Immunohistochemical investigation of gamma-aminobutyric acid ontogeny and transient expression in the central nervous system of Xenopus laevis tadpoles

A cDNA clone encoding the membrane form of guanylyl cyclase was isolated from a Hemicentrotus pulcherrimus testis cDNA library and its nucleotide sequence was determined. The cDNA was 4123 bp long and an open reading frame predicted a protein of 1125 amino acids including an apparent signal peptide of 21 residues; a single transmembrane domain of 25 amino acids which divides the mature protein into an amino-terminal, extracellular domain of 485 amino acids and a carboxyl-terminal, intracellular domain of 594 amino acids. Three potential N-linked glycosylation sites were present in the extracellular domain. Northern blot analysis of poly(A)+RNA from testes, ovaries, eggs and embryos at various developmental stages showed that the cDNA encoding guanylyl cyclase hybridized to a mRNA of 4.4 kb from the testes. We developed a large scale purification method of the phosphorylated (131 kDa) and dephosphorylated (128 kDa) forms of the membrane-bound guanylyl cyclase from H. pulcherrimus spermatozoa. The purified 131 kDa and 128 kDa forms of the guanylyl cyclase contained 26.0 +/- 1.3 and 4.3 +/- 0.7 moles of phosphate per mol protein (mean +/- S.D.; n = 6), respectively. The amino-terminal amino acids of both the 131 kDa and 128 kDa forms of the guanylyl cyclase could not be detected, suggesting that they were blocked.     

Purification and subunit structure of extracellular superoxide dismutase from mouse lung tissue

The first purification of mouse extracellular superoxide dismutase (EC-SOD) and the analysis of the native enzyme are described. Mouse EC-SOD was purified from lung tissues with a high recovery (41%) and a specific polyclonal antibody against the purified enzyme was obtained. The purified enzyme had a strong affinity for, heparin and a molecular mass of 150 kDa (estimated by a gel filtration chromatography). The native mouse EC-SOD was composed of two different sizes of subunits, a M(r) of 33 and 35 kDa (determined by SDS-PAGE). The 35-kDa subunit had an interchain disulfide bond at the C-terminus and existed as a covalent dimer in the molecule, whereas the 33-kDa subunit resulted from the 35-kDa subunit by truncating its C-terminus as a posttranslational modification, with resultant loss of the interchain disulfide bond. These results suggest that the native mouse EC-SOD is a heterotetramer composed of two different dimers, with or without a covalent bond.     

Regulation of ribosomal RNA gene transcription during retinoic acid-induced differentiation of mouse teratocarcinoma cells

The human cytomegalovirus glycoprotein B gene (gB; gpUL55) was truncated at amino acid 692 and recombined into Autographa californica nuclear polyhedrosis virus (baculovirus). Infection of insect cells with the recombinant baculovirus resulted in high-level expression and secretion of the truncated gB protein (gB-S) into the culture medium. Purification of gB-S by monoclonal antibody affinity chromatography yielded a protein of ca. 200 kDa. Characterization of the 200-kDa purification product indicated that the recombinant gB protein retained many structural and functional features of the viral gB. Comparison of electrophoretic migration patterns in reduced versus nonreduced protein samples and immune blotting analysis with antibodies specific for the amino or carboxy-terminus of gB demonstrated that the recombinant protein was composed of disulfide linked 69 kDa amino terminal and 35-kDa carboxy-terminal fragments. In addition, recognition of the 200-kDa gB-S by a conformational-dependent, oligomer-specific monoclonal antibody suggested that gB-S was properly folded and dimeric. Like the viral gB, gB-S had heparin binding ability. One heparin binding site was found to reside within the 35-kDa carboxy-terminal fragment (aa 492-692). Heparin binding was abolished when gB-S was denatured. These data suggest that gB contains a novel heparin binding motif that is at least partially conformational dependent.     

Identification of cysteine pairs within the amino-terminal 5% of apolipoprotein B essential for hepatic lipoprotein assembly and secretion

There is growing evidence that the amino-terminal globular domain of apolipoprotein B (apoB) is essential for lipoprotein particle formation in the hepatic endoplasmic reticulum. To identify the structural requirements for its function in lipoprotein assembly, cysteine (Cys) pairs required to form the seven disulfide bonds within the amino-terminal 21% of apoB were replaced in groups or individually by serine. Substitution of Cys pairs required for formation of disulfide bonds 1-3 or 4-7 (numbered from amino to carboxyl terminus) completely blocked the secretion of apoB28 in transfected HepG2 cells. To identify the specific disulfide bonds required for secretion, Cys pairs were mutated individually. Substitution of Cys pairs required for disulfide bonds 1, 3, 5, 6, or 7 had little or no impact on apoB28 secretion or buoyant density. In contrast, individual substitution of Cys pair 2 (amino acid residues 51 and 70) or 4 (218 and 234) severely inhibited apoB28 secretion and its capacity to undergo intracellular assembly with lipid. The same assembly and secretion defects were observed when these mutations were expressed as part of apoB50. These studies provide direct evidence that the ability of the internal lipophilic regions of apoB to engage in the recruitment and sequestration of lipid during translation is critically dependent upon a structural configuration contained within or affected by the amino-terminal 5% of the protein.     

Putative multiadhesive protein from the marine sponge Geodia cydonium: cloning of the cDNA encoding a fibronectin-, an SRCR-, and a complement control protein module

Sponges (Porifera) representing the simplest metazoan phylum so far have been thought to possess no basal lamina tissue structures. One major extracellular matrix protein that is also a constitutive glycoprotein of the basal lamina is fibronectin. It was the aim of the present study to identify the native protein from the marine sponge Geodia cydonium and to isolate the corresponding cDNA. In crude extracts from this sponge protein(s) of M(r) of approximately 230 and approximately 210 kDa could be visualized by Western-blotting using an anti-fibronectin [human] antibody. By PCR cloning from a cDNA library of G. cydonium we isolated a cDNA comprising one element of fibronectin, the type-III (FN3) module. The cDNA (2.3 kb long), encoding a 701 amino acid [aa] long putative "multiadhesive protein" termed MAP_GEOCY, was found to contain (i) a fibronectin-, (ii) a scavenger receptor cysteine-rich [SRCR]-, and (iii) a short consensus repeat [SCR] module. The 89 aa long fibronectin module comprises the characteristic topology and conserved aa found in fibronectin type-III (FN3) elements. The SRCR module (101 aa) features the characteristics of group B SRCR molecules. The predominant proteins belonging to this group are the mammalian WC1-, M130-, CD6- and CD5 antigens that probably are involved in immunological reactions. The SCR module (54 aa) shows the characteristics of type III SCR modules found in complement receptors. Phylogenetic analyses performed with all three building blocks of the "multiadhesive protein" showed that the respective sponge modules form independent, possibly basal, lineages in trees that include the corresponding modules from higher metazoan animals. In summary, these data demonstrate for the first time that the phylogenetically oldest Metazoa, the sponges, contain protein modules seen in higher animals in proteins of the extracellular matrix and in molecules involved in cell-mediated immune reactions in vertebrates.     

Modulation of cell surface fibronectin assembly sites by lysophosphatidic acid

Lysophosphatidic acid is a product of activated platelets and has diverse actions on cells. We have characterized the effect of lysophosphatidic acid on cell-mediated binding and assembly of fibronectin, an extracellular matrix protein. Serum made from whole blood, but neither platelet-poor plasma nor serum made from platelet-poor plasma, caused enhanced binding of fibronectin to cultured fibroblastic cells. The ability of whole blood serum to enhance binding of fibronectin was abolished by phospholipase B. These results indicate that lysophosphatidic acid derived from platelets is the principal component in whole blood serum that is active in the fibronectin binding assay. 1-oleoyl lysophosphatidic acid, 20-200 nM, was as active as 0.1-0.2% whole blood serum. The stimulatory effect of lysophosphatidic acid on the binding of fibronectin or the amino-terminal 70-kD fragment of fibronectin was rapid, sustained, and lost upon removal of lysophosphatidic acid. The stimulatory effect on binding could not be duplicated by bradykinin, platelet-activating factor, bombesin, or a peptide agonist of the thrombin receptor. Enhanced binding of the 70-kD fragment was due to increases in both the number and affinity of binding sites. Enhanced binding and assembly of fibronectin correlated with changes in cell shape and actin-containing cytoskeleton. The binding sites for fibronectin on lysophosphatidic acid-stimulated cells, as assessed by fluorescence, video, and scanning electron microscopy, were on areas of cell membrane containing numerous filopodia that extended between cells or between cells and substratum. These observations suggest that lysophosphatidic acid functions as a powerful and specific modulator of cell shape and early matrix assembly during wound healing.     

The blood flow characteristics in borderline ovarian tumors based on both color Doppler ultrasound and histopathological analyses

We previously reported that a macrophage response that increased binding to 125I-radiolabeled soluble denatured collagen (gelatin) was induced by preincubation of macrophage with a 70-kDa amino-terminal fibronectin fragment and soluble nonlabeled gelatin [S. F. Penc, F. A. Blumenstock, J. E. Kaplan (1995) J. Leukoc. Biol. 58, 501-509]. We now report that neither protein synthesis nor recycling of receptors between the cell surface and interior were required for this response. However, removal of cell surface components with trypsin demonstrated that induced gelatin binding required native cell surface constituents. It was found that in the presence of the 70-kDa fibronectin fragment and gelatin, matrix metalloprotease-2 (MMP-2) and matrix metalloprotease-9 (MMP-9) activity in the cell layers was significantly decreased or undetectable, respectively. Similar levels of increased gelatin binding could be reproduced after inhibition of matrix-degrading metalloprotease activity with 1'10-phenanthroline. These results demonstrate that a macrophage specific response that decreased gelatinase activity and increased gelatin binding was initiated by interaction with a 70-kDa fibronectin fragment and gelatin.     

MIA ("melanoma inhibitory activity"). Biological functions and clinical relevance in malignant melanoma]

The protein MIA was identified and isolated from the tissue culture supernatant of melanoma cells in vitro by its ability to inhibit thymidine incorporation by melanoma cell lines. After purification and partial sequencing of the peptide, a fragment of the MIA cDNA was cloned by RT-PCR. This cDNA fragment was used to screen phage libraries and subsequently fully encoding human and murine MIA cDNA and genomic DNA clones were obtained. The MIA gene spans a region of approximately 2 kb and is divided into 4 exons. Mapping the MIA gene revealed that the human gene is located on chromosome 19 and the murine gene on chromosome 7. The MIA open reading frame spans 131 (human) or 130 (murine) amino acids. The first 24 (human) or 23 (murine) amino acids represent a signal sequence directing the secretion of MIA into the extracellular compartment. The mature, secreted MIA consists of 107 amino acids and its MW is approximately 11 kDa. Preliminary structural data suggests that MIA is a small globular protein stabilized by two intramolecular disulfide bonds. Expression studies of protein und mRNA levels indicate that MIA is expressed specifically by malignant melanoma cells and chondrocytes. This points to a highly restricted expression pattern which is controlled by the MIA promoter. In addition, MIA provides a clinically useful parameter in patients with malignant melanoma. Enhanced values were measured in the serum of all patients with metastatic melanoma (stage III and IV). In vitro and in vivo experiments using recombinant MIA protein revealed that MIA specifically inhibits attachment of melanoma cells to fibronectin and laminin. Further analysis indicated a direct binding between MIA and the matrix proteins. This finding provides an explanation for the capability of MIA to inhibit proliferation of melanoma cells in vitro. Our studies suggest a putative function of MIA in regulated detachment of melanoma cells from the extracellular matrix which is an important step in metastasis.     

Cancer cells release a covalent complex containing disulfide-linked domains from urinary plasminogen activator, neural cell adhesion molecule, and haptoglobin alpha and beta chains

We have previously reported on the secretion of a family of high Mr plasminogen activators (PAs) by a human lung cancer cell line [Harvey et al. (1991) Biochim. Biophys. Acta 1078, 360-368]. We have now extended these studies to several human cancer cell lines and a human embryonic lung cell line. In the present study with HPL-SK-1 lung cancer, A431 epidermoid cancer, ovarian carcinoma, and embryonic lung cell lines, we show that the 900- and the 660-kDa PAs are disulfide-bonded multiprotein oligomeric complexes. They are functionally and immunologically related to human urinary PA (uPA). Their size and PA activity are not destroyed by strong denaturants such as 8 M urea or 2% sodium dodecyl sulfate (SDS), suggesting that the uPA moiety is covalently associated with the rest of the molecule. It is only under strong denaturing conditions with 1.4 M beta-mercaptoethanol and 2% SDS that the uPA moiety could be released as a 21- to 23-kDa fragment along with two major polypeptide chains of 70 and 40 kDa, respectively. The presence of the uPA active center in the reduced PA660 was demonstrated by [3H]diisopropylphosphorofluoridate labeling and by Western blot using a monoclonal antibody to uPA B chain. N-terminal amino acid sequencing of the 70- and 40-kDa polypeptides, respectively, showed homology to the neural cell adhesion molecule and the beta chain of haptoglobin. A minor fragment of 18 kDa obtained under strong reduction conditions was also sequenced and shown to share homology with the alpha chain of haptoglobin. Western blot analysis of the reduced PAs with monoclonal antibody to the neural cell adhesion molecule and rabbit anti-haptoglobin confirmed the homologies obtained by the sequence data. Further, immobilized monoclonal antibodies to the neural cell adhesion molecule, uPA B chain, and rabbit anti-haptoglobin bound the multiprotein complexes with uPA activity, from A431, ovarian cancer, and embryonic lung cell lines. The bound material, after dissociation, exhibited PA activity that was inhibited by monoclonal antibody to the uPA B chain. These data suggest that in tumor and embryonal cell lines, in addition to proper folding and assembly of proteins by intramolecular disulfide bond formation in the endomembrane compartment, intermolecular disulfide bonds could also occur, producing multiprotein oligomers as in the present case. Formation of such oligomers may have a selective advantage for such cells in the focalization of proteolytic activity through the interaction of the neural cell adhesion molecule domain with the extracellular matrix and in immunosuppression of lymphocytes by the haptoglobin portion of the complex.     

Cloning and deduced amino acid sequence of a novel cartilage protein (CILP) identifies a proform including a nucleotide pyrophosphohydrolase

The cDNA cloning and expression in vitro and in eukaryotic cells of a novel protein isolated from human articular cartilage, cartilage intermediate layer protein (CILP) is described. A single 4. 2-kilobase mRNA detected in human articular cartilage encodes a polypeptide of 1184 amino acids with a calculated molecular mass of 132.5 kDa. The protein has a putative signal peptide of 21 amino acids, and is a proform of two polypeptides. The amino-terminal half corresponds to CILP (molecular mass of 78.5 kDa, not including post-translational modifications) and the carboxyl-terminal half corresponds to a protein homologous to a porcine nucleotide pyrophosphohydrolase, NTPPHase (molecular mass of 51.8 kDa, not including post-translational modifications). CILP has 30 cysteines and six putative N-glycosylation sites. The human homolog of porcine NTPPHase described here contains 10 cysteine residues and two putative N-glycosylation sites. In the precursor protein the NTPPHase region is immediately preceded by a tetrapeptide conforming to a furin proteinase cleavage consensus sequence. Expression of the full-length cDNA in a cell-free translation system and in COS-7 or EBNA cells indicates that the precursor protein is synthesized as a single polypeptide chain that is processed, possibly by a furin-like protease, into two polypeptides upon or preceding secretion.     

Rho-mediated contractility exposes a cryptic site in fibronectin and induces fibronectin matrix assembly

Many factors influence the assembly of fibronectin into an insoluble fibrillar extracellular matrix. Previous work demonstrated that one component in serum that promotes the assembly of fibronectin is lysophosphatidic acid (Zhang, Q., W.J. Checovich, D.M. Peters, R.M. Albrecht, and D.F. Mosher. 1994. J. Cell Biol. 127:1447-1459). Here we show that C3 transferase, an inhibitor of the low molecular weight GTP-binding protein Rho, blocks the binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment to cells and blocks the assembly of fibronectin into matrix induced by serum or lysophosphatidic acid. Microinjection of recombinant, constitutively active Rho into quiescent Swiss 3T3 cells promotes fibronectin matrix assembly by the injected cells. Investigating the mechanism by which Rho promotes fibronectin polymerization, we have used C3 to determine whether integrin activation is involved. Under conditions where C3 decreases fibronectin assembly we have only detected small changes in the state of integrin activation. However, several inhibitors of cellular contractility, that differ in their mode of action, inhibit cell binding of fibronectin and the 70-kD NH2-terminal fibronectin fragment, decrease fibronectin incorporation into the deoxycholate insoluble matrix, and prevent fibronectin's assembly into fibrils on the cell surface. Because Rho stimulates contractility, these results suggest that Rho-mediated contractility promotes assembly of fibronectin into a fibrillar matrix. One mechanism by which contractility could enhance fibronectin assembly is by tension exposing cryptic self-assembly sites within fibronectin that is being stretched. Exploring this possibility, we have found a monoclonal antibody, L8, that stains fibronectin matrices differentially depending on the state of cell contractility. L8 was previously shown to inhibit fibronectin matrix assembly (Chernousov, M.A., A.I. Faerman, M.G. Frid, O.Y. Printseva, and V.E. Koteliansky. 1987. FEBS (Fed. Eur. Biochem. Soc.) Lett. 217:124-128). When it is used to stain normal cultures that are developing tension, it reveals a matrix indistinguishable from that revealed by polyclonal anti-fibronectin antibodies. However, the staining of fibronectin matrices by L8 is reduced relative to the polyclonal antibody when the contractility of cells is inhibited by C3. We have investigated the consequences of mechanically stretching fibronectin in the absence of cells. Applying a 30-35% stretch to immobilized fibronectin induced binding of soluble fibronectin, 70-kD fibronectin fragment, and L8 monoclonal antibody. Together, these results provide evidence that self-assembly sites within fibronectin are exposed by tension.     

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.     

Functional beta1-integrins release the suppression of fibronectin matrix assembly by vitronectin

beta1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., F?ssler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active beta1A is expressed. Third, beta1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, beta1-null cells adherent to vitronectin are flat and lack filopodia, whereas beta1-null cells adherent to fibronectin or beta1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional beta1-integrins.     

Norrie disease protein (norrin) forms disulfide-linked oligomers associated with the extracellular matrix

COS-7 cells transfected with a DNA construct encoding the 133 amino acids in norrin plus six histidine residues at its carboxyl terminus were pulse-labeled with [35S]cysteine, and the labeled norrin was examined in cell lysates, the medium, and the extracellular matrix. SDS-gel electrophoresis under reducing conditions showed that the norrin expressed had an apparent Mr = 14,000 and was present only in cell lysates and the extracellular matrix. Under nonreducing conditions, most of the norrin in the extracellular matrix was oligomers that contained up to approximately 20 monomers. One of the major extracellular species of norrin under reducing conditions after cross-linking of norrin oligomers with bis(sulfosuccinimidyl)suberate had an apparent Mr = 28,000, consistent with covalent cross-linked dimers. Thus the covalently cross-linked dimers are key structural components of norrin oligomers. By site-directed mutagenesis, the codon for half-cystine 95 in norrin was changed to one encoding alanine. The norrin C95A found in the extracellular matrix of cells transfected with this mutant was the size of dimers, indicating that half-cystine 95 is involved in oligomer formation. The corresponding half-cystine residue in human prepro-von Willebrand factor is also involved in interchain disulfide bond formation, which is consistent with the sequence identity of the half-cystine residues in norrin and part of the half-cystine residues in a disulfide-rich domain of von Willebrand factor. Replacement of valine at residue 60 in norrin by glutamic acid, a mutation found in humans with a severe type of Norrie disease, results in a considerable reduction (50%) in the amount of norrin in the extracellular matrix of transfected COS-7 cells. Replacement of arginine at residue 121 by glutamine, which is associated with a less severe type of Norrie disease, results in a reduction in the amount of norrin R121Q in the extracellular matrix (26%). These studies suggest that norrin is a secreted protein that forms disulfide-bonded oligomers that are associated with the extracellular matrix upon secretion from cells. Moreover, the disulfide-rich motif of norrin and prepro-von Willebrand factor promotes interchain disulfide bond formation among polypeptides in which it is found.     

Cardiac geometry and mass changes associated with pacing-induced cardiomyopathy in the dog

Olfactomedin is the major glycoprotein of the extracellular mucous matrix of frog olfactory neuroepithelium. It is responsible for the primary architecture of this extracellular matrix by forming via intermolecular disulfide bonds polymers, which are covered with evenly spaced carbohydrate groups. To study glycosylation of olfactomedin, we raised antibodies against the mature protein and antibodies against a region adjacent to an N-linked glycosylation site near its amino terminus. The latter antibodies cannot bind when this site is glycosylated and reveal precursors of olfactomedin in the perinuclear regions of Bowman's glands. In contrast, antiserum against the mature protein stains acinar regions of glands and the ciliary surface. Enzymatic deglycosylation of olfactomedin shows stepwise removal of carbohydrate and reveals a 51-kDa deglycosylated form. Our results indicate that, prior to secretion, most, if not all, of the six potential N-linked glycosylation sites of olfactomedin are glycosylated with carbohydrate moieties of about 8-10 sugar residues.     

N-linked glycosylation of the human Ca2+ receptor is essential for its expression at the cell surface

The human Ca2+ receptor (hCaR) is a member of the superfamily of G protein-coupled receptors. Its large (approximately 600 residue) amino-terminal extracellular domain contains 9 potential N-linked glycosylation sites. Immunoblot of cell membranes derived from HEK-293 cells, stably transfected with the hCaR, showed two major immunoreactive bands of approximately 150 and 130 kDa, respectively. Complete digestion of the membranes with PN-glycosidase F yielded a single major immunoreactive band of approximately 115 kDa, confirming the presence of N-linked glycosylation. Treatment of these cells with tunicamycin, which blocks N-linked glycosylation, inhibited signal transduction in response to Ca2+. Flow cytometric analysis showed decreased expression of the hCaR on the cell membrane in tunicamycin-treated cells. Immunoblot of tunicamycin-treated cells showed a reduction in the amount of the 150-kDa band and conversion of the 130-kDa band to the presumptively nonglycosylated 115-kDa form. Tunicamycin treatment of cells, transfected with a mutant hCaR complementary DNA containing a nonsense codon at position 599 preceding the 1st transmembrane domain, blocked the secretion of a 95-kDa protein, representing the amino-terminal extracellular domain, into the medium. These results demonstrate that N-linked glycosylation is required for normal expression of the hCaR at the cell surface.     

Solution structure of the glycosylated second type 2 module of fibronectin

Fibronectin is an extracellular matrix glycoprotein that plays a role in a number of physiological processes involving cell adhesion and migration. The modules of the fibronectin monomer are organized into proteolytically resistant domains that in isolation retain their affinity for various ligands. The tertiary structure of the glycosylated second type 2 module (2F2) from the gelatin-binding domain of fibronectin was determined by two-dimensional nuclear magnetic resonance spectroscopy and simulated annealing. The structure is well defined with an overall fold typical of F2 modules, showing two double-stranded antiparallel beta-sheets and a partially solvent-exposed hydrophobic cluster. An N-terminal beta-sheet, that was not present in previously determined F2 module structures, may be important for defining the relative orientation of adjacent F2 modules in fibronectin. This is the first three-dimensional structure of a glycosylated module of fibronectin, and provides insight into the possible role of the glycosylation in protein stability, protease resistance and modulation of collagen binding. Based on the structures of the isolated modules, models for the 1F22F2 pair were generated by randomly changing the orientation of the linker peptide between the modules. The models suggest that the two putative collagen binding sites in the pair form discrete binding sites, rather than combining to form a single binding site.     

Identification and characterization of a novel 9.2-kDa membrane sector-associated protein of vacuolar proton-ATPase from chromaffin granules

Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.     

The von Hippel-Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix

Fibronectin coimmunoprecipitated with wild-type von Hippel-Lindau protein (pVHL) but not tumor-derived pVHL mutants. Immunofluorescence and biochemical fractionation experiments showed that fibronectin colocalized with a fraction of pVHL associated with the endoplasmic reticulum, and cold competition experiments suggested that complexes between fibronectin and pVHL exist in intact cells. Assembly of an extracellular fibronectin matrix by VHL-/- renal carcinoma cells, as determined by immunofluorescence and ELISA assays, was grossly defective compared with VHL+/+ renal carcinoma cells. Reintroduction of wildtype, but not mutant, pVHL into VHL-/- renal carcinoma cells partially corrected this defect. Finally, extracellular fibronectin matrix assembly by VHL-/- mouse embryos and mouse embryo fibroblasts (MEFs), unlike their VHL+/+ counterparts, was grossly impaired. These data support a direct role of pVHL in fibronectin matrix assembly.     

Molecular cloning of Aralar, a new member of the mitochondrial carrier superfamily that binds calcium and is present in human muscle and brain

We have identified a new calcium-dependent subfamily of mitochondrial carrier proteins with members in Saccharomyces cerevisiae, Caenorhabditis elegans, and various mammalian species. The members of this subfamily have a bipartite structure: a carboxyl-terminal half with the characteristic features of the mitochondrial solute carrier superfamily and an amino-terminal extension harboring various EF-hand domains. A member of this subfamily (that we have termed Aralar) was cloned from a human heart cDNA library. The corresponding cDNA comprises an open reading frame of 2037 base pairs encoding a polypeptide of 678 amino acids. The carboxyl-terminal half of Aralar (amino acids 321-678) has high similarity with the oxoglutarate, citrate, and adenine nucleotide carriers (28-29% identity), whereas the amino-terminal half (amino acids 1-320) contains three canonical EF-hands. Aralar amino-terminal half was shown to bind calcium by 45Ca2+ overlay and calcium-dependent mobility shift assays. The subcellular localization of the protein in COS cells transfected with Aralar was exclusively mitochondrial. Antibodies against Aralar amino-terminal fusion protein recognized a 70-kDa protein in brain mitochondrial fractions. Northern blot analysis showed that the protein was expressed in heart, brain, and skeletal muscle. The domain structure, mitochondrial localization, and presence in excitable tissues suggests a possible function of Aralar as calcium-dependent mitochondrial solute carrier.