title: Point mutations in the ERα Gαi binding domain segregate nonnuclear from nuclear receptor function.
abstract: A subpopulation of plasma membrane-associated estrogen receptor (ER)α interact directly with G proteins and mediate nonnuclear receptor signaling. This mechanism underlies numerous processes, including important cardiovascular protective actions of estradiol (E(2)), such as the activation of endothelial NO synthase (eNOS) and endothelial cell growth and migration. In the present work we sought a genetic approach to differentiate nonnuclear from nuclear ERα actions. We generated single alanine substitutions within the Gαi-binding domain of ERα (amino acids 251-260) and tested signaling to eNOS or ERK1,2 and activation of luciferase (Luc) reporters signifying transactivation via direct or indirect ERα-DNA binding in HeLa cells. The point mutants ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A were all incapable of activating eNOS in response to E(2), and ERα-R256A and ERα-D258A also showed loss of ERK1,2 activation. In contrast, ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A all displayed normal capacity to invoke E(2)-induced transactivation of estrogen response element (ERE)-Luc or Sp1-Luc. However, whereas activator protein 1-Luc activation by ERα-R256A and ERα-D258A was intact, ERα-K257A and ERα-R260A were incapable of activator protein 1-Luc activation. In in vitro pull-down assays with the two mutants that lack all nonnuclear functions tested and retain all nuclear functions tested, ERα-R256A and ERα-D258A, there was normal direct interaction between Gαi and ERα-R256A and an absence of interaction between Gαi and ERα-D258A. When expressed in endothelial cells, these two mutants prevented E(2)-induced migration and eNOS activation mediated by endogenous receptor, indicative of dominant-negative action. Thus, the point mutants ERα-R256A and ERα-D258A in the receptor GαI-binding domain provide genetic segregation of nonnuclear from nuclear ERα function.
authors.affiliation: Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wu
authors.forename: Ken
authors.initials: K
authors.lastname: Chambliss
authors.forename: Wan-Ru
authors.initials: WR
authors.lastname: Lee
authors.forename: Ivan S
authors.initials: IS
authors.lastname: Yuhanna
authors.forename: Chieko
authors.initials: C
authors.lastname: Mineo
authors.forename: Philip W
authors.initials: PW
authors.lastname: Shaul
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004958:Estradiol / Q000502:physiology; D047628:Estrogen Receptor alpha / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D016297:Mutagenesis, Site-Directed; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D017354:Point Mutation; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D015533:Transcriptional Activation
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D004958:Estradiol; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D019206:GTP-Binding Protein alpha Subunits, Gi-Go