Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions

Estrogen receptor alpha (ER alpha) activation functions AF-1 and AF-2 classically mediate gene transcription in response to estradiol (E2). A fraction of ER alpha is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological roles of MISS in vivo are poorly understood. We therefore generated a mouse with a point mutation of the palmitoylation site of ER alpha (C451A-ER alpha) to obtain membrane-specific loss of function of ER alpha. The abrogation of membrane localization of ER alpha in vivo was confirmed in primary hepatocytes, and it resulted in female infertility with abnormal ovaries lacking corpora lutea and increase in luteinizing hormone levels. In contrast, E2 action in the uterus was preserved in C451A-ER alpha mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions such as rapid dilatation, acceleration of endothelial repair, and endothelial NO synthase phosphorylation were abrogated in C451AER alpha mice. A complementary mutant mouse lacking the transactivation function AF-2 of ER alpha (ER alpha-AF2 degrees) provided selective loss of function of nuclear ER alpha actions. In ER alpha-AF20, the acceleration of endothelial repair in response to estrogen-dendrimer conjugate, which is a membrane-selective ER ligand, was unaltered, demonstrating integrity of MISS actions. In genome-wide analysis of uterine gene expression, the vast majority of E2-dependent gene regulation was abrogated in ER alpha-AF20, whereas in C451A-ER alpha it was nearly fully preserved, indicating that membrane-to-nuclear receptor cross-talk in vivo is modest in the uterus. Thus, this work genetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.