DOWN-REGULATION OF BETA-ADRENERGIC RECEPTORS BY PINDOLOL IN GS-ALPHA-TRANSFECTED S49 CYC-MURINE LYMPHOMA-CELLS

The role of the alpha subunit of the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase (G(s-alpha)) in the down-regulation of beta-drenergic receptors by pindolol was studied in S49 cyc- cells (normally G(s-alpha)-deficient) transfected to express functional recombinant rat G(s-alpha). An inducible cell line (S49 G(s-alpha)IND) was derived from S49 cyc- cells transfected with a vector containing the full-length coding sequence of G(s-alpha) under the inducible control of the mouse mammary tumor virus long-terminal repeat promoter. G(s-alpha) was not detectable in S49 G(s-alpha)IND cells by immunoblot or by ADP-ribosylation in the presence of cholera toxin and [alpha-P-32]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol-stimulated cyclic AMP accumulation increased within 3 h. After 15 h, G(s-alpha) was present at a level 40-50% of that found in S49 wild-type (WT) cells as measured either by immunoblot analysis or by [alpha-P-32]ADP-ribosylation. Membranes prepared from G(s-alpha)IND cells grown in the presence of dexamethasone bound agonist with high affinity, and this binding was sensitive to guanine nucleotides. A second vector, DzbG(s-alpha)+, contained the coding sequence of G(s-alpha) under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc- cells, and the resulting cells, S49 G(s-alpha)CST cells, expressed G(s-alpha) at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol-stimulated cyclic AMP accumulation in S49 G(s-alpha)CST 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 beta-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 G(s-alpha)IND cells. When G(s-alpha)CST cells were exposed to 50 nM pindolol for 12 h, the density of beta-adrenergic receptors was down-regulated by the same amount as in S49 WT cells. These results suggest that G(s-alpha) is necessary to restore the ability of pindolol to down-regulate beta-adrenergic receptors in S49 cyc- cells and that the protein must be expressed at a level comparable to that found in S49 WT cells.