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

Estrogen receptor alpha (ERa) activation functions AF-1 and AF-2classically mediate gene transcription in response to estradiol (E2).A fraction of ERa is targeted to plasma membrane and elicits membrane-initiated steroid signaling (MISS), but the physiological rolesof MISS in vivo are poorly understood. We therefore generateda mouse with a point mutation of the palmitoylation site of ERa(C451A-ERa) to obtain membrane-specific loss of function of ERa.The abrogation of membrane localization of ERa in vivo was confirmed in primary hepatocytes, and it resulted in female infertilitywith abnormal ovaries lacking corpora lutea and increase in lutei-nizing hormone levels. In contrast, E2 action in the uterus waspreserved in C451A-ERa mice and endometrial epithelial proliferation was similar to wild type. However, E2 vascular actions suchas rapid dilatation, acceleration of endothelial repair, and endo-thelial NO synthase phosphorylation were abrogated in C451A-ERa mice. A complementary mutant mouse lacking the transacti-vation function AF-2 of ERa (ERa-AF20) provided selective loss offunction of nuclear ERa actions. In ERa-AF20, the acceleration ofendothelial 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 generegulation was abrogated in ERa-AF20, whereas in C451A-ERa itwas nearly fully preserved, indicating that membrane-to-nuclearreceptor cross-talk in vivo is modest in the uterus. Thus, this workgenetically segregated membrane versus nuclear actions of a steroid hormone receptor and demonstrated their in vivo tissue-specific roles.