ESTRADIOL-BINDING MECHANISM AND BINDING-CAPACITY OF THE HUMAN ESTROGEN-RECEPTOR IS REGULATED BY TYROSINE PHOSPHORYLATION

We have investigated the effects of tyrosine phosphorylation on the es tradiol-binding mechanism and binding capacity of the human estrogen r eceptor (hER). The wild type hER and a point mutant form of the hER, i n which tyrosine 537 was mutated to phenylalanine (Y537F hER), were ex pressed in Sf9 insect cells. The wild type hER, but not the Y537F hER, reacted with a anti-phosphotyrosine monoclonal antibody, indicating t hat tyrosine 537 was the only tyrosine phosphorylated on the hER. Scat chard and Hill analyses of the the binding interaction of [H-3]estradi ol with the wild type hER indicated that the addition of millimolar ph osphotyrosine, but not tyrosine, phosphate, or phosphoserine, abolishe d the cooperative binding mechanism of the hER. These observations are consistent with the idea that phosphotyrosine blocks dimerization and site-site interactions between the hER monomers. The wild type hER bo und 10-fold more [H-3]estradiol than the Y537F hER. Treatment of the p urified wild type hER with a tyrosine phosphatase decreased the bindin g capacity of the hER by approximately 90%, whereas, a serine/threonin e phosphatase had no effect. The estrogen-binding capacity of the tyro sine-dephosphorylated hER was completely restored by rephosphorylation of tyrosine 537 with p60(c-src), a tyrosine kinase. These results ind icate that p60(c-src) can restore estrogen binding to the tyrosine-dep hosphorylated hER and that dimerization and cooperative site-site inte raction of the hER occur via a phosphotyrosine-binding interaction.