Activation of cAMP-dependent protein kinase is required for heterologous desensitization of adenylyl cyclase in S49 wild-type lymphoma cells.

Abstract

We report here that, contrary to previously reported findings, treatment of S49 wild-type (WT) lymphoma cells with 0-50 nM epinephrine resulted in a heterologous desensitization of adenylyl cyclase (EC 4.6.1.1)-that is, epinephrine and prostaglandin El (PGEI) stimulations of adenylyl cyclase were reduced. Observation of this heterologous desensitization required the assay of adenylyl cyclase with submillimolar concentrations of Mg2+ and low concentrations of epinephrine. Also, whereas previously there had been no evidence for any role of cAMP-dependent protein kinase in the desensitization of the WT fi-adrenergic receptor, our data comparing the characteristics of the desensitization in WT, kin-, and cyc -lymphoma cells [where kin -and cyc -refer to variants of S49 WT cells lacking cAMP-dependent protein kinase activity (kin-) and the a subunit of the stimulatory guanine nucleotide-binding regulatory protein (cyc -)] now suggest that cAMP-dependent protein kinase mediates the heterologous desensitization of adenylyl cyclase. Specifically, we found that only the WT cells exhibited epinephrine-induced heterologous desensitization. The kin -and cyc-cells exhibited only homologous desensitization, and much higher concentrations of epinephrine were required to elicit the homologous desensitization in the variants relative to the heterologous desensitization of the WT. Treatment of WT and cyc-cells with dibutyryl cAMP or treatment of WT with forskolin or PGE, caused the heterologous desensitization of adenylyl cyclase, indicating that neither receptor occupancy nor activation of adenylyl cyclase was necessary for the heterologous desensitization'.For some time now it has been widely thought that epinephrine-induced desensitization of the B-adrenergic receptor in wild-type (WT) S49 lymphoma cells was homologous and that it did not require the a subunit of the stimulatory guanine nucleotide-binding regulatory protein (Gsa), cAMP, or cAMP-dependent protein kinase (1-9). Support for this was derived in large part from studies of the various mutants of the WT defective in components of the cAMP second messenger system. In particular, we demonstrated that homologous, agonist-induced desensitizations of either the pS-adrenergic or the prostaglandin E1 (PGE,)-responsive adenylyl cyclase (EC 4.6.1.1) required rather high levels of receptor occupancy and were indistinguishable in the WT and cyc -mutant (the cyc -S49 mutant lacks Gsa) (4-7).Recently we presented data that, in contrast to earlier studies, revealed a major difference in epinephrine-induced desensitization between WT and cyc -cells and raised once again the possibility that either cAMP, cAMP-dependent protein kinase, or Gsa was involved in the desensitization of S49 lymphoma cells (10). We found that incubation of WT cells with 5-50 nM epinephrine desensitized epinephrine stimulation of the WT adenylyl cyclase but that even 50 nM epinephrine had no effect on the response of cyc -cells.Observation of this difference in the desensitization of adenylyl cyclase...

Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase.

“…However, PKA-mediated desensitization occurs at about 100-fold lower agonist concentrations than p8ARK-mediated desensitization. Similar conclusions regarding the high agonist sensitivity of PKA-mediated desensitization have been drawn from studies using S49 mouse lymphoma cells defective in PKA (10).…”

“…Three separate rapid mechanisms have been delineated in earlier studies, and their quantitative contribution to the overall loss of signaltransduction capacity has been analyzed. These studies have shown that PKA-mediated desensitization occurs in response to remarkably low agonist concentrations (6,8,10), whereas PARK-mediated desensitization and sequestration exactly parallel the receptor occupancy curve; i.e., they are halfmaximal when 50% of the receptors are agonist-occupied (8).…”

Characterization of Agonist Stimulation of cAMP-Dependent Protein Kinase and G Protein-Coupled Receptor Kinase Phosphorylation of the β2-Adrenergic Receptor Using Phosphoserine-Specific Antibodies

“…Previous studies have shown that PKA desensitization of the ␤ 2 AR was heterologous; that is, that any stimulation of cAMP and PKA could lead to a desensitization of epinephrine stimulation of adenylyl cyclase. We and others have also shown that activation of PKC with PMA led to a similar desensitization in a number of cell types (Clark et al, 1988Kunkel et al, 1989;Johnson et al, 1990;Bouvier et al, 1991;Hausdorff et al, 1991;Yuan et al, 1994). Both the PKA-and PKC-mediated desensitizations were shown to be ablated with an S261,262A substitution mutant of the ␤ 2 AR (Johnson et al, 1990;Yuan et al, 1994).…”

“…That is, agonist occupancy was not required; rather any non-␤ 2 AR agonist or drug activation of PKA could result in desensitization of epinephrine stimulation (Clark et al, 1988Kunkel et al, 1989;Hausdorff et al, 1991;Yuan et al, 1994). However, these conclusions were based on the measurement of the desensitization of adenylyl cyclase, and localization of the ␤ 2 AR sites involved in heterologous desensitization based on site-directed mutagenesis.…”

Cargo-Mediated Regulation of a Rapid Rab4-Dependent Recycling Pathway

“…We show that B2AR signaling via PKA also regulates discrete recycling events in a heterologous cell model, suggesting a general significance of this mechanism in diverse cell types. Our results indicate that this regulation occurs by a recurrent signaling loop requiring a specific PKA site in the B2AR itself, suggesting a novel form of cargo-specific regulation of a rapid recycling pathway.PKA-mediated phosphorylation of the B2AR is a wellknown biochemical consequence of B2AR signaling via heterotrimeric G proteins, and PKA-mediated phosphorylation of the B2AR contributes to functional desensitization of receptor-mediated signaling (Bouvier et al, 1988;Clark et al, 1988;Hausdorff et al, 1989;Liggett et al, 1989;Tran et al, 2004). Such functional regulation can occur in the absence of endocytosis, however, and we are not aware of previous evidence linking PKA-dependent phosphorylation of the B2AR to regulation of endocytic recycling.…”