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Notes: Abstract: The role of the a subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (Gsα) in the down-regulation of β-Adrenergic receptors by pindolol was studied in S49 cyc− cells (normally Gsα-deficient) transfected to express functional recombinant rat Gsα. An inducible cell line (S49 GsαIND) was derived from S49 cyc− cells transfected with a vector containing the full-length coding sequence of Gsα under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. Gsα was not detectable in S49 GsαIND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [α-32P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, Gsα was present at a level 40–50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [α-32P]ADP-ribosylation. Membranes prepared from GsαIND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbGsα+, contained the coding sequence of Gsα under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc− cells, and the resulting cells, S49 GsαCST cells, expressed Gsα at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 GsαCST cells was at least as great as in S49 WT cells. When cells were grown in the presence of dexamethasone, exposure to 50 nM pindolol for 12 h down-regulated the density of 0-adrenergic receptors in S49 WT cells to 60% of that in cells grown in the absence of pindolol, but pindolol had no effect on the density of receptors on cyc− or Gsα IND cells. When Gsα CST cells were exposed to 50 nM pindolol for 12 h, the density ofβ-Adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that Gsα is necessary to restore the ability of pindolol to down-regulate β-adrenergic receptors in S49 cyc~ cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.