Abstract
Three separate processes may contribute to rapid beta-adrenergic receptor
desensitization: functional uncoupling from the stimulatory guanine
nucleotide-binding protein Gs, mediated by phosphorylation of the
receptors by two distinct kinases, the specific beta-adrenergic receptor
kinase (beta ARK) and the cyclic AMP-dependent protein kinase A (PKA),
as well as a spatial uncoupling via sequestration of the receptors
away from the cell surface. To evaluate the relative importance and
potential role of the various processes in different physiological
situations, a kinetic analysis of these three mechanisms was performed
in permeabilized A431 epidermoid carcinoma cells. To allow a separate
analysis of each mechanism, inhibitors of the various desensitization
mechanisms were used: heparin to inhibit beta ARK, the PKA inhibitor
peptide PKI to inhibit PKA, and concanavalin A treatment to prevent
sequestration. Isoproterenol-induced phosphorylation of beta 2 receptors
in these cells by beta ARK occurred with a t1/2 of less than 20 sec,
whereas phosphorylation by PKA had a t1/2 of about 2 min. Similarly,
beta ARK-mediated desensitization of the receptors proceeded with
a t1/2 of less than 15 sec, and PKA-mediated desensitization with
a t1/2 of about 3.5 min. Maximal desensitization mediated by the
two kinases corresponded to a reduction of the signal-transduction
capacity of the receptor/adenylyl cyclase system by about 60% in
the case of beta ARK and by about 40% in the case of PKA. Receptor
sequestration was much slower (t1/2 of about 10 min) and involved
no more than 30% of the cell surface receptors. It is concluded that
beta ARK-mediated phosphorylation is the most rapid and quantitatively
most important factor contributing to the rapid desensitization.
This rapidity of the beta ARK-mediated mechanism makes it particularly
well suited to regulate beta-adrenergic receptor function in rapidly
changing environments such as the synaptic cleft.
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