Conformational change in the vinculin C-terminal depends on a critical histidine residue (His-906).

A phospholipid-controlled interaction between the N-terminal and C-terminal domains of vinculin is thought to be a major mechanism that regulates binding activities of the protein. To probe the mechanisms underlying these interactions we used chemical modification and site-directed mutagenesis directed at histidine residues. Diethylpyrocarbonate (DEPC) modification of the C-terminal, but not the N-terminal, domain greatly decreased affinity of the N-terminal-C-terminal binding, implicating histidine residues in the C-terminal. Mutation of either or both C-terminal histidines (at positions 906 and 1026), however, did not affect N-C binding at neutral pH. The H906A mutation did prevent DEPC effects and also prevented the normal decrease in binding affinity for the N-terminal at lower pH. We found that the wild type C-terminal domain, but not the H906A mutant, underwent a conformational change at pH 6.5, reflected in an altered circular dichroism spectrum and apparent oligomerization. Phospholipid also induced conformational changes in the wild type C-terminal domain but not in the H906A mutant, even though the mutant protein did bind to the phospholipid. Finally, the sensitivity of the N-C interaction to phospholipid was much reduced by the H906A mutation. These results show that H906 plays a key role in the conformational dynamics of the C-terminal domain and thus the regulation of vinculin.

Interaction of the N- and C-terminal domains of vinculin. Characterization and mapping studies.

The vinculin head to tail intramolecular self-association controls its binding sites for other components of focal adhesions. To study this interaction, the head and tail domains were expressed, purified, and assayed for various characteristics of complex formation. Analytical centrifugation demonstrated a strong interaction in solution and formation of a complex more asymmetric than either of the individual domains. A survey of binding conditions using a solid-phase binding assay revealed characteristics of both electrostatic and hydrophobic forces involved in the binding. In addition, circular dichroism of the individual domains and the complex demonstrated that conformational changes likely occur in both domains during association. The interaction sites were more closely mapped on the protein sequence by deletion mutagenesis. Amino acids 181-226, a basic region within the acidic head domain, were identified as a binding site for the vinculin tail, and residues 1009-1066 were identified as sufficient for binding the head. Moreover, mutation of an acidic patch in the tail (residues 1013-1015) almost completely eliminated its ability to interact with the head domain further supporting the significance of ionic interactions in the binding. Our data indicate that the interaction between the head and tail domains of vinculin occurs through oppositely charged contact sites and results in conformational changes in both domains.

Expression and membrane assembly of a transmembrane region from Neu.

Transmembrane domains of receptor tyrosine kinases are increasingly seen as key modulatory elements in signaling pathways. The present work addresses problems surrounding expression, isolation, secondary structure recovery, and assembly into membranes, of the relatively large quantities of transmembrane peptides needed to investigate these pathways by NMR spectroscopy. We demonstrate significant correspondence between SDS-PAGE behavior of such peptides and their (2)H NMR spectra in lipid bilayer membranes. A 50-residue peptide, Neu(exp), containing the transmembrane portion of the receptor tyrosine kinase, Neu, was designed for expression in Escherichia coli. The sequence also contained 11-12 amino acids from each side of the transmembrane domain. The common problem of low expressivity of transmembrane peptides was encountered-likely associated with membrane toxicity of the desired gene product. This difficulty was overcome by expressing the peptide as a TrpE fusion protein in a pATH vector to target expression products to inclusion bodies, and subsequently removing the TrpE portion by cyanogen bromide cleavage. Inclusion bodies offered the additional benefits of reduced proteolytic degradation and simplified purification. The presence of a hexa-His tag allowed excellent recovery of the final peptide, while permitting use of denaturing solvents and avoiding the need for HPLC with its attendant adsorption losses. Isolated expressed peptides were found to be pure, but existed as high oligomers rich in beta-structure as evidenced by CD spectroscopy and SDS-PAGE behavior. Dissolution in certain acidic organic solvents led to material with increased alpha-helix content, which behaved in detergent as mixtures of predominantly monomers and dimers-a situation often considered to exist in cell membranes. For purposes of NMR spectroscopy, peptide alanine residues were deuterated in high yield during expression. The same acidic organic solvents used to dissolve and dissociate expressed transmembrane peptides proved invaluable for their assembly into lipid bilayers. Analogous transmembrane peptides from the human receptor tyrosine kinase, ErbB-2, demonstrated related phenomena.

Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants.

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.

Developmental expression of the collagen-binding heat-shock protein GP46 and collagen types I and IV in rat tissues.

The temporal expression of protein and mRNA encoding the collagen-binding heat-shock glycoprotein, gp46, were determined in the heart, kidney, and lung during early rat postnatal development. The steady-state levels of collagen types I and IV mRNA expression were also examined to determine if gp46 and these collagen types are co-regulated during ontogenesis. Western blot analysis using a monoclonal antibody to gp46 revealed that gp46 levels are developmentally regulated. In heart and kidney, gp46 levels were high on days 3 and 8, reduced significantly on day 25, and low to undetectable on day 69. Protein levels of gp46 in the lung exhibited a similar temporal pattern except on day 3, when very low levels of gp46 were detected. mRNA expression of gp46 during early postnatal development did not correlate with gp46 protein accumulation in these tissues, suggesting a complex pre- and post-translational regulatory scheme. In the heart, protein levels of gp46 correlated well with collagen type I alpha 1(I) mRNA expression but not with collagen type IV alpha 1(IV). In contrast, gp46 protein levels closely paralleled alpha 1(IV) expression in the kidney. Gp46 levels exhibited no apparent correlation with either alpha 1(I) or alpha 1(IV) levels in the lung. These results show that gp46 is developmentally regulated at both the protein and mRNA levels in a tissue specific manner. The relationship between gp46 and collagen alpha 1(I) and alpha 1(IV) chain mRNA expression also has been shown to be tissue specific.

Purification and properties of thyroid hormone receptor beta 1 expressed in Escherichia coli as a fusion protein.

Thyroid hormone receptor binds to specific DNA sequences and acts as a hormone-dependent transcriptional regulator. The protein can form homodimers, or heterodimers with the related 9-cis-retinoic acid receptor (RXR) or retinoic acid receptor (RAR) receptor families, leading to complex patterns of regulation. To obtain relatively large quantities of the receptor for biochemical studies, we have inserted the cDNA for human thyroid receptor beta into a variant of the pGEX vector (pGEX-KG) and produced the protein in Escherichia coli as a fusion with glutathione-S-transferase. Conditions for protein production, isolation on glutathione agarose, and thrombin cleavage to generate active receptor were developed. Final yields were approximately 1 mg/liter of culture. Scatchard plots of 125I-triiodothyronine binding data revealed a single class of sites with a Kd of 0.1 nM. An overlay assay was established to measure protein-protein binding and used to show a direct interaction with bacterially expressed RXR receptor. Binding of the purified receptor to DNA response elements measured in a DNA binding assay was increased by RXR to different extents, depending on the DNA sequence. This preparation will be useful in exploring the mechanisms of receptor activity.

Interaction of procollagen I and other collagens with colligin.

Colligin is a collagen-binding glycoprotein of molecular mass 46000 Da localized to the endoplasmic reticulum (ER) of diverse kinds of cells that produce collagen I. In order to help define its role in collagen biosynthesis and to study the interaction of colligin with procollagen I in detail, the binding characteristics of colligin purified from L6 myoblasts have been studied. A total of 3 mol were found to bind/mol of procollagen I, with a Kd of about 25 nM. Both pure and separated pro alpha 1(I) and procollagen alpha 2 (I) chains were able to compete with procollagen I for binding to colligin. However, colligin binds to pro alpha 2 (I) with higher affinity than to pro alpha 1 (I). To find if the binding activity of colligin was altered during purification, an assay to measure colligin binding to procollagen in crude myoblast cell extracts was developed. This procedure gave the same binding parameters as did the highly purified colligin. Among different collagen types, colligin was found to bind to collagen I and collagen IV, but not to collagen III. In order to examine whether glycosylation or phosphorylation of colligin were required for the binding of colligin to procollagen I and to obtain enough colligin for further studies, recombinant protein was produced in Escherichia coli. An immunoaffinity purification scheme was used to get virtually pure protein in milligram yields. Comparison of the recombinant colligin with that isolated from L6 myoblasts showed that both types existed in solution as monomers and dimers. In addition, both types of colligins showed identical properties with regard to their binding to procollagen I and the isolated pro alpha 1(I) and pro alpha 2(I) chains. Post-translational modifications of colligin were thus not essential for binding to procollagen I.

Inhibition of procollagen I degradation by colligin: a collagen-binding serpin.

Colligin is a collagen-binding glycoprotein localized to the endoplasmic reticulum (ER) and has been proposed to play a role in collagen biosynthesis. Its membership in the serpin family prompted us to examine its effect on procollagen degradation. We first showed that procollagen degradation can take place in the ER of L6 myoblasts by using brefeldin A to block transit from the ER. This degradation could be prevented by the serine protease inhibitors N-tosyl-L-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). To examine procollagen degradation in vitro, isolated liver microsomes were incubated with procollagen. Intact microsomes were unable to degrade labeled procollagen I, fibronectin, or the cytoplasmic proteins, phosphorylase b and the RI subunit of the cAMP-dependent protein kinase. However, when the microsomes were permeabilized by treatment with detergent, they became capable of degrading procollagen and fibronectin, but not the cytoplasmic proteins. The degrading activity was not due to cross-contamination by lysosomal or cytoplasmic, multicatalytic proteases. The proteolysis of procollagen chains in the treated microsomes was partially inhibited by TPCK, TLCK, and leupeptin. The most effective inhibitor was, however, colligin. In its presence, the breakdown of procollagen I, but not of fibronectin, was specifically inhibited. Colligin itself was not degraded by the microsomal preparations. The protein degrading activity was localized to the microsomal membranes, and showed a pH optimum of about 8.0. From these studies it is inferred that one of the roles of colligin may be to protect the procollagen I chains in the ER from degradation prior to their transport to the cis-Golgi compartment.

A novel very high molecular mass protein located in the membrane skeleton.

A monoclonal antibody generated from a mouse immunized with L6 rat myoblast cells was found to react with a major 700-kDa band and a minor 500-kDa band in immunoblots. Immunofluorescence microscopy demonstrated a submembranous location in tissue sections and an exclusion from stress fiber regions in cultured cells. Further, permeabilization of cultured cells with nonionic detergent prior to fixation changed the diffuse pattern of fluorescence to a web. These findings are characteristic of membrane skeletal proteins. In muscle tissue, the protein was much more abundant in fast twitch fibers and was found in internal locations as well as at the membrane. The protein could be solubilized in the absence of detergents and, hence, is not transmembrane. Although initially discovered in myoblast cells, the protein is present in a variety of tissue types, including brain, kidney, heart, liver, and lung. Pulse-chase labeling of the two bands suggested that the 500-kDa band was not a breakdown product of the 700-kDa protein. The protein appears to be a previously undiscovered membrane skeletal constituent for which the name "endossin" is proposed.

Protein kinase A regulation of cAMP phosphodiesterase expression in rat skeletal myoblasts.

We have been studying cAMP signaling in L6 myoblasts because of its potential role in regulating the differentiation of these cells into multinucleate myotubes. Previous studies have shown that treatment of L6 myoblasts with cAMP analogs causes an increase in cAMP phosphodiesterase activity. To assess the role of protein kinase A in this cAMP-mediated increase in cAMP phosphodiesterase activity, L6 myoblasts were transfected with a plasmid containing the cDNA for a mutant regulatory subunit of protein kinase A, which functions as a dominant negative inhibitor of this enzyme. The cDNA was under control of the metallothionein promoter in the construct. Induction of the mutant regulatory subunit with Zn2+ decreased cAMP-dependent protein kinase activity by 90%. Zn2+ treatment was also able to completely block the cAMP-mediated increase in phosphodiesterase activity, showing that this effect is mediated by protein kinase A. The activity of the cAMP-induced phosphodiesterase was inhibited by low concentrations of RO 20-1724, showing that it was a member of the type IV low Km cAMP phosphodiesterase family of enzymes. We used the polymerase chain reaction and consensus primers designed to amplify phosphodiesterase sequences to show that L6 myoblasts also contain mRNA for a type IV low Km cAMP phosphodiesterase designated PDE3.1. The levels of this mRNA were increased greatly by treatment with dibutyryl cAMP or forskolin in L6 myoblasts and also in differentiated L6 myotubes. Run-off transcription assays showed that this increase in PDE mRNA was regulated, at least in part, by an increase in the rate of transcription of the PDE3 gene. The induction of PDE3 message by cAMP was blocked when the L6 transfectants were treated with Zn2+ to induce protein kinase A inhibition. Therefore, some of the cAMP-mediated increase in phosphodiesterase activity seen in L6 myoblasts is due to a protein kinase A-mediated increase in PDE3 mRNA. This pathway may serve as a feedback mechanism to modulate the inhibitory effects of cAMP on myogenesis.

A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors.

Several cDNA clones encoding a 46-kDa collagen-binding glycoprotein (gp46) from rat skeletal myoblasts were isolated and sequenced. The cDNA encoded a 17-amino acid signal peptide and a 400-amino acid mature protein, containing three potential N-linked oligosaccharide attachment sites. The cDNA sequence of gp46 shows 93% identity in the coding region with J6, a retinoic acid-inducible gene coding for a protein of unknown function described from embryonal carcinoma F9 cells. The first 41 NH2-terminal amino acids of the predicted J6 sequence are, however, different from the gp46 sequence as a result of a 7-base pair insertion in the gp46 cDNA. In addition, the NH2-terminal amino acid sequence of hsp47, a collagen-binding protein found in chick embryo fibroblasts, shows 64% identity to gp46 over 36 residues. Interestingly, this alignment begins 10 residues inward from the first amino acid in the mature form of gp46. A significant sequence similarity was observed between gp46 and members of the serine protease inhibitor (serpin) family. Unlike other serpins, however, gp46 is both a heat shock and a collagen-binding protein and is localized to the lumen of the endoplasmic reticulum, as suggested by the presence of the RDEL sequence at the COOH terminus. This sequence is similar to other proposed endoplasmic reticulum retention signals.

Partial cleavage mapping of the cytoskeletal protein vinculin. Antibody and talin binding sites.

Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there. The use of [14C]cyanide in conjunction with DTNB showed which peptides contained the original amino terminus. Secondary cleavage with N-chlorosuccinimide, a tryptophan-specific reagent, helped locate fragments, although it led to apparent increases in molecular weight of the products. These experiments revealed the location of 10 of the major DTNB fragments on the sequence. This map was used to locate binding sites. The site of interaction between vinculin and the focal contact protein talin was mapped by binding labeled talin to the separated fragments. The binding site was found to be in the amino-terminal 325 amino acids. The binding site of a commercially obtained monoclonal antivinculin antibody was mapped using Western blotting of cleaved vinculin. It proved to bind in the central area of the molecule between amino acid residues 545 and 737. Thus the cysteine cleavage reaction products provide a map of general utility for locating features on the vinculin molecule.

Two stress proteins of the endoplasmic reticulum bind denatured collagen.

A differentiation-related gelatin-binding 46 kilodalton (kDa) glycoprotein in myoblasts (GP46, colligin) shares several properties with the 78-kDa glucose-regulated protein (GRP78), including location in the endoplasmic reticulum and related C-terminal sequences. These similarities extend to stress inducibility, since we find that GP46 is a heat-shock protein; its synthesis is elevated at 42 degrees C, resulting in a two- to three-fold increase in protein level. Further, GRP78 is a gelatin-binding protein; together with GP46 it is retained on gelatin-Sepharose beads. GRP78 and GP46 do not interact; each protein can be individually eluted, GP46 at low pH and GRP78 by ATP. These results suggest that the proteins have distinct roles in the synthesis of collagen and point to a simple method for purification.

Partial characterization of a collagen-binding, differentiation-related glycoprotein from skeletal myoblasts.

A 46-kDa glycoprotein, gp46, which binds collagen has been purified to homogeneity from L6 rat skeletal myoblasts. The procedure involves extraction of crude myoblast membranes with 1% sodium dodecyl sulfate followed by concanavalin A affinity chromatography and preparative gel electrophoresis. The sequence of 15 N-terminal amino acids had some resemblance to a sequence in myosin light chains. The oligosaccharide chains of the glycoprotein can be released by treatment with endoglycosidase H, suggesting that gp46 has high-mannose type of glycans. Galactose and sialic acid are not detected in the purified protein. gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in several fibroblastic and myogenic cell lines, but not in a hematopoietic cell line. Undifferentiated F9 embryonal carcinoma cells lacked gp46 but the glycoprotein was induced when the cells were made to differentiate in the presence of retinoic acid. Broad survey of gp46 in different cell lines also suggests that it is present mainly in those cell lines which attach to the substratum and produce collagens. Although the function of gp46 is not yet known, the evidence suggests that it is developmentally regulated and is probably involved in the synthesis or assembly of collagen in the endoplasmic reticulum.

Ethyl-3,4-dihydroxybenzoate inhibits myoblast differentiation: evidence for an essential role of collagen.

To study the role of (pro)collagen synthesis in the differentiation of rat L6 skeletal myoblasts, a specific inhibitor of collagen synthesis, ethyl-3,4-dihydroxybenzoate (DHB), was utilized. It is shown that DHB reversibly inhibits both morphological and biochemical differentiation of myoblasts, if it is added to the culture medium before the cell alignment stage. The inhibition is alleviated partially by ascorbate, which along with alpha-ketoglutarate serves as cofactor for the enzyme, prolyl hydroxylase. DHB drastically decreases the secretion of procollagen despite an increase in the levels of the mRNA for pro alpha 1(I) and pro alpha 2(I) chains. Probably, the procollagen chains produced in the presence of DHB, being underhydroxylated, are unable to fold into triple helices and are consequently degraded in situ. Along with the inhibition of procollagen synthesis, DHB also decreases markedly the production of a collagen-binding glycoprotein (gp46) present in the ER. The results suggest that procollagen production and/or processing is needed as an early event in the differentiation pathway of myoblasts.

Phosphorylation of high molecular weight proteins in platelets treated with 12-O-tetradecanoylphorbol-13-acetate.

Changes in the phosphorylation of three high molecular weight cytoskeletal proteins in platelets (actin binding protein, platelet talin and myosin heavy chain) were investigated after treatment with a phorbol ester. All three showed rapid increases in phosphate incorporation, reaching near-maximal values within three minutes. Phosphopeptide maps of the proteins before and after phorbol treatment revealed a single new site in myosin heavy chain, two new peptides in actin binding protein, and multiple sites in talin. These results point to multiple cytoskeletal targets of protein kinase C and suggest complex mechanisms for reorganizing microfilaments.

Inhibition of protein kinase C by sphingosine correlates with the presence of positive charge.

The role of the 2-amino group of sphingosine on the in vitro inhibition of protein kinase C was investigated by comparing protein kinase C activity in the presence and absence of sphingosine at various pH's. Inhibition by sphingosine was found to be pH dependent. Above pH 7.75, sphingosine has little or no inhibitory effect. In fact, at pH 8.5, sphingosine slightly enhances enzyme activity above that which occurs when the enzyme is stimulated by diacylglycerol and phosphatidylserine. After correcting for electrostatic repulsion, we find that the intrinsic pK for sphingosine in Triton micelles is 8.5. Inhibition of protein kinase C by sphingosine at physiological pH's therefore correlates with the presence of a positive charge.

Studies on the mechanism of action of a bilayer stabilizing inhibitor of protein kinase C: cholesterylphosphoryldimethylethanolamine.

Cholesterylphosphoryldimethylethanolamine is a zwitterionic compound which is a good bilayer stabilizer. As has been found with many other compounds having these properties, cholesterylphosphoryldimethylethanolamine is found to be a potent inhibitor of protein kinase C in both vesicle and micelle assay systems. The kinetics of the inhibition in Triton X-100 micelles was non-competitive with respect to ATP, histone, diolein, phorbol ester and Ca2+. It has a Ki of about 30 microns. The inhibition kinetics as a function of phosphatidylserine concentration is more complex but suggestive of competitive inhibition. Cholesterylphosphoryldimethylethanolamine does not prevent the partitioning of protein kinase C into the membrane. This inhibitor lowers the Ca2+-phosphatidylserine-independent phosphorylation of protamine sulfate by protein kinase C and directly affects the catalytic segment of the enzyme generated by tryptic hydrolysis. Thus, this zwitterionic bilayer stabilizing inhibitor of protein kinase C both competes with the binding of phosphatidylserine as well as affects the active site of protein kinase C.

A collagen-binding protein involved in the differentiation of myoblasts recognizes the Arg-Gly-Asp sequence.

We had earlier demonstrated that a 46-kDa glycoprotein is involved in the differentiation of rat skeletal myoblasts. We now show that the binding of this glycoprotein to collagen and gelatin is disrupted by Arg-Gly-Asp (RGD) containing peptide but not by Arg-Gly-Glu (RGE). The former peptide also selectively elutes the 46-kDa glycoprotein bound to gelatin-Sepharose. Since all other proteins which bind RGD sequences have been found at the cell surface, we attempted to localize the 46-kDa glycoprotein by means of immuno fluorescent staining and radioiodine labeling. Surprisingly, the majority of the protein was found to be localized in the endoplasmic reticulum. Protease treatment of a microsomal fraction revealed that the protein is in the interior of the reticulum. Immunoprecipitation experiments, using a polyclonal antibody against the 46-kDa protein, demonstrated that no closely related proteins exist in myoblasts and also confirmed that the protein was not a fragment of a cell-surface localized protein. These findings suggest that the RGD sequence is also used in protein recognition within the cell.