Site-specific ERα phosphorylation determines sex-dependent metabolic, reproductive, and body-compositional phenotypes in mice.

Estrogen receptor alpha (ERα) phosphorylation regulates receptor activity and tissue-specific gene expression. We generated serine (S) to alanine (A) phosphorylation-deficient knock-in mice targeting two conserved ERα sites, S171 and S216, to examine their physiological roles. ERα S216A females were subfertile, with ∼30% smaller litters and diminished uterine growth in response to estradiol (E2). Single-cell spatial transcriptomics revealed a disrupted E2-regulated transcriptome in the myometrium. Metabolic profiling revealed the suppression of glycolytic and redox pathways in ERα S216A mice, with males exhibiting reduced adiposity and increased lean mass. Skeletal analysis revealed opposing effects: ERα S216A females exhibited reduced femoral bone density, while ERα S171A females showed an increase. These data demonstrate critical roles for site-specific ERα phosphorylation in modulating receptor levels and activity, as well as gene expression, which have a profound impact on murine body composition, fertility, and metabolism.