Endocrinology, Vol 106, 1715-1727, Copyright © 1980 by Endocrine Society
Estrogen receptor cleavage and plasminogen activation by enzymes in human breast tumor cytosol
MR Sherman, FB Tuazon and LK Miller
Estrogen receptors in cytoplasmic extracts of breast tumors from more than
40 patients were separately analyzed by gel filtration and/or
ultracentrifugation under diverse conditions. Resultant patterns are
presented for specimens from 11 women with infiltrating duct carcinoma and
are representative of results obtained in all samples of sufficient size
and receptor content (approximately 40 fmol/mg cytosol protein) for
accurate determination of hydrodynamic parameters. Estradiol- binding
components of intracellular origin were distinguished from the serum
contaminant, sex hormone-binding globulin by their high affinity for
diethylstilbestrol and negligible affinity for 5 alpha-
dihydrotestosterone. The predominant molecular forms of the receptors, but
not the steroid specificity, varied dramatically with experimental factors,
including the duration of the fractionation procedure, ionic strength, and
the presence of protease inhibitors, particularly the bacterial tripeptides
N-acetyl- and N-propionyl-L-leucyl-L-leucyl-DL- arginine aldehydes
(leupeptin). At least three discrete forms of the intracellular receptors
were detected. The smallest labeled complex, the mero-receptor, with a
sedimentation coefficient of about 3S and a Stokes radius of about 24 A,
was formed during prolonged analysis of control cytosol in hypotonic or
hypertonic buffers. Complexes with an intermediate sedimentation
coefficient (approximately 5S) and Stokes radius (approximately 34A) were
detected when control cytosol was analyzed rapidly in hypotonic buffer or
when cytosol containing 50 nM leupeptin was analyzed in hypertonic buffer.
The largest receptor form (10.5S, 71A) was predominant in cytosol prepared
with 50 mM leupeptin and analyzed in hypotonic buffer. In this small series
of patients, there was no obvious correlation between the molecular form of
the receptors and the clinical status or eventual responsiveness to
endocrine therapy. Preliminary studies of endogenous proteolytic enzymes in
breast tumor cytosol that may be involved in mero-receptor formation
included assays of plasminogen activators (EC 3.4.21.-) by fibrinolytic and
spectrofluorometric techniques. The detection of high concentrations of
plasminogen activators in several tumor cytosols and the inhibition of this
activity by leupeptin, which stabilizes the large receptor forms in this
and other systems, are consistent with a possible role of these enzymes in
receptor cleavage.
