Net 114a

SR121463B is a V2R antagonist and known pharmacoperone that was used in the current study, after being generously provided by Dr. Claudine Serradeil at Sanofi-Aventis and used as received. Other pharmacoperones were identified by us by high throughput screening of a large chemical library [22 (link), 23 (link)]. Several reagents were used as obtained from indicated vendors: 3-Isobutyl-1-methylxanthine (IBMX, Sigma Aldrich, St. Louis, MO), vasopressin (Tocris Biosciences, Bristol, England UK), fetal calf serum (FCS, Hyclone, Logan, UT), Dulbecco’s MEM (DMEM), PBS (GIBCO, Invitrogen). pTRE2-Hygromycin vector (Invitrogen, San Diego, CA), human arginine-vasopressin 2 receptor (V2R; Gene Bank Accession Number: AY242131), Gq plasmid (Gene Bank Accession Number: U43083) [24 (link)]; both plasmids from cDNA Resource Center; www.cdna.org;), myo-[2-³H(N)]-inositol (NET-114A; PerkinElmer, Waltham, MA), vasopressin (8-L-arginine), [phenylalaninyl-3,4,5-³H(N)- (NET800, specific activity = 66.3 Ci/mmol; PerkinElmer, Waltham, MA), and unbound 125-Iodine (016303710; MP Biomedicals, Santa Ana, CA).

MCF-7 cells stably expressing mGPR30 or HEK293 cells transiently expressing hGPR30 were incubated with 1 µCi/ml myo-[³H] inositol (NET114A, PerkinElmer) in growth medium for 24 h at 37 °C and 5% CO₂. Subsequently, cells were washed and incubated with 20 mM LiCl in HBSS for 20 min at 37 °C. Cells were then treated with vehicle or ligands for 30 min, and inositol phosphates were extracted using 5% perchloric acid. After neutralisation with 0.72 M KOH/0.6 M KHCO₃, centrifugation at 20,400 × g for 10 min, and separation on Micro Bio-Spin™ Chromatography Columns (7326204, Bio-Rad) packed with AG 1-X8 Anion Exchange Resin (1432446, Bio-Rad), total inositol phosphates were eluted with a solution containing 0.1 M formic acid and 1 M formate. The radioactivity of the myo-[³H] inositol-containing phospholipids was measured using a liquid scintillation counter (Tri-Carb 5110TR, PerkinElmer).