Differential modulation of cell phenotype by different molecular weight forms of basic fibroblast growth factor: possible intracellular signaling by the high molecular weight forms

To study possible functional differences of the 18-kD and high molecular weight forms of basic fibroblast growth factor (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, 22, 22.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (22, 22.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. Growth in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways.     

Preferential binding of high molecular weight forms of von Willebrand factor to fibrillar collagen

The time- and concentration-dependent binding of von Willebrand factor to fibrillar collagen was examined by following the disappearance from plasma of ristocetin cofactor activity and factor VIII-related antigen, the functional and immunologic determinants of von Willebrand factor. Examination of both bound and unbound factor VIII-related antigen by crossed immunoelectrophoresis revealed a preferential binding of the higher molecular weight forms of von Willebrand factor to fibrillar collagen.     

Similar high molecular weight forms of growth hormone-releasing hormone are found in rat brain and testis

We have utilized a new radioimmunoassay for rat growth hormone-releasing hormone (GHRH) to investigate the presence of GHRH in different organ systems of adult rat, and specifically the rat central nervous system (CNS). The highest concentration of GHRH was found, as expected, in the hypothalamus, but significant amounts were also located in the brain cortex, predominantly the frontal cortex, as well as in the testis. Smaller amounts were identified in the cerebellum and brain stem. Sephadex and reversed phase high performance liquid chromatography demonstrated that while hypothalamic GHRH exclusively eluted at the position of rat GHRH (1-43), in testis and brain the major form was predominantly (testis) or wholly (brain) of a higher molecular weight. While this molecular species has yet to be further characterized, the data suggest the similar GHRH-like species exist in the CNS as well as the testis.     

High molecular weight forms of adrenocorticotropic hormone are glycoproteins.

Mouse pituitary tumor cells (AtT-20/D-16v) were incubated in medium containing [3H] glucosamine or [3H] mannose. By analyzing immunoprecipitates of cell extracts and culture medium it was shown that [3H] glucosamine and [3H] mannose were incorporated into all three high molecular weight forms of ACTH; label was not incorporated into Mr=4,500 ACTH (which is thought to be similar to the 39 amino acid polypeptide form of ACTH, alpha(1-39)). Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the apparent molecular weights of these glycoprotein forms of ACTH were 31,000, 23,000, and 13,000. Gel filtration in 6 M guanidine HCl indicated that the molecular weights of these forms of ACTH were substantially lower; sodium dodecyl sulfate-polyacrylamide gel electrophoresis has often been found to overestimate the molecular weight of glycoproteins. A significant fraction of the high molecular weight ACTH in tumor cell extracts binds to columns of concanavalin A-agarose and can be eluted with 0.2 M alpha-methyl-D-mannopyranoside; porcine alpha(1-39) does not bind to concanavalin A-agarose. High molecular weight glycoprotein ACTH can be detected in extracts of mouse and bovine pituitary by using concavalin A affinity chromatography.     

Higher molecular weight immunoreactive species of luteinizing hormone releasing hormone: possible precursors of the hormone.

Separation of extracts of sheep hypothalami on Sephadex G-25 gave three peaks exhibiting luteinizing hormone releasing hormone immunoreactivity. One peak corresponded in elution volume with luteinizing hormone releasing hormone but the others (I and II) eluted earlier, indicating that they are of higher molecular weight. Elution volumes were unaffected by 8 M urea treatment. Incubation of I and II with hypothalamic peptidases produced a small quantity of immunoreactive material eluting in the luteinizing hormone releasing hormone region. Digestion of I with trypsin resulted in a marked increase in total immunoreactivity and the production of material with the same elution volume as II. Tryptic digestion of II gave rise to a small quantity of immunoreactive peptide eluting in the luteinizing hormone releasing hormone region. The amount of I and II relative to luteinizing hormone releasing hormone was lower in the median eminence than in the supra optic chiasmatic and basal hypothalamic regions.     

Steroidogenic activity of high molecular weight forms of corticotropin.

The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Mammalian high molecular weight and monomeric forms of valyl-tRNA synthetase

Valyl-tRNA synthetase from rat liver sediments at 15.5 S with a Stokes radius of 90 A, corresponding to a native molecular weight of 585,000. Purification of valyl-tRNA synthetase to homogeneity by a combination of conventional and affinity column chromatography yields a fully active monomeric form of valyl-tRNA synthetase with a sedimentation coefficient of 7.7 S and a Stokes radius of 45 A. The subunit molecular weight of the monomeric valyl-tRNA synthetase is 140,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the presence of 400 mM KCl, the purified monomeric valyl-tRNA synthetase associates to a high molecular weight form. The high molecular weight valyl-tRNA synthetase in the homogenate can be readily converted to the monomeric form by controlled trypsinization. The kinetic parameters of the two forms are nearly identical. The results suggest that the high molecular weight valyl-tRNA synthetase is a homotypic tetramer and converts to the monomeric valyl-tRNA synthetase after the cleavage of a small peptide.     

Purification of a high molecular weight somatomedin binding protein from human plasma

The somatomedins insulin-like growth factor I and II (1,2) are in serum bound to high-molecular weight binding proteins (6,7,8). By use of a four step chromatographic procedure a somatomedin binding protein was isolated from outdated human plasma. Exclusion chromatography on Sephadex G-200 disclosed a molecular weight of 150 kDa. After lyophilization however, the binding activity was found in a lower molecular weight range of 35-45 kDa. A partial amino acid sequence analysis of the lyophilized material revealed a possible N-terminal sequence of Ala-Pro-Trp. This sequence is identical to the N-terminal sequence of the 35 kDa somatomedin binding protein previously isolated from human amniotic fluid (16).     

Atrial natriuretic factor: atrial conversion of high to low molecular weight forms

The atrial natriuretic factor elutes by gel filtration in high and low molecular weight fractions. Extraction and elution of rat atria in 1.0 M acetic acid yielded a predominance of the high molecular weight form(s); whereas when these procedures were carried out in 0.1 M acetic acid, there was a predominance of the low molecular weight forms. When partially purified high molecular weight natriuretic activity was eluted in 0.1 M acetic acid, the high molecular weight form(s) remained intact. When partially purified high molecular weight natriuretic activity was mixed with crude atrial extract in 0.1 M acetic acid, there was an apparent conversion to the low molecular weight forms. Extraction of rat atria in boiling 0.1 M acetic acid blocked this conversion. It is concluded that rat atria contain a heat labile factor that converts high molecular weight natriuretic activity to the low molecular weight forms.     

Mapping of caldesmon: relationship between the high and low molecular weight forms

Caldesmon is a widely distributed contractile protein that occurs in both a high molecular weight [120-150-kilodalton (kDa)] and a low molecular weight (71-80-kDa) form, depending on the tissue. The structural relationship between these two forms was examined by mapping techniques. Partial cyanogen bromide cleavage in conjunction with sodium dodecyl sulfate gel electrophoresis was used to construct a map of the cleavage points and determine the relative position of the fragments in a high molecular weight caldesmon from chicken gizzard (caldesmon125). By use of this map, markers for different regions of the protein were obtained: Antibodies directed toward certain areas were prepared by affinity purification, and specific 125I-labeled tryptic peptides were found to originate from terminal cyanogen bromide fragments. Mapping of a lower molecular weight form of caldesmon (caldesmon72 from chicken liver) revealed the presence of sequences located in both ends of caldesmon125. A terminal 38-kDa fragment of both proteins was apparently identical on the basis of arrangement of cleavage sites, antibody reactivity, and iodopeptide mapping. Fragments from the other end of both proteins exhibited an identical pattern of peptides. These results show that it is sequences located in the central area of caldesmon125 which are missing in caldesmon72, indicating that the smaller molecule is not simply a proteolytic product of the larger. The two forms of caldesmon may be derived from separate genes or by alternative splicing from a single gene.     

The thyroid hormone receptors: molecular basis of thyroid hormone resistance.

Major progress has been achieved in the mechanism of action of thyroid hormones thanks to the identification of the T3 receptor as the product of the proto-oncogene c-erbA. Recognition of subsets of receptors with and without T3-binding properties and of the interaction of different receptors with each other leads to new insights in cell regulation and development. In thyroid hormone resistance, distinct mutations in the T3-binding domain of thyroid hormone receptor (TR)beta have been identified in unrelated families. No correlation between the type of mutation and tissue resistance has been established. Mutant TRs bind to thyroid hormone response elements (TREs) on both negative or positive T3-controlled genes. Subjects with heterozygous TR beta gene deletion are not affected, supporting the hypothesis that mutant TRs act through a dominant negative effect. In generalized thyroid hormone resistance, mutated TR beta may interfere through competition for TREs and/or formation of inactive dimers. Finally, deficiency in T3 receptor auxiliary protein or other accessory proteins or competition between mutant and normal TRs for these factors is not excluded.     

Calcium-activated potassium transport and high molecular weight forms of calpromotin.

Investigations of human red blood cells show that a cytoplasmic protein called calpromotin is involved in the regulation of calcium-activated potassium transport. Calpromotin associates with the membrane in the presence of calcium and undergoes a chemical transformation. High performance gel filtration and gel electrophoresis show that the cytoplasmic and membrane-bound calpromotin can exist in both low and high molecular weight forms. The biochemical properties of the high molecular weight membrane-bound calpromotin are not the same as the high molecular weight cytoplasmic calpromotin. The high molecular weight membrane forms of calpromotin are increased by leupeptin and diminished by iodoacetic acid. Therefore, the leupeptin enhancement and iodoacetic inhibition of calcium-activated potassium transport may involve the high molecular weight forms of membrane-bound calpromotin.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

Studies on FGF-2: Nuclear localization and function of high molecular weight forms and receptor binding in the absence of heparin

Multiple forms of FGF-2 have been shown to exist in many cell types. These different species of molecular masses of 18, 21.5, 22, and 24 kDa are all translated via the use of alternate initiation codons. The three forms of HMW FGF-2 initiate at CUGs codons, whereas the 18 kDa form initiates at an AUG codon. The entire 18 kDa sequence is contained within the larger forms of HMW FGF-2 as the AUG codon is 3′ to the CUG codons. Although the 18 kDa form FGF-2 is localized primarily in the cytosol, a significant fraction of the HMW FGF-2 has a nuclear location. The nuclear localization of HMW FGF-2 is determined by amino acid residues in the amino-terminal extended sequence. The residues required for nuclear localization appear to be RG repeats that are found at multiple sites within the amino-terminal extension of HMW FGF-2. The nuclear localization of HMW FGF-2 suggested that these species may have unique properties. By selecting permanent transfectants of 3T3 cells expressing HMW, 18 kDa FGF-2, or all forms of FGF-2, we have found that HMW FGF-2 can endow cells with a phenotype different from that of cells expressing 18 kDa FGF-2. These cells are transformed by what appears to be the intracellular action of HMW FGF-2. The interaction of FGF-2 with heparin has also been examined. Contrary to other reports claiming that FGF-2 required heparin or heparan-sulfate for interaction with its high-affinity receptor, we have found that FGF-2 binds to its receptor in the absence of glycosaminoglycans, and that this binding activates the receptor. © 1994 Wiley-Liss, Inc.     

Identification of lipopolysaccharide binding site on high molecular weight kininogen

Plasma kallikrein kinin system (KKS) activation along with its cellular receptors expression are increased after injury and in patients with septic shock, hypotensive bacteremia and rhesus monkey infected with Salmonella typhimurium. KKS signaling cascade is activated by activated factor XII (FXIIa, Hageman factor)- and prolylcarboxypeptidase (PRCP)-dependent pathways on endothelial cells. Among the many entities that comprise the KKS, high molecular weight kininogen (HK), a bradykinin precursor, is critical in the assembly and activation of this system. HK is primarily expressed in the liver and secreted into the bloodstream. The activation of the KKS influences the permeability of the endothelium by liberating bradykinin (BK) from HK. BK is a potent inflammatory peptide which stimulates constitutive bradykinin B2 and inducible B1 receptors to release nitric oxide and prostacyclin. Regardless of the triggers, PK can only be activated on HK bound to the artificial negatively charged or to cell membrane surfaces. Since LPS has a negatively charged moiety and the ability to induce inflammatory responses in human, we determined the interaction between LPS and HK. HKH19 (HK cell binding site) and heparin inhibited LPS binding to HK with IC₅₀s of 15 nM and 20 μg/ml, respectively. C1-inhibitor and N-acetylglucosamine glycan inhibited LPS binding to HK with IC₅₀s of about 10 μg/ml and 10 mM, respectively. This novel study underscores the implication of HK in infection. We propose that HKH19, heparin, and C1-inhibitor present therapeutic potential for the treatment of sepsis and hypotensive bacteremia.     

Novel structure of a high molecular weight FK506 binding protein fromArabidopsis thaliana

We have isolated clones of an Arabidopsis gene (ROF1, forrotamaseFKBP) encoding a high molecular weight member of the FK506 binding protein (FKBP) family. The deduced amino acid sequence of ROF1 predicts a 551-amino acid, 62 kDa polypeptide which is 44% identical to human FKBP59 — a 59 kDa FKBP which binds to the 90 kDa heat shock protein and is associated with inactive steroid hormone receptors. ROF1 contains three FKBP12-like domains in the N-terminal portion of the protein (in contrast to two domains in mammalian FKBP59), an internal repeat structure associated with protein-protein interactions (tetratricopeptide repeats), and a putative calmodulin binding domain near the C-terminal region of the protein. No sequences associated with protein translocation out of the cytosol were found in ROF1.ROF1 mRNA was found at equivalent low levels in light-grown roots, stems, and flowers and at slightly higher levels in leaves. The abundance ofROF1 mRNA increased several-fold under stress conditions such as wounding or exposure to elevated NaCl levels.