title: Alpha 2A adrenergic receptors inhibit cAMP accumulation in embryonic stem cells which lack Gi alpha 2.
abstract: alpha 2A adrenergic receptors are thought to inhibit adenylyl cyclase primarily through Gi alpha 2. We tested the requirement for Gi alpha 2 to inhibit cAMP accumulation by stable expression of alpha 2A adrenergic receptors in mouse embryonic stem cells. Host lines consisted of wild-type CCE cells, and CCE cells with targeted disruption of the Gi alpha 2 gene by two-stage homologous recombination (Mortensen, R. M., Zubiuar, M., Neer, E. J., and Seidman, J. G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7036-7040; Mortensen, R. M., Conner, D. A., Chao, S., Geisterfer-Lowrance, A. A., and Seidman, J. G. (1992) Mol. Cell. Biol. 12, 2391-2395). Knockouts were confirmed by Northern blot and immunoblot. We studied three clones derived from wild-type CCE cells (2, 6, and 8) expressing 450 +/- 50, 3000 +/- 120, and 150 +/- 20 fmol of receptor/mg of protein, respectively, and two Gi alpha 2-null clones (7 and 18) expressing 2100 +/- 250 and 300 +/- 40 fmol of receptor/mg of protein. The specific agonist UK14304 caused an inhibition of cAMP accumulation in clones 2, 6 and 8 (58 +/- 16%, 62 +/- 7%, and 52 +/- 12%) and in clones 7 (47 +/- 3%) and 18 (40 +/- 5%), but not in nontransfected CCE cells. IC50 values were similar for all clones (approximately 200 nM). The effect was attenuated by pertussis toxin and the antagonist rauwolscine. These studies show that expression of Gi alpha 2 is not required for alpha 2A adrenergic receptors to inhibit cAMP accumulation.
authors.affiliation: Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: J R
authors.initials: JR
authors.lastname: Raymond
authors.forename: J M
authors.initials: JM
authors.lastname: Arthur
authors.forename: S J
authors.initials: SJ
authors.lastname: Casañas
authors.forename: C L
authors.initials: CL
authors.lastname: Olsen
authors.forename: T W
authors.initials: TW
authors.lastname: Gettys
authors.forename: R M
authors.initials: RM
authors.lastname: Mortensen
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018341:Receptors, Adrenergic, alpha-2 / Q000502:physiology*; D013234:Stem Cells / Q000378:metabolism*
chemical_list: D018341:Receptors, Adrenergic, alpha-2; D000242:Cyclic AMP; D019204:GTP-Binding Proteins