The globular domains of type VI collagen are related to the collagen-binding domains of cartilage matrix protein and von Willebrand factor.

Type VI collagen is a transformation-sensitive glycoprotein of the extracellular matrix of fibroblasts. We have isolated and sequenced several overlapping cDNA clones (4153 bp) which encode the entire alpha 2 subunit of chicken type VI collagen. The deduced amino acid sequence predicts that the alpha 2(VI) polypeptide consists of 1015 amino acid residues that are arranged in four domains: a hydrophobic signal peptide of 20 residues, an amino-terminal globular domain of 228 residues, a collagenous segment of 335 residues and a carboxy-terminal globular domain of 432 residues. The collagenous domain contains seven Arg-Gly-Asp tripeptide units, some of which are likely to be used as cell-binding sites. The globular domains contain three homologous repeats with an average length of 180 amino acid residues. These repeats show a striking similarity to the collagen-binding motifs found in von Willebrand factor and cartilage matrix protein. We therefore speculate that the globular domains of the alpha 2(VI) polypeptide may interact with collagenous structures.     

EHD1--an EH-domain-containing protein with a specific expression pattern.

A cDNA that is a member of the eps15 homology (EH)-domain-containing family and is expressed differentially in testis was isolated from mouse and human. The corresponding genes map to the centromeric region of mouse chromosome 19 and to the region of conserved synteny on human chromosome 11q13. Northern analysis revealed two RNA species in mouse. In addition to the high levels in testis, expression was noted in kidney, heart, intestine, and brain. In human, three RNA species were evident. The smaller one was predominant in testis, while the largest species was evident in other tissues as well. The predicted protein sequence has an EH domain at its C-terminus, including an EF, a Ca2+ binding motif, and a central coiled-coil structure, as well as a nucleotide binding consensus site at its N-terminus. As such, it is a member of the EH-domain-containing protein family and was designated EHD1 (EH domain-containing 1). In cells in tissue culture, we localized EHD1 as a green fluorescent protein fusion protein, in transferrin-containing, endocytic vesicles. Immunostaining of different adult mouse organs revealed major expression of EHD1 in germ cells in meiosis, in the testes, in adipocytes, and in specific retinal layers. Results of in situ hybridization to whole embryos and immunohistochemical analyses indicated that EHD1 expression was already noted at day 9.5 in the limb buds and pharyngeal arches and at day 10.5 in sclerotomes, at various elements of the branchial apparatus (mandible and hyoid), and in the occipital region. At day 15.5 EHD1 expression peaked in cartilage, preceding hypertrophy and ossification, and at day 17.5 there was no expression in the bones. The EHD1 gene is highly conserved between nematode, Drosophila, mouse, and human. Its predicted protein structure and cellular localization point to the possibility that EHD1 participates in ligand-induced endocytosis.     

Regulation of collagen VI expression in fibroblasts. Effects of cell density, cell-matrix interactions, and chemical transformation

Collagen VI expression was studied in cultured human skin fibroblasts and mouse 3T3 cells using cDNA probes specific for alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. A 2-3-fold increase of these mRNAs was observed when fibroblasts were grown at increasing densities while only minimal changes occurred for the mRNA levels of collagens I and III, fibronectin, and beta-actin. Changes in mRNA correlated well with an increased production of corresponding proteins as determined by immunological assays. A comparable increase of alpha 1(VI) and alpha 2(VI) but not of alpha 3(VI) chain mRNAs was found for fibroblasts grown in a three-dimensional collagen gel after gel contraction. These conditions resulted, however, in a decrease of steady-state levels of collagens I and III and actin mRNAs. Transformation of 3T3 cells by phorbol ester did not change collagen VI mRNAs but caused a 3-5-fold reduction in mRNA levels for the other extracellular matrix proteins. These data strongly imply different regulatory mechanisms for the expression of collagen VI compared with collagens I and III and fibronectin. The differences may be correlated to changes in cell shape and reflect the requirement for collagen VI as a cell-binding protein.     

Chemical shift assignments of the C-terminal Eps15 homology domain-3 EH domain

The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation.     

The complete primary structure of type XII collagen shows a chimeric molecule with reiterated fibronectin type III motifs, von Willebrand factor A motifs, a domain homologous to a noncollagenous region of type IX collagen, and short collagenous domains with an Arg-Gly-Asp site

Extracellular matrix molecules are generally categorized as collagens, elastin, proteoglycans, or other noncollagenous structural/cell interaction proteins. Many of these extracellular proteins contain distinctive repetitive modules, which can sometimes be found in other proteins. We describe the complete primary structure of an alpha 1 chain of type XII collagen from chick embryonic fibroblasts. This large, structurally chimeric molecule identified by cDNA analysis combines previously unrelated molecular domains into a single large protein 3,124 residues long (approximately 340 kD). The deduced chicken type XII collagen sequence starts at the amino terminus with one unit of the type III motif of fibronectin, which is followed by one unit homologous to the von Willebrand factor A domain, then one more fibronectin type III module, a second A domain from von Willebrand factor, 6 units of type III motif and a third A domain, 10 consecutive units of type III motif and a fourth A domain, a domain homologous to the NC4 domain peptide of type IX collagen, and finally two short collagenous regions previously described as part of the partially sequenced collagen type XII molecule; an Arg-Gly-Asp potential cell adhesive recognition sequence is present in a hydrophilic region at the terminus of one collagenous domain. Antibodies raised to type XII collagen synthesized in a bacterial expression system recognized not only previously reported bands (220 kD et cetera) in tendons, but also bands with apparently different molecular sizes in fibroblasts and 4-d embryos. The antibodies stained a wide variety of extracellular matrices in embryos in patterns distinct from those of fibronectin or interstitial collagens. They prominently stained extracellular matrix associated with certain neuronal tissues, such as axons from dorsal root ganglia and neural tube. These studies identify a novel chimeric type of molecule that contains both adhesion molecule and collagen motifs in one protein. Its structure blurs current classification schemes for extracellular proteins and underscores the potentially large diversity possible in these molecules.     

Role of EHD1 and EHBP1 in perinuclear sorting and insulin-regulated GLUT4 recycling in 3T3-L1 adipocytes

Insulin stimulates glucose transport in muscle and adipose tissues by recruiting intracellular membrane vesicles containing the glucose transporter GLUT4 to the plasma membrane. The mechanisms involved in the biogenesis of these vesicles and their translocation to the cell surface are poorly understood. Here, we report that an Eps15 homology (EH) domain-containing protein, EHD1, controls the normal perinuclear localization of GLUT4-containing membranes and is required for insulin-stimulated recycling of these membranes in cultured adipocytes. EHD1 is a member of a family of four closely related proteins (EHD1, EHD2, EHD3, and EHD4), which also contain a P-loop near the N terminus and a central coiled-coil domain. Analysis of cultured adipocytes stained with anti-GLUT4, anti-EHD1, and anti-EHD2 antibodies revealed that EHD1, but not EHD2, partially co-localizes with perinuclear GLUT4. Expression of a dominant-negative construct of EHD1 missing the EH domain (DeltaEH-EHD1) markedly enlarged endosomes, dispersed perinuclear GLUT4-containing membranes throughout the cytoplasm, and inhibited GLUT4 translocation to the plasma membranes of 3T3-L1 adipocytes stimulated with insulin. Similarly, small interfering RNA-mediated depletion of endogenous EHD1 protein also markedly dispersed perinuclear GLUT4 in cultured adipocytes. Moreover, EHD1 is shown to interact through its EH domain with the protein EHBP1, which is also required for insulin-stimulated GLUT4 movements and hexose transport. In contrast, disruption of EHD2 function was without effect on GLUT4 localization or translocation to the plasma membrane. Taken together, these results show that EHD1 and EHBP1, but not EHD2, are required for perinuclear localization of GLUT4 and reveal that loss of EHBP1 disrupts insulin-regulated GLUT4 recycling in cultured adipocytes.     

EHD2 and the novel EH domain binding protein EHBP1 couple endocytosis to the actin cytoskeleton

Here we identified two novel proteins denoted EH domain protein 2 (EHD2) and EHD2-binding protein 1 (EHBP1) that link clathrin-mediated endocytosis to the actin cytoskeleton. EHD2 contains an N-terminal P-loop and a C-terminal EH domain that interacts with NPF repeats in EHBP1. Disruption of EHD2 or EHBP1 function by small interfering RNA-mediated gene silencing inhibits endocytosis of transferrin into EEA1-positive endosomes as well as GLUT4 endocytosis into cultured adipocytes. EHD2 localizes with cortical actin filaments, whereas EHBP1 contains a putative actin-binding calponin homology domain. High expression of EHD2 or EHBP1 in intact cells mediates extensive actin reorganization. Thus EHD2 appears to connect endocytosis to the actin cytoskeleton through interactions of its N-terminal domain with membranes and its C-terminal EH domain with the novel EHBP1 protein.     

EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids

The four Eps15 homology (EH) domain-containing proteins, EHD1-EHD4, have recently been ascribed roles in the regulation of the recycling of distinct receptor molecules and are often found associated with tubular structures. Here, we report the analysis of all four EHD proteins with regard to tissue distribution, intracellular localization and lipid binding properties. Specific antibodies reveal distinct expression profiles for the individual proteins in tissues and at intracellular locations, where they potentially interact with specific phospholipids. Moreover, EHD proteins colocalize with vesicular and tubular structures, implying roles in transport processes and cytoskeletal dynamics. Protein variants carrying mutations in the N-terminal nucleotide-binding P-loop region are no longer associated with phospholipids or membrane compartments, while deletion of the C-terminal EH domain affects targeting to tubular structures. All EHD proteins are able to bind to phospholipids, but localizations differ for each protein.     

AtEHDs in endocytosis

Endocytosis regulates many important and diverse processes in eukaryotic life. EH domain containing proteins function as regulators of endocytosis through protein-protein interactions. Several interactors of mammalian EHDs were identified, including clathrin machinery components. The four human EHD proteins share high homology at the protein level and possess similar domains, but appear to be involved in different stages of intracellular trafficking. EHD1 regulates recycling through the endocytic recycling compartment (ERC). EHD2 has been found to inhibit internalization in mammalians when overexpressed.We have recently investigated the importance of EH domain containing proteins in plant endocytosis. We were able to show that both of the Arabidopsis EHD homologs, termed AtEHD1 and AtEHD2, play important roles in plant endocytosis. Knockdown of AtEHD1 delayed internalization, and overexpression of AtEHD2 inhibited endocytosis. Thus, the function of plant EHDs is highly homologous to that of mammalian EHDs.Key words: endocytosis, endosome, EH domain, EHD1, EHD2, recycling     

Identification of binding partners interacting with the α1-N-propeptide of type V collagen

The predominant form of type V collagen is the [α1(V)]?α2(V) heterotrimer. Mutations in COL5A1 or COL5A2, encoding respectively the α1(V)- and α2(V)-collagen chain, cause classic EDS (Ehlers-Danlos syndrome), a heritable connective tissue disorder, characterized by fragile hyperextensible skin and joint hypermobility. Approximately half of the classic EDS cases remain unexplained. Type V collagen controls collagen fibrillogenesis through its conserved α1(V)-N-propeptide domain. To gain an insight into the role of this domain, a yeast two-hybrid screen among proteins expressed in human dermal fibroblasts was performed utilizing the N-propeptide as a bait. We identified 12 interacting proteins, including extracellular matrix proteins and proteins involved in collagen biosynthesis. Eleven interactions were confirmed by surface plasmon resonance and/or co-immunoprecipitation: α1(I)- and α2(I)-collagen chains, α1(VI)-, α2(VI)- and α3(VI)-collagen chains, tenascin-C, fibronectin, PCPE-1 (procollagen C-proteinase enhancer-1), TIMP-1 (tissue inhibitor of metalloproteinases-1), MMP-2 (matrix metalloproteinase 2) and TGF-β1 (transforming growth factor β1). Solid-phase binding assays confirmed the involvement of the α1(V)-N-propeptide in the interaction between native type V collagen and type VI collagen, suggesting a bridging function of this protein complex in the cell-matrix environment. Enzymatic studies showed that processing of the α1(V)-N-propeptide by BMP-1 (bone morphogenetic protein 1)/procollagen C-proteinase is enhanced by PCPE-1. These interactions are likely to be involved in extracellular matrix homoeostasis and their disruption could explain the pathogenetic mechanism in unresolved classic EDS cases.     

Recycling to the plasma membrane is delayed in EHD1 knockout mice

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.     

EHD1 and Eps15 interact with phosphatidylinositols via their Eps15 homology domains

The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.     

EHD proteins associate with syndapin I and II and such interactions play a crucial role in endosomal recycling

EHD proteins were shown to function in the exit of receptors and other membrane proteins from the endosomal recycling compartment. Here, we identify syndapins, accessory proteins in vesicle formation at the plasma membrane, as differential binding partners for EHD proteins. These complexes are formed by direct eps15-homology (EH) domain/asparagine proline phenylalanine (NPF) motif interactions. Heterologous and endogenous coimmunoprecipitations as well as reconstitutions of syndapin/EHD protein complexes at intracellular membranes of living cells demonstrate the in vivo relevance of the interaction. The combination of mutational analysis and coimmunoprecipitations performed under different nucleotide conditions strongly suggest that nucleotide binding by EHD proteins modulates the association with syndapins. Colocalization studies and subcellular fractionation experiments support a role for syndapin/EHD protein complexes in membrane trafficking. Specific interferences with syndapin-EHD protein interactions by either overexpression of the isolated EHD-binding interface of syndapin II or of the EHD1 EH domain inhibited the recycling of transferrin to the plasma membrane, suggesting that EH domain/NPF interactions are critical for EHD protein function in recycling. Consistently, both inhibitions were rescued by co-overexpression of the attacked protein component. Our data thus reveal that, in addition to a crucial role in endocytic internalization, syndapin protein complexes play an important role in endocytic receptor recycling.     

EH domain of EHD1

EHD1 is a member of the mammalian C-terminal Eps15 homology domain (EH) containing protein family, and regulates the recycling of various receptors from the endocytic recycling compartment to the plasma membrane. The EH domain of EHD1 binds to proteins containing either an Asn-Pro-Phe or Asp-Pro-Phe motif, and plays an important role in the subcellular localization and function of EHD1. Thus far, the structures of five N-terminal EH domains from other proteins have been solved, but to date, the structure of the EH domains from the four C-terminal EHD family paralogs remains unknown. In this study, we have assigned the 133 C-terminal residues of EHD1, which includes the EH domain, and solved its solution structure. While the overall structure resembles that of the second of the three N-terminal Eps15 EH domains, potentially significant differences in surface charge and the structure of the tripeptide-binding pocket are discussed.     

A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.

A new SH2-containing sequence, SHC, was isolated by screening cDNA libraries with SH2 representative DNA probes. The SHC cDNA is predicted to encode overlapping proteins of 46.8 and 51.7 kd that contain a single C-terminal SH2 domain, and an adjacent glycine/proline-rich motif with regions of homology with the alpha 1 chain of collagen, but no identifiable catalytic domain. Anti-SHC antibodies recognized three proteins of 46, 52, and 66 kd in a wide range of mammalian cell lines. These SHC proteins complexed with and were phosphorylated by activated epidermal growth factor receptor. The physical association of SHC proteins with activated receptors was recreated in vitro by using a bacterially expressed SHC SH2 domain. NIH 3T3 mouse fibroblasts that constitutively overexpressed SHC acquired a transformed phenotype in culture and formed tumors in nude mice. These results suggest that the SHC gene products couple activated growth factor receptors to a signaling pathway that regulates the proliferation of mammalian cells.     

Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones

Pepsin-solubilized collagen VI was prepared from human placenta and used to separate three constituent chains for determining partial amino acid sequences. Antibodies raised against the chains assisted in the identification and purification of several cDNA clones from three expression lambda gt11 libraries. Most of the clones hybridized to either a 3.5-kb or 4.2-kb mRNA species which by matching peptide and nucleotide sequences could be identified as coding for the alpha 2(VI) or alpha 1(VI) chain, respectively. Other clones hybridized to either an 8.5-kb mRNA which very likely encoded the alpha 3(VI) chain or to an unknown 2.0-kb mRNA. Northern blots revealed a considerable variation in the mRNA levels for each collagen VI chain in both skin and cornea fibroblasts and in several tumor cell lines. Limited sequence data generated from peptides and cDNA clones demonstrated a characteristic cysteine pattern at the junction between N-terminal globular domain and triple helix in all three chains. In addition, the data showed occasional interruptions of triplet sequences within the triple-helical domain and the presence of two Arg-Gly-Asp sequences which are potential cell-binding structures.     

WARP Interacts with Collagen VI-Containing Microfibrils in the Pericellular Matrix of Human Chondrocytes

Collagen VI and WARP are extracellular structural macromolecules present in cartilage and associated with BM suprastructures in non-skeletal tissues. We have previously shown that in WARP-deficient mice, collagen VI is specifically reduced in regions of the peripheral nerve ECM where WARP is expressed, suggesting that both macromolecules are part of the same suprastructure. The object of this study was to conduct a detailed analysis of WARP-collagen VI interactions in vitro in cartilage, a tissue rich in WARP and collagen VI. Immunohistochemical analysis of mouse and human articular cartilage showed that WARP and collagen VI co-localize in the pericellular matrix of superficial zone articular chondrocytes. EM analysis on extracts of human articular cartilage showed that WARP associates closely with collagen VI-containing suprastructures. Additional evidence of an interaction is provided by immunogold EM and immunoblot analysis showing that WARP was present in collagen VI-containing networks isolated from cartilage. Further characterization were done by solid phase binding studies and reconstitution experiments using purified recombinant WARP and isolated collagen VI. Collagen VI binds to WARP with an apparent K_d of approximately 22 nM and the binding site(s) for WARP resides within the triple helical domain since WARP binds to both intact collagen VI tetramers and pepsinized collagen VI. Together, these data confirm and extend our previous findings by demonstrating that WARP and collagen VI form high affinity associations in vivo in cartilage. We conclude that WARP is ideally placed to function as an adapter protein in the cartilage pericellular matrix.     

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast- specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole- like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.