to type XII collagen (32). The disappearance of EHD4 from the epiphysial cartilage prior to the appearance of hypertrophic chondrocytes is evidence of extensive remodeling of the epiphysial cartilage matrix prior to the onset of overt secondary ossification and has been also observed in the distribution of the exon 8-containing ␣1 chains of type XI collagen (34).
The extracellular localization of EHD4 in cartilage and in fibroblast culture suggests a function for this protein that is distinct from other members of the EH domain-containing family, all of which are intracellular proteins. Within the EHD group, the localization of EHD1 suggests that it too is intracellular, although the data presented did not rule out an extracellular location in some tissues such as cartilage. The localizations of EHD2 and EHD3 have yet to be determined. We propose that EHD4 is a tightly bound component of the ECM and that the binding is calcium-dependent, similar to thrombospondin 4 (35) and mediated by the calcium-binding EH domain. This suggests the possibility of a structural role for EHD4, either through self-assembly or heterologous interactions, although other functions, including growth factor modulation, cannot be excluded at this time.  Fig. 6, lane 3 was applied to a DEAE anion-exchange column, and the bound fraction was collected. The chromatographic profile shown is from the separation of the EHD4 containing DEAE fraction on a Sephacryl S-400 molecular sieve column (2.6 ϫ 110 cm) equilibrated with 40 mM Tris-HCl, pH 6.8, containing 2 M urea and 0.2 M sodium sulfate. The inset shows the Western blot (pAb 1306) on the left, and the Coomassie Blue-stained 7.5% polyacrylamide gel is on the right. The numbers of the lanes on the gel correspond to the numbered fractions on the profile. All samples were unreduced.
FIG. 8. Type VI collagen affinity blot. Bovine fetal cartilage EDTA extract was separated on a 7.5% SDS-PAGE gel without reduction and transferred to a polyvinylidene difluoride membrane. In lane 1 the membrane was treated with biotinylated type VI collagen tetramer, washed, and visualized with a type VI collagen monoclonal antibody. In lane 2 the membrane was developed using pAb 1050 to mark the position of EHD4. The positions of the Bio-Rad high molecular weight protein standards (see Fig. 6) are indicated on the left, and the molecular masses of the EHD4 bands are on the right.