Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.
Abstract
DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain
alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to
stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting
phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase,
leads to a marked attenuation of the ability of alpha 1-receptor agonists such as
norepinephrine to stimulate the turnover of inositol phospholipids. This turnover
was measured by determining the 32P content of phosphatidylinositol and phosphatidic
acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated
cells also displayed a decrease in binding affinity of cellular alpha 1 receptors
for agonists with no change in antagonist affinity. By using affinity chromatography
on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately
equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As
assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor
ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol
of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate
per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated
inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same
rapid time course with a t1/2 less than 2 min. These results indicate that calcium-
and phospholipid-dependent protein kinase may play an important role in regulating
the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating
and deactivating them.
Type
Journal articleSubject
AnimalsCell Line
Cricetinae
Enzyme Activation
Male
Molecular Weight
Norepinephrine
Phorbols
Phosphatidylinositols
Phosphoproteins
Phosphorylation
Protein Kinase C
Protein Kinases
Receptors, Adrenergic, alpha
Tetradecanoylphorbol Acetate
Vas Deferens
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https://hdl.handle.net/10161/7880Collections
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Marc G. Caron
James B. Duke Distinguished Professor of Cell Biology
Studies of the mechanisms of action and regulation of hormones and neurotransmitters
at the cellular and molecular levels constitute the main goals our of research activities.
G protein-coupled receptors (GPCR) mediate the actions of signaling molecules from
unicellular organisms to man. We have used adrenergic and dopamine receptors to characterize
the structure/function and regulation mechanisms of these prototypes of G protein-coupled
receptors. Another approach has been to characterize
This author no longer has a Scholars@Duke profile, so the information shown here reflects
their Duke status at the time this item was deposited.
Robert J. Lefkowitz
James B. Duke Distinguished Professor of Medicine
Dr. Lefkowitz’s memoir, A Funny Thing Happened on the Way to Stockholm, recounts his
early career as a cardiologist and his transition to biochemistry, which led to his
Nobel Prize win.
Robert J. Lefkowitz, M.D. is James B. Duke Professor of Medicine and Professor of
Biochemistry and Chemistry at the Duke University Medical Center. He has been an Investigator
of the
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