title: Assessment of cell line competence for studies of pharmacological GPR30 modulation.
abstract: OBJECTIVE
  Cell lines used to study the role of the G protein-coupled receptor 30 (GPR30) or G protein-coupled estrogen receptor (GPER) as a mediator of estrogen responses have yielded conflicting results. This work identified a simple assay to predict cell line competence for pharmacological studies of GPR30.
  
  
  METHODS
  The phosphorylation or expression levels of ERK1/2, Akt, c-Fos and eNOS were evaluated to assess GPR30 activation in response to known agonists (17β-estradiol and G-1) in MCF-7 and T-47D breast cancer cell lines and in bovine aortic endothelial cells. GPR30 expression was analyzed by qRT-PCR and Western blot with two distinct antibodies directed at its carboxy and amino terminals.
  
  
  RESULTS
  None of the agonists, at any of the concentrations tested, activated any of those target proteins. Additional experiments excluded the disruption of the signaling pathway, interference of phenol red in the culture medium and constitutive proteasome degradation of GPR30 as possible causes for the lack of response of the three cell lines. Analysis of receptor expression showed the absence of clearly detectable GPR30 species of 44 and 50-55 kDa previously identified in cell lines that respond to 17β-estradiol and G-1.
  
  
  CONCLUSIONS
  Cells that do not express the 44 and 50-55 kDa species do not respond to GPR30 agonists. Thus, the presence or absence of these GPR30 species is a simple and rapid manner to determine whether a given cell line is suitable for pharmacological or molecular studies of GPR30 modulation.
authors.affiliation: a Faculty of Pharmacy , University of Coimbra , Coimbra , Portugal.|b Center for Neuroscience and Cell Biology, University of Coimbra , Coimbra , Portugal.
authors.forename: Cátia
authors.initials: C
authors.lastname: Sousa
authors.affiliation: a Faculty of Pharmacy , University of Coimbra , Coimbra , Portugal.|b Center for Neuroscience and Cell Biology, University of Coimbra , Coimbra , Portugal.
authors.forename: Madalena
authors.initials: M
authors.lastname: Ribeiro
authors.affiliation: a Faculty of Pharmacy , University of Coimbra , Coimbra , Portugal.|b Center for Neuroscience and Cell Biology, University of Coimbra , Coimbra , Portugal.
authors.forename: Ana Teresa
authors.initials: AT
authors.lastname: Rufino
authors.affiliation: a Faculty of Pharmacy , University of Coimbra , Coimbra , Portugal.|b Center for Neuroscience and Cell Biology, University of Coimbra , Coimbra , Portugal.
authors.forename: Alcino Jorge
authors.initials: AJ
authors.lastname: Leitão
authors.affiliation: a Faculty of Pharmacy , University of Coimbra , Coimbra , Portugal.|b Center for Neuroscience and Cell Biology, University of Coimbra , Coimbra , Portugal.
authors.forename: Alexandrina Ferreira
authors.initials: AF
authors.lastname: Mendes
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002417:Cattle; D003517:Cyclopentanes / Q000008:administration & dosage; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004958:Estradiol / Q000008:administration & dosage; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D004967:Estrogens / Q000819:agonists / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D061986:MCF-7 Cells; D051058:Oncogene Protein v-akt / Q000096:biosynthesis; D010766:Phosphorylation; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D011804:Quinolines / Q000008:administration & dosage; D011960:Receptors, Estrogen / Q000096:biosynthesis* / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C517943:1-(4-(6-bromobenzo(1,3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta(c)quinolin-8-yl)ethanone; D003517:Cyclopentanes; D047628:Estrogen Receptor alpha; D004967:Estrogens; C109584:GPER1 protein, human; D016760:Proto-Oncogene Proteins c-fos; D011804:Quinolines; D011960:Receptors, Estrogen; D043562:Receptors, G-Protein-Coupled; D004958:Estradiol; D051058:Oncogene Protein v-akt
keywords: Agonist; GPER; GPR30; antagonist; breast cancer cell line; endothelial cell