Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors

Exposure of beta-adrenergic receptors (beta ARs) to agonists causes rapid desensitization of the receptor-stimulated adenylyl cyclase response. Three main mechanisms have been implicated in this process: phosphorylation of the receptors by the cAMP-dependent protein kinase (PKA), phosphorylation by the specific agonist-dependent beta AR kinase, and sequestration of the receptors away from the cell surface. By applying inhibitors of these processes to digitonin-permeabilized A431 cells we investigated their contributions to beta AR desensitization. Each process could be selectively inhibited: PKA-dependent phosphorylation by an inhibitor peptide (amino acids 1-24 of the heat-stable inhibitor of PKA (PKI], beta AR kinase-dependent phosphorylation by heparin, and sequestration by concanavalin A. In permeabilized cells, heparin plus PKI completely blocked agonist-induced phosphorylation of the beta ARs. Desensitization was assessed by quantitating the signal transduction efficacy of the system. At high agonist concentrations (approximately 1 microM) up to 70% desensitization occurred. Complete blockade of this desensitization required the concurrent inhibition of all three pathways. When individual pathways were blocked it could be demonstrated that either the PKA or beta AR kinase mechanisms alone resulted in 40-50% desensitization whereas sequestration alone caused 20-30% desensitization. At low agonist concentrations (approximately 10 nM), the PKA pathway was selectively activated. These data indicate that while desensitization mediated via the three different mechanisms can occur independently, the quantitative contributions are not additive. Such findings suggest distinct but overlapping physiological roles for each mechanism in controlling receptor function.