DECREASE OF HORMONE-BINDING CAPACITY OF ESTROGEN-RECEPTOR BY CALCIUM

We have adressed the question as to whether calcium may modify the [H- 3]estradiol ([H-3]E-2) binding properties of the estrogen receptor (ER ). A human recombinant full length ER (yER) expressed in yeast was use d to limit the potential interference of ER-associated proteins and pr oteases present in the target tissues. Ca++ (0.1-10 mM) always produce d an important loss of [H-3]E-2 binding capacity without any effect on the hormone binding affinity of residual receptors. This loss was ref lected in a decrease of immunoreactivity for monoclonal antibodies rai sed against the hormone binding domain. An ER recombinant expressing s olely this domain confirmed that the ion operated at this level. Bindi ng of [I-125]Z-17 alpha-(2-iodovinyl)-11 beta-chloromethyl estradiol-1 7 beta (an compound with very high selectivity for ER) as well as [I-1 25]tamoxifen aziridine were similarly affected. Size-exclusion chromat ography failed to reveal the emergence of any ER isoforms of low molec ular weight rejecting the hypothesis of a Ca++-induced proteolysis. In agreement with this conclusion, EDTA reversed the loss of [H-3]E-2 bi nding capacity. Phosphoamino acids (PY, PT and PS) partly antagonized the effect of Ca++ suggesting its interaction with phosphoamino acid r esidues. Worthy of note, the effect of Ca++ appeared more marked when assessed by DCC than HAP assay. The phosphocalcic nature of the HAP ma trix may explain this phenomenon which was observed with cytosolic ER from various origins.