Specific sequence elements are required for the expression of functional tumor necrosis factor-alpha-converting enzyme (TACE).

The tumor necrosis factor-alpha-converting enzyme (TACE) is a membrane-anchored zinc metalloprotease involved in precursor tumor necrosis factor-alpha secretion. We designed a series of constructs containing full-length human TACE and several truncate forms for overexpression in insect cells. Here, we demonstrate that full-length TACE is expressed in insect cells inefficiently: only minor amounts of this enzyme are converted from an inactive precursor to the mature, functional form. Removal of the cytoplasmic and transmembrane domains resulted in the efficient secretion of mature, active TACE. Further removal of the cysteine-rich domain located between the catalytic and transmembrane domains resulted in the secretion of mature catalytic domain in association with the precursor (pro) domain. This complex was inactive and function was only restored after dissociation of the complex by dilution or treatment with 4-aminophenylmercuric acetate. Therefore, the pro domain of TACE is an inhibitor of the catalytic domain, and the cysteine-rich domain appears to play a role in the release of the pro domain. Insect cells failed to secrete a deletion mutant encoding the catalytic domain but lacking the inhibitory pro domain. This truncate was inactive and extensively degraded intracellularly, suggesting that the pro domain is required for the secretion of functional TACE.     

Interactions of the Cytoplasmic Domain of P-Selectin with Clathrin-coated Pits Enhance Leukocyte Adhesion under Flow

Flowing leukocytes tether to and roll on P-selectin, a receptor on endothelial cells that is rapidly internalized in clathrin-coated pits. We asked whether the association of P-selectin with clathrin-coated pits contributes to its adhesive function. Under flow, rolling neutrophils accumulated efficiently on CHO cells expressing wild-type P-selectin or a P-selectin construct with a substitution in the cytoplasmic domain that caused even faster internalization than that of the wild-type protein. By contrast, far fewer rolling neutrophils accumulated on CHO cells expressing P-selectin constructs with a deletion or a substitution in the cytoplasmic domain that impaired internalization. Neutrophils rolled on the internalization-competent constructs with greater adhesive strength, slower velocity, and more uniform motion. Flowing neutrophils tethered equivalently to internalization-competent or internalization-defective P-selectin, but after tethering, they rolled further on internalization-competent P-selectin. Confocal microscopy demonstrated colocalization of α-adaptin, a component of clathrin-coated pits, with wild-type P-selectin, but not with P-selectin lacking the cytoplasmic domain. Treatment of CHO cells or endothelial cells with hypertonic medium reversibly impaired the clathrin-mediated internalization of P-selectin and its ability to support neutrophil rolling. Interactions of the cytoplasmic domain of P-selectin with clathrin-coated pits provide a novel mechanism to enhance leukocyte adhesion under flow.     

Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells

In addition to the mature 50 amino acid transforming growth factor alpha (TGF alpha), some transformed cells appear to produce multiple higher molecular weight forms. The structure and derivation of most of these larger soluble TGF alpha species remain to be established. We previously reported that a chemically induced rat hepatocellular carcinoma cell line, JM1, secreted acid-stable proteins which bind to epidermal growth factor receptors and stimulate DNA synthesis in primary cultures of normal adult rat hepatocytes. Purification and characterization of these hepatoma-derived growth factors have indicated their relationship to TGF alpha. Two EGF-competing activities of apparent Mr 30K and 10K were separated by gel filtration of concentrated JM1-conditioned medium and further purified by ion-exchange chromatography and reverse-phase HPLC. Both growth factors were detected by a radioimmunoassay specific for TGF alpha. Western blotting with antibodies to the 50 amino acid TGF alpha revealed that the lower molecular weight factor comigrated with the synthetic 6-kDa rat TGF alpha. The higher molecular weight TGF alpha appeared on immunoblots as a diffuse band of 18-21 kDa, which converted to the mature 6-kDa form upon digestion with elastase, confirming a precursor-product relationship. However, the 18-21-kDa proteins did not react with antibodies directed against the carboxy-terminal cytoplasmic segment of the transmembrane TGF alpha precursor. Enzymatic deglycosylation of the 18-21-kDa TGF alpha species by sequential removal of sialic acids and O- and N-linked carbohydrate reduced the molecular weight to 11K. The size and soluble nature of this polypeptide suggest that it represents the extracellular domain of the transmembrane TGF alpha precursor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Cysteine Residue in the Carboxyl-Terminal Domain of the m2 Muscarinic Acetylcholine Receptor Is Not Required for Receptor-Mediated Inhibition of Adenylate Cyclase

Muscarinic acetylcholine receptors (mAChRs) share with many other receptors of the guanine nucleotide-binding protein-coupled receptor family a highly conserved cysteine residue in the putative cytoplasmic carboxyl-terminal region of the protein. Because elimination of this cysteine in the beta 2-adrenergic receptor has been reported to decrease functional responsiveness, we determined if this cysteine residue is essential for mAChR-effector coupling by replacing Cys457 of the m2 mAChR with glycine and expressing wild-type and mutant receptor in Chinese hamster ovary (CHO) cells. The mutant and wild-type receptors exhibited similar affinities for binding of muscarinic ligands. In addition, the mutation did not affect cell surface localization or receptor-mediated inhibition of adenylate cyclase. These results indicate that the cysteine residue in the carboxyl-terminal domain of the m2 mAChR is not required for ligand binding or mAChR-mediated inhibition of adenylate cyclase in CHO cells.     

Tissue polarity genes of Drosophila regulate the subcellular location for prehair initiation in pupal wing cells

The purpose of this study was to explore the functional role of the cytoplasmic domain of the alpha subunit of the alpha 5/beta 1 integrin, a fibronectin receptor. Mutant CHO cells that express very low levels of endogenous hamster alpha 5 subunit (CHO clone B2) were transfected with an expression vector containing full-length or truncated human alpha 5 cDNAs to form chimeric human alpha 5/hamster beta 1 integrins. Three transfectants were examined: B2a27 expresses a full-length human alpha 5 subunit with 27 amino acids in the cytoplasmic domain; B2a10 expresses an alpha 5 with a 17-amino acid cytoplasmic truncation; B2a1 expresses an alpha 5 with a 26-amino acid truncation. Levels of alpha 5/beta 1 surface expression in B2a27 and B2a10 cells were similar to that in wild type CHO cells. The expression of alpha 5/beta 1 in B2a1 cells was less, amounting to 15-20% of WT levels, despite message levels that were three to five times greater than those of B2a27. The transfectants were used to examine the role of the alpha 5 cytoplasmic domain in cell adhesion, cell motility, cytoskeletal organization, and integrin-mediated tyrosine phosphorylation. The adhesion characteristics of B2a27 and B2a10 cells on fibronectin substrata were similar to each other and to wild type CHO cells. B2a1 cells displayed slight reductions in the strength and rate of adhesion to fibronectin. Cell motility in the presence of fibronectin was similar for B2a27, B2a10, and wild type CHO cells, while the B2a1 cells were substantially less motile. Comparable degrees of cell spreading and extensive organization of actin filaments were observed for B2a27, B2a10, and wild type CHO cells on fibronectin substrata. The B2a1 cells spread to a lesser degree, and some organization of actin was observed; the untransfected B2 cells remained round on fibronectin substrata and showed no actin reorganization. Since the reduced motility and cell spreading observed in the B2a1 cells might be due either to reduced surface expression of alpha 5/beta 1 or to the truncation in the alpha 5 cytoplasmic domain, we used flow cytometric cell sorting to select populations of B2a1 and B2a27 cells expressing similar levels of cell surface alpha 5. The deficits in spreading and motility were present in B2a1 cells expressing high levels of alpha 5. Thus the region of the alpha 5 cytoplasmic domain adjacent to the membrane seems to play an important role in cytoskeletal organization and cell motility. We also examined whether alpha subunit truncation would affect integrin- mediated tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1

A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta 1 (TGF beta 1) encoded by simian TGF beta 1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF beta 1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF beta 1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF beta 1, when expressed as the full-length pre-pro-TGF beta 1, appeared to block secretion. By contrast, if the pro domain (designated beta 1-latency-associated peptide [beta 1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110-210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF beta 1 and biological assays indicate that residues 50-85 of beta 1-LAP are required for association with mature TGF beta 1.     

Transforming growth factor alpha in arterioles: cell surface processing of its precursor by elastases.

Analysis of the transforming growth factor alpha (TGF alpha) cDNA predicts that the mature TGF alpha polypeptide is cleaved from the extracellular domain of its precursor, which is an integral membrane protein. Furthermore, the cleavage sites for the release of this mitogen are compatible with the participation of an elastaselike protease. We have immunohistochemically localized TGF alpha to the vascular smooth muscle cells in the arterioles. To investigate whether polymorphonuclear (PMN) leukocytic elastase, a blood-borne protease, could process the cell surface TGF alpha, NR6 cells were transfected with the rat TGF alpha cDNA. The cDNA encoded the entire open reading frame, and its expression was under the control of the mouse metallothionein I promoter. A cloned transfectant, termed 1B2, synthesized the TGF alpha precursor in a zinc-inducible manner, and the precursor was localized to the cell surface. Western blot (immunoblot) analysis indicated that treatment of the zinc-induced 1B2 cells with either PMN leukocytic or pancreatic elastase resulted in the release of the mature TGF alpha polypeptide. The released TGF alpha was bioactive, as it was capable of both competing with epidermal growth factor for binding to its receptor and stimulating [3H]thymidine incorporation in the mitogenic assay. Formaldehyde fixation of the 1B2 cells eliminated basal release of TGF alpha but allowed normal processing by both PMN leukocytic and pancreatic elastase to occur. However, human cathepsin G, bovine pancreatic alpha 1-chymotrypsin, collagenase, trypsin, subtilisin, and plasmin failed to release any detectable fragments of the TGF alpha precursor from the fixed cells. The location of TGF alpha in the arterioles and ability of PMN leukocytic elastase to process the membrane-bound TGF alpha precursor suggests a novel role for this elastase at the wound site.     

Proteolytic processing of TGFα redirects its mitogenic activity: the membrane-anchored form is autocrine,the secreted form is paracrine

Wild-type transforming growth factor α (TGFα) expression in lactotrope cells in the pituitary gland led to lactotrope-specific pituitary hyperplasia and adenomata. To indicate whether the EGF receptor is involved in this TGFα-mediated phenotype, we bred TGFα mice with mice expressing the cytoplasmic truncated-EGF receptor (EGFR-tr), which is dominant-negative in other models. These bitransgenic mice developed pituitary pathology despite expression of the dominant-negative receptor. To further characterize this observation, we generated two lineages of transgenic mice that overexpress mutant forms of TGFα: a processed soluble form (s TGFα) and a cytoplasmic-deleted form (TGFαΔC). While sTGFα expression in lactotrope cells failed to induce autocrine lactotrope hyperplasia, the pituitary became very enlarged due to proliferation of neighboring interstitial cells. In contrast, the TGFαΔC mice did not develop a phenotype, although the mRNA and protein were present in the pituitary and this form of TGFα was confirmed to be biologically active and targeted properly to the plasma membrane of cultured CHO cells. The results suggest that the cytoplasmic domain of TGFα is required for autocrine parenchymal tumor formation in the pituitary gland. This signal cannot be inhibited by the EGFR-tr. Conversely, the released form of TGFα appears to have primarily paracrine activity.     

Rational design of a chimeric toxin: an intramolecular location for the insertion of transforming growth factor alpha within Pseudomonas exotoxin as a targeting ligand.

To investigate the potential utility of Pseudomonas exotoxin (PE) in forming rationally designed chemotherapeutic agents, we inserted a cDNA encoding transforming growth factor alpha (TGF alpha) at several locations in a gene encoding a mutant full-length PE (PE4E) which does not bind to the PE receptor. After expression in Escherichia coli, we purified the chimeric toxins to near homogeneity and showed that they were specifically cytotoxic to human epidermoid, ovarian, colon, and hepatocellular carcinoma lines. Like the previously reported TGF alpha-PE40, one of the new molecules (TGF alpha-PE4E) contains the ligand at the amino terminus. Two additional chimeras (PE4E-TGF alpha and PE4E-TGF alpha-598-613) each contain TGF alpha inserted near the carboxyl terminus of PE. We show that preservation of the correct PE carboxyl-terminal amino acid sequence, REDLK, allows the toxins containing TGF alpha carboxyl inserts to retain significant cytotoxicity against target cells, since another molecule (PE4E-TGF alpha-ILK) containing a nonfunctional carboxyl-terminal sequence was over 100-fold less active. The chimeric toxins with TGF alpha had the same binding affinity for the EGF receptor whether the ligand occupied the amino or carboxyl position. Molecules with TGF alpha near the carboxyl position were consistently less active against target cells but also less toxic to mice than those with TGF alpha at the amino terminus, indicating both types of molecules might be therapeutically effective. Our results establish that a ligand can be placed near the carboxyl terminus of PE, within the portion of the toxin that translocates to the cytosol. The amino-terminal position in such molecules is then available for the placement of other targeting ligands.     

Integrin cytoplasmic domains mediate inside-out signal transduction

We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.     

Latent forms of transforming growth factor-beta (TGF beta) derived from bone cultures: identification of a naturally occurring 100-kDa complex with similarity to recombinant latent TGF beta

Transforming growth factor-beta (TGF beta) is produced by most tissues, including bone, as a complex that is biologically inert. Release of TGF beta homodimer from this latent complex is necessary for TGF beta to exert effects on target cells. Thus, the nature of the latent complex and the mechanisms responsible for TGF beta release are the key to understanding TGF beta actions. We have found that murine calvarial bone cultures secrete multiple latent forms of TGF beta. Using analytical chromatography and Western blot analysis, we have compared bone latent TGF beta with the previously characterized latent complex present in platelets and with simian TGF beta precursor, which is stably expressed in a latent form by Chinese hamster ovarian (CHO) cells. A major component of the bone material appears to be a latent complex of 100 kDa, consisting of mature TGF beta (25-kDa homodimer). Like the recombinant TGF beta precursor, it elutes from a Mono-Q fast pressure liquid chromatography anion exchange column at 0.2 M NaCl and shows a very similar banding pattern on Western blots. Thus, this bone complex closely resembles recombinant TGF beta precursor expressed in a latent form by CHO cells and differs from the naturally occurring platelet complex, which has an additional 135-kDa binding protein that is bound through disulfide bonds to the precursor proregion. Western blot analysis also indicates that, like CHO cells, which express recombinant TGF beta precursor, but unlike other cell types, the bone cultures secrete detectable amounts of uncleaved TGF beta precursor. The bone calvarial culture is the first example of a naturally occurring system that expresses the 100-kDa latent TGF beta complex.     

The Tetraspanin CD9 Influences the Adhesion, Spreading, and Pericellular Fibronectin Matrix Assembly of Chinese Hamster Ovary Cells on Human Plasma Fibronectin

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and na?ve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.     

Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.     

Low binding capacity and altered O-linked glycosylation of low density lipoprotein receptor in a monensin-resistant mutant of Chinese hamster ovary cells

We have studied function and structure of the low density lipoprotein (LDL) receptors in a monensin-resistant (Monr-31) mutant isolated from Chinese hamster ovary (CHO) cells. To assay the ability of the receptor to bind LDL, we employed three methods, 125I-LDL binding to the cells at 4 degrees C, 125I-LDL binding to the receptor-phospholipid complex (Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1980) J. Biol. Chem. 255, 11442-11447), and ligand blotting (Daniel, T.O., Schneider, W.J., Goldstein, J.L., and Brown, M.S. (1983) J. Biol. Chem. 258, 4606-4611). The LDL receptor number was similar in both CHO and Monr-31, but the binding affinity was reduced in the mutant. The semi-quantitative immunoblotting assay with an antibody directed against the COOH-terminal 14 amino acids and the ligand-blotting assay with LDL also showed that the relative steady-state level of the receptor in Monr-31 was comparable to that in CHO, whereas the binding capacity of the receptor in Monr-31 was lower than that in CHO. The precursor and degradation forms of the LDL receptors produced in the mutant cells were similar in size to those in the parental cells, but the apparent molecular mass of the mature receptor protein in sodium dodecyl sulfate-polyacrylamide gels was reduced about 5000 daltons in the mutant. These results suggest a structural change at the NH2-terminal LDL binding domain. Tests of the effects of tunicamycin, endo-alpha-N-acetylgalactosaminidase (O-glycanase), and sialidase (neuraminidase) on the molecular size of the mature receptors indicated that the reduced size of the receptor in the mutant cells resulted from altered oligosaccharide chain(s) linked to serine/threonine residues in the binding domain. We compared the molecular sizes and binding activity of human LDL receptors in several clones derived from CHO and Monr-31 cells which were transfected with human LDL receptor cDNA. The human LDL receptors produced in the transfected clones of Monr-31 were also smaller in molecular size and lower in binding capacity than those produced in the transfected clones of CHO. These results suggest that both structural and functional alteration of the LDL receptor of Monr-31 is not caused by a mutation in the structural gene of the LDL receptor but by altered processing or maturation of the receptor. The correlation of the decrease in molecular size and reduced binding capacity of the LDL receptor is discussed.     

Regulation of glycoprotein Ib-IX-von Willebrand factor interaction by cAMP-dependent protein kinase-mediated phosphorylation at Ser 166 of glycoprotein Ib(beta)

The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates initial platelet adhesion and activation. It is known that the cytoplasmic domain of GPIbbeta is phosphorylated at Ser(166) by cAMP-dependent protein kinase (PKA). To understand the physiological role of GPIbbeta phosphorylation, a GPIb-IX mutant replacing Ser(166) of GPIbbeta with alanine (S166A) and a deletion mutant lacking residues 166-181 of GPIbbeta (Delta165) were constructed. These mutants, expressed in Chinese hamster ovary (CHO) cells, showed an enhanced VWF-binding function compared with wild type GPIb-IX. Treatment of CHO cells expressing wild type GPIb-IX with a PKA inhibitor, PKI, reduced Ser(166) phosphorylation and also enhanced VWF binding to GPIb-IX. Furthermore, cells expressing S166A or Delta165 mutants showed a significantly enhanced adhesion to immobilized VWF under flow conditions. Consistent with the studies in CHO cells, treatment of platelets with PKI enhanced VWF binding to platelets. In contrast, a PKA stimulator, forskolin, reduced VWF binding and VWF-induced platelet agglutination, which was reversed by PKI. Thus, PKA-mediated phosphorylation of GPIbbeta at Ser(166) negatively regulates VWF binding to GPIb-IX and is one of the mechanisms by which PKA mediates platelet inhibition.     

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.     

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.     

The cytoplasmic domain of the alpha1 integrin subunit influences stress fiber formation via the conserved GFFKR motif

Integrins are heterodimeric transmembrane proteins that mediate substrate adhesion and migration but also the bidirectional transfer of information across the plasma membrane via their cytoplasmic domains. We addressed the question of whether the very short cytoplasmic tail of the alpha1 integrin subunit of alpha1beta1 integrin is required for alpha1beta1-specific adhesion, spreading, and migration. For this purpose we transfected the alpha1 integrin subunit and two cytoplasmically truncated alpha1 subunits into Chinese hamster ovary (CHO) cells. Elimination of the entire cytoplasmic domain of the alpha1 subunit does not affect adhesion but leads to inhibition of spreading and stress fiber formation. The defect in spreading could not be rescued by lysophosphatidic acid, which has been reported to stimulate actin stress fiber formation via Rho. Additionally, deletion of the entire cytoplasmic domain of the alpha1 subunit abolishes migration toward alpha1beta1-specific substrates. Migration and stress fiber formation are similar in CHO-alpha1 cells and CHO cells carrying an alpha1 subunit still containing the conserved GFFKR motif. So, the GFFKR motif of the alpha1 subunit is essential and sufficient for these processes.     

Contrasting roles for integrin beta 1 and beta 5 cytoplasmic domains in subcellular localization, cell proliferation, and cell migration

To carry out a detailed comparison of the roles of integrin beta 1 and beta 5 cytoplasmic domains, we expressed both wild type beta 1 and chimeric beta 1/5 constructs in CHO cells. In the latter, the cytoplasmic domain of beta 1 was replaced with that of beta 5. The human beta 1 and beta 1/5 constructs appeared at similar levels at the cell surface (mostly as alpha 5 beta 1 heterodimers) and contributed equally to CHO cell adhesion to fibronectin. However, beta 1 but not beta 1/5 localized to focal adhesion-like structures when CHO cells were spread on fibronectin. Furthermore, only the beta 1-CHO cells showed increased proliferation in response to fibronectin plus an integrin-activating anti-beta 1 antibody, and showed increased appearance of 32P-labeled protein (p90) that correlated with proliferation. In sharp contrast, the beta 1/5-CHO cells were notably more migratory than beta 1-CHO cells in a transwell haptotactic migration assay. These results indicate that the beta 1 and beta 5 integrin subunit cytoplasmic domains can translate similar adhesive information into highly contrasting subsequent events. Thus, we have established that "inside-out" and "outside-in" integrin signaling pathways are regulated by fundamentally distinct mechanisms. In addition, we suggest that the same properties of the beta 1 cytoplasmic domain that promote recruitment to visible focal adhesion-like structures may also be conductive to cell proliferation. Conversely, the properties of the beta 5 tail that make it less likely to localize into focal adhesion-like structures may contribute to enhanced cell migration.     

Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.