Regulation of cellular Gsα levels and basal adenylyl cyclase activity by expression of the β2-adrenoceptor in neuroblastoma cell lines

  • Graeme Milligan
  • Gun-Do Kim
  • Ian Mullaney
  • Elaine J. Adie
Part of the Developments in Molecular and Cellular Biochemistry book series (DMCB, volume 15)

Abstract

Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human β2 adrenoceptor cDNA under the control of the β actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (βN22) but not one expressing only low levels of the receptor (βN17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg2+ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn2+. The elevated basal activity was partially reversed by addition of the β-adrenoceptor antagonist propranolol. Agonist activation of βN22 but not βN17 cells led to a large selective down-regulation of cellular Gsα levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [3H] forskolin was achieved with substantially greater potency (some 30 fold) in βN22 cell membranes than in βN17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [3H] forskolin binding which was equipotent in each of βN22, βN17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of Gsα down-regulation in each cell type. Expression of an epitope tagged variant of Gsα in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type Gsα. Isolation of clones of NCB20 cells expressing high levels of the β2 adrenoceptor also resulted in a specific agonist-induced down-regulation of G sα.

Key words

guanine nucleotide binding protein adenylyl cyclase cyclic AMP prostagladin adrenoceptor 

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Copyright information

© Springer Science+Business Media Dordrecht 1995

Authors and Affiliations

  • Graeme Milligan
    • 1
  • Gun-Do Kim
    • 1
  • Ian Mullaney
    • 1
  • Elaine J. Adie
    • 1
  1. 1.Molecular Pharmacology Group, Department of BiochemistryUniversity of GlasgowGlasgowScotland, UK

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