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Down‐Regulation of β‐Adrenergic Receptors by Pindolol in G sα ‐Transfected S49 cyc − Murine Lymphoma Cells
Author(s) -
Gonzales Jerry M.,
O'Donnell J. Kevin,
Stadel Jeffrey M.,
Sweet Ray W.,
Molinoff Perry B.
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb09367.x
Subject(s) - transfection , microbiology and biotechnology , cholera toxin , pertussis toxin , g protein , gs alpha subunit , cell culture , adp ribosylation , biology , receptor , pindolol , chemistry , nad+ kinase , biochemistry , endocrinology , genetics , enzyme
The role of the a subunit of the guanine nucleotide‐binding regulatory protein that stimulates adenylyl cyclase (G sα ) in the down‐regulation of β‐Adrenergic receptors by pindolol was studied in S49 cyc − cells (normally G sα ‐deficient) transfected to express functional recombinant rat G sα . An inducible cell line (S49 G sα IND) was derived from S49 cyc − cells transfected with a vector containing the full‐length coding sequence of G sα under the inducible control of the mouse mammary tumor virus long‐terminal repeat promoter. G sα was not detectable in S49 G sα IND cells by immunoblot or by ADP‐ribosylation in the presence of cholera toxin and [α‐ 32 P]NAD. When cells were grown in 100 nM dexamethasone, isoproterenol‐stimulated cyclic AMP accumulation increased within 3 h. After 15 h, G sα 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‐ 32 P]ADP‐ribosylation. Membranes prepared from G sα 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 sα +, contained the coding sequence of G sα under the constitutive regulatory control of the SV40 early promoter. This vector was introduced into cyc − cells, and the resulting cells, S49 G sα CST cells, expressed G sα at a level comparable to that found in S49 WT cells as measured by immunoblot analysis. Isoproterenol‐stimulated cyclic AMP accumulation in S49 G sα 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 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 G sα IND cells. When G sα 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 G sα 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.