Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Domain and functional analysis of a novel platelet-endothelial cell surface protein, SCUBE1

SCUBE1 (signal peptide-CUB-EGF domain-containing protein 1) is a novel, secreted, cell surface glycoprotein expressed during early embryogenesis and found in platelet and endothelial cells. This protein is composed of an N-terminal signal peptide sequence followed by nine tandemly arranged epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeat motifs, and one CUB domain at the C terminus. However, little is known about its domain and biological function. Here, we generated a comprehensive panel of domain deletion constructs and a new genetic mouse model with targeted disruption of Scube1 (Scube1(Delta cub/Delta cub)) to investigate the domain function and biological significance. A number of cell-based assays were utilized to define the critical role of the spacer region for membrane association and establish that the EGF-like repeats 7-9 are sufficient for the formation of SCUBE1-mediated homophilic adhesions in a calcium-dependent fashion. Biochemical and molecular analyses showed that the C-terminal cysteine-rich motifs and CUB domain could directly bind and antagonize the bone morphogenetic protein activity. Furthermore, genetic ablation of this C-terminal region resulted in brain malformation in the Scube1(Delta cub/Delta cub) embryos. Together, our results support the dual roles of SCUBE1 on brain morphogenesis and cell-cell adhesions through its distinct domain function.     

Molecular cloning of 114/A10, a cell surface antigen containing highly conserved repeated elements, which is expressed by murine hemopoietic progenitor cells and interleukin-3-dependent cell lines

Monoclonal antibody 114/A10, raised against the murine multipotential hemopoietic progenitor cell line B6SUtA, identifies an antigen highly expressed by primary myeloid progenitor cells, the myelomonocytic leukemia cell line WEHI-3, and various interleukin-3 (IL-3)-dependent cell lines. Western blotting studies indicate that the 114/A10 antigen has a mean relative molecular mass of 150,000 but varies greatly in size range between different cell types. cDNA clones encoding this protein were isolated from a plasmid-based expression vector library prepared from B6SUtA RNA. Three clones corresponding in size to the two major mRNA species detected in IL-3-dependent cell lines (3.0 and 2.2 kilobase pairs) and differing in their utilization of alternative polyadenylation signals were obtained. These clones contain a single long open reading frame of 573 amino acids possessing the typical characteristics of an integral membrane protein. A particularly striking feature of this sequence is the presence at the extracellular amino terminus of a series of eight highly conserved 27-amino acid, serine/threonine-rich (55%) tandem repeats that may serve as sites of extensive glycosylation. The extracellular domain also contains three epidermal growth factor-like cysteine-rich repeats. The distribution and structural characteristics of the 114/A10 antigen suggest a possible regulatory role in the cellular response to IL-3.     

Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans.

The glp-1 gene encodes a membrane protein required for inductive cell interactions during development of the nematode Caenorhabditis elegans. Here we report the molecular characterization of 15 loss-of-function (lf) mutations of glp-1. Two nonsense mutations appear to eliminate glp-1 activity; both truncate the glp-1 protein in its extracellular domain and have a strong loss-of-function phenotype. Twelve missense mutations and one in-frame deletion map to sites within the repeated motifs of the glp-1 protein (10 epidermal growth factor [EGF]-like and 3 LNG repeats extracellularly and 6 cdc10/SWI6, or ankyrin, repeats intracellularly). We find that all three types of repeated motifs are critical to glp-1 function, and two individual EGF-like repeats may have distinct functions. Intriguingly, all four missense mutations in one phenotypic class map to the N-terminal EGF-like repeats and all six missense mutations in a second phenotypic class reside in the intracellular cdc10/SWI6 repeats. These two clusters of mutations may identify functional domains within the glp-1 protein.     

Domain and functional analysis of a novel breast tumor suppressor protein, SCUBE2

Signal peptide CUB (complement proteins C1r/C1s, Uegf, and Bmp 1)-EGF domain-containing protein 2 (SCUBE2) is a secreted, membrane-associated multidomain protein composed of five recognizable motifs: an NH(2)-terminal signal peptide sequence, nine copies of epidermal growth factor (EGF)-like repeats, a spacer region, three cysteine-rich repeats, and one CUB domain at the COOH terminus. Our previous clinical study showed that SCUBE2 may act as a novel breast tumor suppressor gene and serve as a useful prognostic marker. However, the specific domain responsible for its tumor suppressor activity and the precise mechanisms of its anti-tumor effect remain unknown. Using a combination of biochemical, molecular, and cell biology techniques, we further dissected the molecular functions and signal pathways mediated by the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain of SCUBE2. Independent overexpression of the NH(2)-terminal EGF-like repeats or COOH-terminal CUB domain resulted in suppression of MCF-7 breast cancer cell proliferation and reduced MCF-7 xenograft tumor growth in nude mice. Molecular and biochemical analyses revealed that the COOH-terminal CUB domain could directly bind to and antagonize bone morphogenetic protein activity in an autocrine manner, whereas the NH(2)-terminal EGF-like repeats could mediate cell-cell homophilic adhesions in a calcium-dependent fashion, interact with E-cadherin (a master tumor suppressor), and decrease the β-catenin signaling pathway. Together, our data demonstrate that SCUBE2 has growth inhibitory effects through a coordinated regulation of two distinct mechanisms: antagonizing bone morphogenetic protein and suppressing the β-catenin pathway in breast cancer cells.     

MEGF9: a novel transmembrane protein with a strong and developmentally regulated expression in the nervous system

MEGF9 [multiple EGF (epidermal growth factor)-like-domains 9], a novel transmembrane protein with multiple EGF-like repeats, is predominantly expressed in the developing and adult CNS (central nervous system) and PNS (peripheral nervous system). The domain structure of MEGF9 consists of an N-terminal region with several potential O-glycosylation sites followed by five EGF-like domains, which are highly homologous with the short arms of laminins. Following one single pass transmembrane domain, a highly conserved short intracellular domain with potential phosphorylation sites is present. The protein was recombinantly expressed and characterized as a tissue component. To study the expression pattern further, immunohistochemistry was performed and staining was detected in Purkinje cells of the cerebellum and in glial cells of the PNS. Additional expression was observed in the epidermal layer of skin, papillae of the tongue and the epithelium of the gastrointestinal tract. By immunoelectron microscopy, MEGF9 was detected in glial cells of the sciatic nerve facing the basement membrane. MEGF9 represents a novel putative receptor, expressed in neuronal and non-neuronal tissues, that is regulated during development and could function as a guidance or signalling molecule.     

Sequence-specific 1H NMR assignments, secondary structure, and location of the calcium binding site in the first epidermal growth factor like domain of blood coagulation factor IX.

Factor IX is a blood clotting protein that contains three regions, including a gamma-carboxyglutamic acid (Gla) domain, two tandemly connected epidermal growth factor like (EGF-like) domains, and a serine protease region. The protein exhibits a high-affinity calcium binding site in the first EGF-like domain, in addition to calcium binding in the Gla domain. The first EGF-like domain, factor IX (45-87), has been synthesized. Sequence-specific resonance assignment of the peptide has been made by using 2D NMR techniques, and its secondary structure has been determined. The protein is found to have two antiparallel beta-sheets, and preliminary distance geometry calculations indicate that the protein has two domains, separated by Trp28, with the overall structure being similar to that of EGF. An NMR investigation of the calcium-bound first EGF-like domain indicates the presence and location of a calcium binding site involving residues on both strands of one of the beta-sheets as well as the N-terminal region of the peptide. These results suggest that calcium binding in the first EGF-like domain could induce long-range (possibly interdomain) conformational changes in factor IX, rather than causing structural alterations in the EGF-like domain itself.     

Functional Integration of the Conserved Domains of Shoc2 Scaffold

Shoc2 is a positive regulator of signaling to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2). Shoc2 is also proposed to interact with RAS and Raf-1 in order to accelerate ERK1/2 activity. To understand the mechanisms by which Shoc2 regulates ERK1/2 activation by the epidermal growth factor receptor (EGFR), we dissected the role of Shoc2 structural domains in binding to its signaling partners and its role in regulating ERK1/2 activity. Shoc2 is comprised of two main domains: the 21 leucine rich repeats (LRRs) core and the N-terminal non-LRR domain. We demonstrated that the N-terminal domain mediates Shoc2 binding to both M-Ras and Raf-1, while the C-terminal part of Shoc2 contains a late endosomal targeting motif. We found that M-Ras binding to Shoc2 is independent of its GTPase activity. While overexpression of Shoc2 did not change kinetics of ERK1/2 activity, both the N-terminal and the LRR-core domain were able to rescue ERK1/2 activity in cells depleted of Shoc2, suggesting that these Shoc2 domains are involved in modulating ERK1/2 activity.     

Purification, cloning, and characterization of an acidic ectoprotein phosphatase differentially expressed in the infectious bloodstream form of Trypanosoma brucei

We purified an ecto-phosphatase of 115 kDa (TryAcP115) specifically expressed by bloodstream forms of Trypanosoma brucei. The corresponding gene coded for a 45-kDa protein potentially including a signal peptide, a membrane-spanning domain and an N-terminal domain containing 8 N-glycosylation sites. There was no significant sequence homology with other phosphatases. Antiserum to the Escherichia coli recombinant N-terminal domain, Petase7, recognized a protein of 55 kDa in Western blots after deglycosylation of the TryAcP115 protein by N-glycosidase F. Immunofluorescence and trypsin treatment of living parasites showed that TryAcP115 was localized to the surface of the parasite and that its N-terminal domain was oriented extracellularly. The recombinant N-terminal domains, expressed in E. coli and Leishmania amazonensis, harbored phosphatase activity against Tyr(P)-Raytide, Ser(P)-neurogranin, and ATP. The enzymatic properties of native TryAcP115 and the recombinant proteins for the substrate Tyr(P)-Raytide were virtually identical and included: (i) K(m) and V(max) values of 15 nM and 200 pmol/min/mg, (ii) no requirement for divalent cations, and (iii) sensitivity to vanadate, sodium fluoride, and tartrate, but insensitivity to okadaic acid and tetramisole. Although the function of TryAcP115 remains unknown, a differentially expressed, unique ecto-phosphatase could regulate growth or influence parasite-host interactions and might provide a useful target for chemotherapy.     

Chain termini cross-talk in the swapping process of bovine pancreatic ribonuclease

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1-13) and/or the C-terminal β-strand (residues 116-124), thus forming a variety of oligomers, including two different dimers. Cis-trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis-trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.     

Opacity factor activity and epithelial cell binding by the serum opacity factor protein of Streptococcus pyogenes are functionally discrete

Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFDeltaFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFDeltaFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFDeltaFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFDeltaFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFDeltaFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone.     

Multiple domains of the large fibroblast proteoglycan, versican.

The primary structure of a large chondroitin sulfate proteoglycan expressed by human fibroblasts has been determined. Overlapping cDNA clones code for the entire 2389 amino acid long core protein and the 20-residue signal peptide. The sequence predicts a potential hyaluronic acid-binding domain in the amino-terminal portion. This domain contains sequences virtually identical to partial peptide sequences from a glial hyaluronate-binding protein. Putative glycosaminoglycan attachment sites are located in the middle of the protein. The carboxy-terminal portion includes two epidermal growth factor (EGF)-like repeats, a lectin-like sequence and a complement regulatory protein-like domain. The same set of binding elements has also been identified in a new class of cell adhesion molecules. Amino- and carboxy-terminal portions of the fibroblast core protein are closely related to the core protein of a large chondroitin sulfate proteoglycan of chondrosarcoma cells. However, the glycosaminoglycan attachment regions in the middle of the core proteins are different and only the fibroblast core protein contains EGF-like repeats. Based on the similarities of its domains with various binding elements of other proteins, we suggest that the large fibroblast proteoglycan, herein referred to as versican, may function in cell recognition, possibly by connecting extracellular matrix components and cell surface glycoproteins.     

Structure and function of the receptor-like protein kinases of higher plants

Cell surface receptors located in the plasma membrane have a prominent role in the initiation of cellular signalling. Recent evidence strongly suggests that plant cells carry cell surface receptors with intrinsic protein kinase activity. The plant receptor-like protein kinases (RLKs) are structurally related to the polypeptide growth factor receptors of animals which consist of a large extracytoplasmic domain, a single membrane spanning segment and a cytoplasmic domain of the protein kinase gene family. Most of the animal growth factor receptor protein kinases are tyrosine kinases; however, the plant RLKs all appear to be serine/threonine protein kinases. Based on structural similarities in their extracellular domains the RLKs fall into three categories: the S-domain class, related to the self-incompatibility locus glycoproteins of Brassica; the leucine-rich repeat class, containing a tandemly repeated motif that has been found in numerous proteins from a variety of eukaryotes; and a third class that has epidermal growth factor-like repeats. Distinct members of these putative receptors have been found in both monocytyledonous plants such as maize and in members of the dicotyledonous Brassicaceae. The diversity among plant RLKs, reflected in their structural and functional properties, has opened up a broad new area of investigation into cellular signalling in plants with far-reaching implications for the mechanisms by which plant cells perceive and respond to extracellular signals.     

Structural and dynamical comparison of alpha, beta and gamma forms of murine epidermal growth factor

Reversed-phase high-performance liquid chromatography of mouse epidermal growth factor (mEGF) purified by the method of Savage and Cohen allows resolution of four forms of the protein hormone: alpha, beta, gamma and delta. alpha-mEGF, the major form isolated by HPLC, is the parent mEGF originally sequenced by Savage and Cohen, and beta-mEGF is des-asparaginyl1-alpha-mEGF. Proton nuclear magnetic resonance spectroscopy has been used to investigate structural and dynamical differences among the alpha, beta and gamma forms of the peptide. Based on these data, gamma-mEGF can be tentatively identified as des-Asn1, Ser2-mEGF. Comparative nuclear Overhauser experiments on amide and aromatic proton resonances suggest that there are significant conformational changes in the peptide structure on cleavage of the N-terminal residues. Backbone amide proton/deuteron exchange rates in gamma-mEGF and beta-mEGF are significantly faster than those in alpha-mEGF suggesting that structural dynamics are enhanced in the minor forms; this interpretation is supported by the decrease in Tyr(2,6)-(3,5) intraresidue NOE magnitudes on going from alpha to beta to gamma forms. These data suggest that the average conformations of beta and gamma-mEGF favor a more open or denatured state of the protein and that the N terminus is critical to the structural integrity of the parent protein.     

Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor.

We have determined the complete nucleotide and deduced amino acid sequence of the major protein core of the human heparan sulfate proteoglycan HSPG2/perlecan of basement membranes. Eighteen overlapping cDNA clones comprise 14.35 kilobase pairs (kb) of contiguous sequence with an open reading frame of 13.2 kb. The mature protein core, without the signal peptide of 21 amino acids, has a M(r) of 466,564. This large protein is composed of multiple modules homologous to the receptor of low density lipoprotein, laminin, neural cell adhesion molecules, and epidermal growth factor. Domain I, near the amino terminus, appears unique for the proteoglycan since it shares no significant homology with any other proteins. It contains three Ser-Gly-Asp sequences that could act as attachment sites for heparan sulfate glycosaminoglycans. Domain II is highly homologous to the LDL receptor and contains four repeats with perfect conservation of all 6 consecutive cysteines. Next is domain III which shares homology to the short arm of laminin A chain and contains four cysteine-rich regions intercalated among three globular domains. Domain IV, the largest module with greater than 2000 residues, contains 21 repeats of the immunoglobulin type as found in neural cell adhesion molecule. Near the beginning of this domain, there is a stretch of 29 hydrophobic amino acids which could allow the molecule to interact with the plasma membrane. Domain V, similar to the carboxyl-terminal globular G-domain of laminin A and to the related protein merosin, contains three globular regions and four EGF-like repeats. In situ hybridization and immunoenzymatic studies show a close association of this gene product with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Our studies show that this proteoglycan is present in all vascularized tissues and suggest that this unique molecule has evolved from the utilization of modular structures with adhesive and growth regulatory properties.     

Structure of the extracellular domain of Tie receptor tyrosine kinases and localization of the angiopoietin-binding epitope

Angiogenesis is essential for tissue repair and regeneration during wound healing but also plays important roles in many pathological processes including tumor growth and metastasis. The receptor protein tyrosine kinase Tie2 and its ligands, the angiopoietins, have important functions in the regulation of angiogenesis. Here, we report a detailed structural and functional characterization of the extracellular region of Tie2. Sequence analysis of the extracellular domain revealed an additional immunoglobulin-like domain resulting in a tandem repeat of immunoglobulin-like domains at the N terminus of the protein. The same domain organization was also found for the Tie1 receptor that shares a high degree of homology with Tie2. Based on structural similarities to other receptor tyrosine kinases and cell adhesion molecules, we demonstrate that the N-terminal two immunoglobulin-like domains of Tie2 harbor the angiopoietin-binding site. Using transmission electron microscopy we furthermore show that the extracellular domain of Tie receptors consists of a globular head domain and a short rod-like stalk that probably forms a spacer between the cell surface and the angiopoietin-binding site. Mutational analysis demonstrated that the head domain consists of the three immunoglobulin-like domains and the three epidermal growth factor-like modules and that the stalk is formed by the three fibronectin type III repeats. These findings might be of particular interest for drug development because Tie receptors are potential targets for treatment of angiogenesis-associated diseases.     

A tryptic peptide from beta-casein depresses protein synthesis and degradation and enhances ureogenesis in primary cultures of rat hepatocytes

1. A peptide which enhances ureogenesis in primary cultured hepatocytes of rats was isolated from a tryptic digest of bovine beta-casein. 2. The structure of the peptide was Ala-Val-Pro-Tyr-Pro-Gln-Arg which is located from 177th to 183rd residues from N-terminal of beta-casein. 3. The peptide also showed the activity to inhibit protein synthesis and protein degradation. 4. It also inhibited DNA synthesis of hepatocytes induced by insulin and/or epidermal growth factor.     

The heparin-binding domain of amphiregulin necessitates the precursor pro-region for growth factor secretion.

The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.     

The cellular response to neuregulins is governed by complex interactions of the erbB receptor family.

Deregulated signaling by the four members of the epidermal growth factor receptor tyrosine kinase family (erbB family) is implicated in the genesis or progression of human cancers. However, efforts to analyze signaling by these receptors have been hampered by the diversity of ligands and extensive interreceptor cross talk. We have expressed the four human erbB family receptors, singly and in pairwise combinations, in a pro-B-lymphocyte cell line (Ba/F3) and investigated the range of interactions activated by the epidermal growth factor homology domain of the agonist neuregulin beta. The results provide the first comprehensive analysis of the response of this receptor family to a single peptide agonist. This peptide induced complex patterns of receptor tyrosine phosphorylation and regulation of Ba/F3 cell survival and proliferation. These data demonstrate the existence of several previously undocumented receptor interactions driven by neuregulin.