Abstract
Binding of the catecholamine agonists epinephrine and norepinephrine
to the beta-adrenergic receptor (BAR) rapidly activates adenylate
cyclase via the stimulatory guanine nucleotide regulatory protein
Gs, and results in rises in cellular levels of cAMP. However, continuous
exposure to these agonists leads within minutes to a dampening of
the enzymatic response. Both in vivo and in vitro studies have implicated
agonist-induced phosphorylation of BAR in this process. These results
include the isolation of a novel beta-adrenergic receptor kinase
(BARK), which has been shown to preferentially phosphorylate receptors
that are occupied by agonist when assessed in vitro. Recent studies
in our laboratory have examined the desensitization process in intact
cells to determine where on the receptor molecule functionally relevant
phosphorylation occurs, and to identify the kinase(s) involved. In
one set of studies, site-specific mutagenic techniques with the cloned
gene for the human beta 2-adrenergic receptor were utilized to delete
putative sites of phosphorylation by BARK and/or the cAMP-dependent
protein kinase (PKA). Following expression of the mutated receptors
in mammalian cells, the cells were challenged with different concentrations
of agonist for 10-15 min and the functional and phosphorylation properties
of the mutant receptors were then assessed. In another set of studies
human A431 cells were permeabilized with low concentrations of digitonin
and treated with selective inhibitors of both BARK and PKA. The cells
were then exposed to desensitizing concentrations of agonist, and
similar measurements performed. Taken together, the results from
both sets of studies suggest that exposure of cells to low (nanomolar)
concentrations of agonist leads to phosphorylation of the receptor
on one or both consensus sites for PKA, and that the predominant
effect of this phosphorylation on the adenylyl cyclase response is
a loss in sensitivity of the receptor to further stimulation by the
agonist. In contrast, exposure of cells to higher (micromolar) concentrations
of agonist leads to BAR phosphorylation by both PKA and BARK, the
latter on the carboxyl terminal region of the receptor. Phosphorylation
of the receptor by both kinases appears to be required for the full
desensitization effect seen with the high concentration of agonist,
which includes both losses in sensitivity and in the maximal responsiveness
of the adenylyl cyclase response upon subsequent challenge with the
agonist. Such a dual kinase control of BAR phosphorylation may have
important implications for understanding the regulation of desensitization
under different physiological circumstances.
Users
Please
log in to take part in the discussion (add own reviews or comments).