The alpha v beta 3 integrin associates with a 190-kDa protein that is phosphorylated on tyrosine in response to platelet-derived growth factor.

Integrins are cell surface heterodimers that mediate cell adhesion to the extracellular matrix. We show that in mouse 3T3 fibroblasts the alpha v beta 3 integrin (vitronectin receptor) coprecipitates with a tyrosine-phosphorylated 190-kDa protein, as detected by antibodies to phosphotyrosine. Three different antibodies to the vitronectin receptor, all of which precipitate the alpha/beta complex, coprecipitated a 190-kDa protein. The three antibodies were raised against the purified placental vitronectin receptor, the platelet alpha IIb beta 3 integrin, and the cytoplasmic domain of the alpha v subunit. The association was specific for the vitronectin receptor, since an antibody to the alpha 5 beta 1 integrin (fibronectin receptor) did not coprecipitate any tyrosine-phosphorylated protein. The phosphorylation of the 190-kDa protein was observed only following cell activation by platelet-derived growth factor, which is known to stimulate tyrosine kinase activity and to modulate cell adhesion. Antibodies raised against the platelet-derived growth factor alpha and beta receptors (M(r) = 170,000 and 190,000, respectively) did not recognize the 190-kDa, integrin-associated phosphorylated protein. Occupancy of the vitronectin receptor by one of its ligands, vitronectin, resulted in an increased amount of tyrosine phosphorylation of the 190-kDa protein. Our data suggest that the association of tyrosine-phosphorylated proteins with integrins may play an important role in growth factor-mediated modulation of cell adhesion.

Disruption of microtubules inhibits the stimulation of tissue plasminogen activator expression and promotes plasminogen activator inhibitor type 1 expression in human endothelial cells.

The expression of certain proteolytic enzymes involved in cell migration (collagenase, urokinase) can be enhanced by the disruption of cellular cytoskeletal organization, suggesting an association between cell shape and gene expression. We have examined the effect of cytoskeleton-disrupting agents on the production and secretion of another proteolytic enzyme, tissue plasminogen activator (tPA), and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), in human endothelial cells. Addition of 1 x 10(-6) M colchicine, 5 x 10(-6) M cytochalasin B, 10(-6) M nocodazole, or 10(-6) M tubulazole had no effect on the constitutive rate of release of tPA. However, the three microtubule-disrupting agents--colchicine, nocodazole, and tubulazole--depressed the stimulation of tPA secretion by phorbol myristate acetate (PMA) by 50- to 65%. Disruption of microfilament structure by cytochalasin B had no effect. In contrast, microtubule disruption in the absence or presence of PMA stimulated PAI-1 secretion by 2.5 and 2 times, respectively. The depression of tPA secretion was not due to inhibition of the secretory function since tPA did not accumulate intracellularly during colchicine treatment. Nor did colchicine affect the PMA activation of protein kinase C-alpha, upon which stimulation of tPA is dependent; neither translocation of the kinase nor phosphorylation of the protein kinase C substrate protein, P80, was inhibited. Measurement of tPA mRNA levels demonstrated that the increase which precedes PMA-enhanced tPA secretion was also inhibited by colchicine by 50%. However, tPA gene transcriptional activity was only reduced 13%, suggesting that a post-transcriptional event was affected by microtubule disruption. PAI-1 mRNA levels and transcription rates were elevated 3.5 times. This study suggests that the changes that occur in endothelial cells during PMA-induced signal transmission leading to enhanced tPA mRNA levels and tPA antigen production can be partly blocked by agents that disrupt microtubule organization.

Identification of a protein transiently phosphorylated by activators of endothelial cell function as the heat-shock protein HSP27. A possible role for protein kinase C.

Several agonists of endothelial cell function (thrombin, histamine, dioctanoylglycerol, phorbol 12-myristate 13-acetate, interleukin-1) have previously been shown to enhance the level of phosphorylation of an undefined 29,000-M(r) protein (P29). Comparison of this protein with other phosphoproteins suggested that it may be related to the mammalian heat-shock protein HSP27. Immunoprecipitation and immunoblot analysis with antibodies specific for human HSP27 demonstrated that P29 was immunochemically identical with HSP27. Further characterization of agonist-induced phosphorylation of HSP27 indicated that phosphorylation occurred exclusively on serine residues, and phosphopeptide analysis of tryptic- and chymotryptic-cleavage products demonstrated that the phosphopeptides generated were identical for each agonist and okadaic acid. Down-regulation of protein kinase C-alpha by prolonged treatment with phorbol esters eliminated the ability of phorbol 12-myristate 13-acetate, dioctanoylglycerol, thrombin and histamine to phosphorylate HSP27 above background levels and deceased interleukin-1-stimulated HSP27 phosphorylation by 60%. These data suggest that the various agonists employed stimulate HSP27 phosphorylation through similar mechanisms and that protein kinase C is probably involved.

Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. Sequence similarity to the vertebrate transferrins.

An iron-binding glycoprotein of Mr = 77,000 has been isolated from hemolymph of the adult sphinx moth Manduca sexta. Since this protein binds ferric ion both in vivo and in vitro and has a secondary structure similar to that of human serum transferrin and human lactoferrin as judged by CD spectra, we decided to clone its cDNA in order to determine its relationship to the vertebrate transferrins. Antiserum generated against this protein was used to screen a larval fat body cDNA library. A 2.0 kilobase clone was isolated that selects an mRNA which, when translated in vitro, produces an immunoprecipitable 77-kDa protein. When the library was rescreened using the 2.0-kilobase clone as a probe, three full-length clones were isolated, and the complete nucleotide sequence of one 2,183-base pair insert was determined. The deduced protein sequence contains an 18-amino acid signal sequence and a mature protein sequence of 663 amino acids with a calculated Mr of 73,436. The sequence was used to search the National Biomedical Research Foundation (NBRF) protein database, revealing significant similarity to the vertebrate transferrins, a family of 80-kDa glycoproteins which transport and sequester iron in the blood and other body fluids. A multiple sequence alignament shows the greatest areas of similarity to be around the two iron binding sites, although the insect protein seems to contain only one such functional site. Moreover, 23 of the 24 cysteine residues in the insect protein occupy identical positions as compared with the other transferrins, indicating a similar overall tertiary structure. Comparison of the two halves of the insect sequence indicates that the protein may have arisen as a result of gene duplication. The similarity of the M. sexta sequence to the vertebrate transferrins may provide important clues to transferrin evolution.