Jacqueline Friedman

Characterization of β2-adrenergic receptor dephosphorylation: comparison with the rate of resensitization

Dephosphorylation of the cyclic AMP-dependent protein kinase (PKA) site phosphoserine 262 and the G protein-coupled receptor kinase (GRK) site phosphoserines 355 and 356 of the β2-adrenergic receptor (β2AR) were characterized in both intact human embryonic kidney 293 cells and subcellular fractions and were correlated with the rate of resensitization of isoproterenol stimulation of adenylyl cyclase after treatment with isoproterenol and blockade by antagonist. Dephosphorylation of the PKA site after stimulation with 300 pM isoproterenol occurred with a t½ of 9 min (k = 0.08 ± 0.016/min) in intact cells in the absence of internalization. Dephosphorylation of the GRK sites in intact cells after treatment with 1.0 μM isoproterenol for 5 min exhibited a lag phase of ≈ 5 min, after which dephosphorylation proceeded slowly with a t½ of 18 min (k = 0.039 ± 0.006/min). Consistent with the slow rate of GRK site dephosphorylation, the phosphatase inhibitors calyculin A and okadaic acid failed to augment phosphorylation in intact cells during continuous agonist stimulation indicating that GRK site dephosphorylation was minimal. However, both inhibited dephosphorylation of the GRK sites after the addition of antagonist. Slow GRK site dephosphorylation after antagonist treatment was also demonstrated by the relative stability of internalized phosphorylated β2AR in cells as observed both by immunofluorescence microscopy using a phospho-site-specific antibody and by studies of the subcellular localization of the GRK-phosphorylated β2AR on sucrose gradients that revealed nearly equivalent levels of GRK site phosphorylation in the plasma membrane and vesicular fractions. In addition, dephosphorylation of the GRK sites by intrinsic phosphatase activity occurred only in the heavy vesicle fractions. In contrast to the slow rates of dephosphorylation, the rate of resensitization of isoproterenol stimulation of adenylyl cyclase was 5- and 10-fold faster (k = 0.43 ± 0.009/min; t½ = 1.6 min), than PKA and GRK site dephosphorylation, respectively, clearly dissociating the rapid phase of resensitization (0-5 min) from dephosphorylation.

Cell-free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP-dependent protein kinase.

We have examined the cell‐free heterologous desensitization of adenylyl cyclase in plasma membrane preparations from S49 wild‐type (WT) and kin− cells (which lack cAMP‐dependent protein kinase) incubated with purified catalytic subunit of cAMP‐dependent protein kinase (cA·PKc). cA·PKc caused a rapid (t1/2 = 40 s) decrease in the hormone responsiveness of adenylyl cyclase in the WT membrane preparations that mimicked the intact cell heterologous desensitization; that is, there was an increase in the Kact for both epinephrine and prostaglandin E1 (PGE1) stimulations of adenylyl cyclase induced at the receptor level because neither forskolin‐ nor NaF‐stimulated activity was affected. The desensitization was independent of agonist occupancy of the receptor, and the effects were blocked both by the active fragment (amino acids 5‐22) of the specific inhibitor of cA · PK and by p[NH]ppA. cA · PKc treatment of kin− membranes resulted in a heterologous desensitization that resembled the effects on WT adenylyl cyclase, with the exception that forskolinstimulated activity was also reproducibly decreased by 24%. cA·PKc had no effect on WT membranes isolated from cells that had previously undergone maximal heterologous desensitization during treatment with 10 μM forskolin. In contrast, cA · PKc‐induced heterologous desensitization of kin− membranes was additive with the epinephrine‐induced homologous desensitization of intact cells. Cell‐free desensitizations were reversed by incubation of membranes with cA·PKc and ADP, conditions that drive the kinase reaction backward. The similarities of our cell‐free cA · PKc‐mediated heterologous desensitization of adenylyl cyclase with the intact cell desensitization support our hypothesis that heterologous desensitization of the WT lymphoma cells is mediated by cA·PK via a mechanism independent of homologous desensitization.—Kunkel, M. W.; Friedman, J.; Shenolikar, S.; Clark, R. B. Cell‐free heterologous desensitization of adenylyl cyclase in S49 lymphoma cell membranes mediated by cAMP‐dependent protein kinase. FASEB J. 3: 2067‐2074; 1989.

Beta-adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg2+.

Treatment with low physiological concentrations of epinephrine (5‐50 nM) rapidly desensitizes β‐adrenergic stimulation of cAMP formation in S49 wild‐type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell‐free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg2+ concentrations in the adenylate cyclase assays to <1.0 mm unmasked this rapid phase of desensitization of the WT cells, and that high Mg2+ concentrations (5‐10 mm) largely obscured the desensitization. Submillimolar Mg2+ conditions also revealed a two‐ to threefold decrease in the affinity of epinephrine binding to the β‐adrenergic receptor after desensitization with 20 nm epinephrine. Detection of 4β‐phorbol 12‐myristate 13‐acetate (PMA) desensitization of the WT β‐adrencrgic receptor was also dependent on low Mg2+ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc− cells were pretreated with 50 nm epinephrine, the β‐adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg2+ effect on epinephrine‐ and PMA‐induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the β‐adrenergic receptors. Our data indicate that cAMP‐dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc−. For the PMA‐induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).—Clark, R. B.; Friedman, J.; Johnson, J. A.; Kunkel, M. W. β‐Adrenergic receptor desensitization of wild‐type but not cyc lymphoma cells unmasked by submillimolar Mg2+. FASEB J. 1: 289‐297; 1987.

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.

Biochemical and Cellular Specificity of Peptide Inhibitors of G Protein-Coupled Receptor Kinases

Identifying novel allosteric inhibitors of G protein-coupled receptor kinases (GRKs) would be of considerable use in limiting both the extent of desensitization of GPCRs as well as downstream positive regulation through GRKs. Several peptides have previously been identified as inhibitors of specific GRKs, but to date there have been few comparisons of the selectivities of these materials on the seven GRKs, modifications to allow cell penetration, or off-target activities. The goal of this study was to determine if a panel of peptides mimicking domains on either GPCRs or GRKs would exhibit selective inhibition of GRKs 2, 5, 6 and 7 phosphorylation of rhodopsin. Peptides included sequences from GRK5; helices 3, 9, and 10 (α3, α9, and α10) in the RH domain, and the N-terminal peptide (N-Ter), as well as the intracellular loop 1 (iL1) of the β2-adrenergic receptor (β2AR), and the Gα transducin C-tail (TCT). While some selectivity for individual GRKs was found, overall selectivity was limited and often not reflective of structural predictions. Off-target effects were probed by determining peptide inhibition of adenylyl cyclase (AC) and PKA, and while peptides had no effect on AC activity, N-Ter, iL1, and α10 were potent inhibitors of PKA. To probe inhibition of GRK activity in intact cells, we synthesized TAT-tagged peptides, and found that TAT-α9-R169A and TAT–TCT inhibited isoproterenol-stimulated GRK phosphorylation of the β2AR; however, the TAT peptides also inhibited isoproterenol and forskolin stimulation of AC activity. Our findings demonstrate potent peptide inhibition of GRK activities in vitro, highlight the differences in the environments of biochemical and cell-based assays, and illustrate the care that must be exercised in interpreting results of either assay alone.

Role of Protein Kinases in the Desensitization-Sensitization of β-Adrenergic Receptor and Prostaglandin Receptor Stimulation of Adenylyl Cyclase

Desensitization of hormonal stimulation of adenylyl cyclase in the S49 wild type (WT) lymphoma cells is complex. At least five types of desensitization of the β-adrenergic receptor response have been identified (fig 1) at the receptor level. These include: (1) cAMPdependent protein kinase (cA•PK)-mediated heterologous desensitization of hormonal stimulation which occurs rapidly in response to low concentrations (EC50 of about 10 nM) of either epinephrine or PGE1, or any drug which increases cAMP (Clark et al. 1988); (2) homologous desensitization associated with the sequestration and internalization of the β-adrenergic receptor which occurs rapidly in response to high concentrations of epinephrine (EC50 about 200 nM), is dependent on substantial agonist occupation of the receptor, and is independent of Gs, cAMP or cA•PK (Green et al. 1981, Clark et al. 1985) and has been suggested to involve multiple phosphorylations by β-adrenergic receptor kinase (Benovic et al. 1987); (3) a protein kinase C (PKC)-mediated heterologous desensitization which occurs in response to relatively high concentrations of the phorbol ester PMA with an EC50 of about 100 nM (Johnson et al. 1986); (4) a slow homologous down regulation of the β-adrenergic receptor, characterized by the loss of antagonist binding, which proceeds with a half-life of about 2 hr in the WT and 6 hr in the cyc mutant (Clark et al. 1985); and (5) a slow homologous desensitization of the β-adrenergic receptor which occurs in response to treatment of cells with low concentrations of epinephrine (3–5 nM) for prolonged periods of time (half-life about 2–4 hr) and which may be, in part, mediated by cA•PK (Butcher et al. 1987).