Contortrostatin, a homodimeric disintegrin, binds to integrin alphavbeta5

Contortrostatin is a homodimeric disintegrin from snake venom. We have shown that contortrostatin binds to integrins alphaIIbbeta3, alpha5beta1, and alphavbeta3. We now use several criteria to demonstrate the binding of contortrostatin to alphavbeta5. First, incubation of T24 cells, which express alphavbeta3 and alphavbeta5, with antibody against alphavbeta3 failed to completely inhibit adhesion of cells to vitronectin. However, pretreatment of the cells with contortrostatin or the combination of antibodies against alphavbeta3 and alphavbeta5 completely blocked adhesion to vitronectin. By contrast, either anti-alphavbeta5 alone or contortrostatin blocked adhesion of an alphavbeta3-negative T24 subline. Second, contortrostatin as well as anti-alphavbeta5 inhibits invasion of OVCAR-5, which express only alphavbeta5. Third, contortrostatin binds to purified alphavbeta5 in a saturable manner. Finally, radioligand binding assays yielded a K(d) value of 24 nM for [(125)I]contortrostatin binding to alphavbeta5. This investigation identifies alphavbeta5 as a binding site for contortrostatin. Blockage of alphavbeta5 by contortrostatin inhibits alphavbeta5-mediated adhesion and invasion.     

Tissue-type plasminogen activator variants with domain duplications and rearrangements

The interactions between tPA domains that are important for catalysis are poorly understood. We have probed the function of interdomain interactions by generating tPA variants in which domains are duplicated or rearranged. The proteins were expressed in a transient mammalian expression system and tested in vitro for their ability to activate plasminogen, induce fibrinolysis and bind to a forming fibrin clot. Duplication of the heavy chain domains of tPA produced enzymatically active tPA variants, many of which demonstrated similar in vitro amidolytic and fibrinolytic activity and similar fibrin affinity to the parent molecule. Zymographic analysis of the domain duplication tPA variants showed one major active species for each variant. Selection of the residues duplicated and the interdomain spacing were found to be critical considerations in the design of tPA variants with duplicated domains. We also rearranged the domains of tPA such that kringle 1 replaced the second kringle domain and vice versa. An analysis of these variants indicates that the first kringle domain can confer fibrin affinity to a tPA variant and function in place of kringle 2. Therefore, in wild-type tPA, the functions of kringle 1 and kringle 2 must be dependent partially on their orientation within the heavy chain of the protein. The functional autonomy of the heavy and light chains of tPA is demonstrated by the activity of a tPA variant in which the order of the heavy and light chains was reversed.     

The primary structure of crotalase,a thrombin-like venom enzyme,exhibits closer homology to kallikrein than to other serine proteases

Amino acid sequences of crotalase, totaling 98 residues or about 37% of the molecule, were determined by Edman degradation and were compared with the published sequences of nine serine proteases. Homologous alignment could be found for all crotalase sequences except one decapeptide. Comparison between crotalase and porcine pancreatic kallikrein showed the largest number of identical amino acids (36%). This finding has led to experiments which demonstrate that crotalase has specific enzymatic properties resembling kallikrein.     

Clonal variants of a melanoma cell line sensitive to growth inhibition by dexamethasone

We have isolated and characterized two clones of the RPMI 3460 Syrian hamster melanoma cell line which exhibit different responses to the synthetic glucocorticoid dexamethasone. In the presence of 10 nM dexamethasone, one clone (clone 6) exhibits the growth inhibition, morphological alterations, and reduction in final cell density observed in the parental RPMI 3460 cell line. In contrast, the other clone (clone 5), although exhibiting a reduction in final cell density, fails to exhibit the growth inhibition and morphological alterations. Thus, the effect of dexamethasone on growth and morphology can be expressed separately from the effect of dexamethasone on final cell density in these cells. This observation suggests that the two sets of responses can be controlled separately and that glucocorticoids may exert their influence through different or divergent biological pathways.In vitro receptor assays suggest that the different phenotypes of clone 5 compared with clone 6 cells cannot be explained by an absence of or reduction in cytosolic glucocorticoid receptor, in clone 5 cells. Additional receptor characterization suggests that the different responses to dexamethasone of clone 5 and clone 6 cells do not reflect changes in the ability of receptor to exist in a stably activated form. Differences in the accumulation or depletion of extracellular components in the growth medium also do not seem to be responsible for the altered phenotype of clone 5 vis-à-vis clone 6 cells.     

Steroid hormone receptor characterization of several histologic variants of a rat prostatic adenocarcinoma

Several histologic variants of the transplantable R-3327 prostatic adenocarcinoma carried in male Copenhagen rats have been characterized and the histologic types have been correlated with steroid hormone receptor content. One type is clearly an adenocarcinoma; this tumor is hormonally responsive and contains substantial amounts of both androgen and estrogen receptors. In contrast, another histologic type, a fibrosarcoma, is hormonally nonresponsive and does not contain either receptor. A third histologic variant is classified as a carcinosarcoma and contains histological elements of both adenocarcinoma and fibrosarcoma and is also hormonally responsive. This tumor contains lower receptor levels than the adenocarcinomas but more than the fibrosarcomas. The androgen receptor appears to be identical in the different histologic forms of the tumor; the sedimentation coefficient is 7.8S and the dissociation constant for methyltrienolone is 4 X 10^?9 M. Similarly, the estrogen receptor from the different histologic forms of the tumor has a sedimentation coefficient of 8.3S and the dissociation constant for estradiol is 7 X 10^?10 M. These findings clearly distinguish the cytosol binding macromolecules from plasma binding proteins, and classify them as steroid hormone receptors. Further, rat serum was devoid of androgen and estrogen binding in the 8S region. Normal prostate tissue from Copenhagen rats contained low levels of an androgen receptor, but no estrogen receptor. It is possible that during growth and/or passage of the R-3327 tumor, the hormonally responsive adenocarcinoma cells do not survive and there is a gradual emergence of the nonresponsive fibrosarcoma. If, as we suspect, the receptors are found in the epithelial cells and not the stromal cells, there clearly should be considerable variation of receptor content in the different intermediary histologic forms of the tumor.