Protein Kinase C Deficiency Blocks Recovery from Agonist-induced Desensitization*

Protein phosphorylation is central to agonist-induced attenuation of the function of G-protein-linked receptors. Stable expression of RNA antisense to specific pro- tein kinase mRNAs permitted analysis of loss-of-func-tion mutants of A431 human epidermoid carcinoma cells, lacking protein kinase A, protein kinase C, or (cid:98) -ad-renergic receptor kinase. Deficiency of protein kinase C, but not the others, amplified rather than attenuated agonist-induced desensitization. In wild-type cells, the t 1 ⁄ 2 for recovery from desensitization was (cid:59) 25 min following removal of agonist. In the protein kinase C-deficient cells, no resensitization was observed even 60 min after agonist removal. Like protein kinase C-deficiency, inhi- bition of protein kinase C with bisindolylmaleimide or calphostin C blocked resensitization. Resensitization was suppressed by FK506, an inhibitor of protein phosphatase 2B, mimicking protein kinase C-deficiency, but in a non-additive manner. The data reveal protein kinase C and protein phosphatase 2B to be critical ele- ments of resensitization.

Protein phosphorylation is central to agonist-induced attenuation of the function of G-protein-linked receptors. Stable expression of RNA antisense to specific protein kinase mRNAs permitted analysis of loss-of-function mutants of A431 human epidermoid carcinoma cells, lacking protein kinase A, protein kinase C, or ␤-adrenergic receptor kinase. Deficiency of protein kinase C, but not the others, amplified rather than attenuated agonist-induced desensitization. In wild-type cells, the t 1 ⁄2 for recovery from desensitization was ϳ25 min following removal of agonist. In the protein kinase C-deficient cells, no resensitization was observed even 60 min after agonist removal. Like protein kinase C-deficiency, inhibition of protein kinase C with bisindolylmaleimide or calphostin C blocked resensitization. Resensitization was suppressed by FK506, an inhibitor of protein phosphatase 2B, mimicking protein kinase C-deficiency, but in a non-additive manner. The data reveal protein kinase C and protein phosphatase 2B to be critical elements of resensitization.
Chronic stimulation of G-protein-linked receptors (GPLRs) 1 provokes attenuation of the receptor-mediated signal, or desensitization (1). Protein phosphorylation is a critical element of agonist-induced desensitization involving at least three prominent kinase activities (2), cAMP-dependent protein kinase (protein kinase A), calcium and phospholipid-sensitive protein kinase (protein kinase C), and members of the G-protein-linked receptor kinase family, like the ␤-adrenergic receptor kinase. ␤ 2 -Adrenergic receptors (␤ 2 AR) are substrates for protein kinase A, protein kinase C, G-protein-coupled receptor kinase, as well as growth factor receptors with intrinsic tyrosine kinase activity (3,4). Study of the complex roles of protein kinases in the functional regulation of these multiply phosphorylated receptor substrates has been accelerated through the use of lossof-function mutant cells in which target protein kinases have been suppressed by antisense oligodeoxynucleotides (2) and dominant negative mutant kinase (5), in addition to protein kinase inhibitors (6), receptor mutagenesis (7,8), and reconstitution of purified elements in vitro (9).
Recently, we reported cell type-specific roles of various protein kinases in agonist-induced desensitization using oligode-oxynucleotides to suppress these enzymes transiently (2). Suppression of protein kinase C, but not protein kinase A or ␤-adrenergic receptor kinase, amplified rather than attenuated agonist-induced desensitization in a variety of cell types. In the current work we explore this novel role of protein kinase C. Protein kinase C is shown to be obligate for resensitization of GPLR, its action blocked by protein kinase C-deficiency, by the protein kinase C inhibitor bisindolylmaleimide, and mimicked by FK506, a protein phosphatase 2B inhibitor.
Construction of the pLNC Retroviral Vectors-The antisense sequences, 5Ј-TTGGCTTTGGCTAAGAATTCTTTCACGCTCTCC-3Ј, 5Ј-TCGGCCAGCACCGCCTCCAGGTCCGCCAT-3Ј, and 5Ј-CTGCAGAA-GGTGGGCTGCTTGAAGAAGCG-3Ј derived from the complementary sequences of mouse protein kinase A catalytic ␤-subunit (10), human ␤-adrenergic receptor kinase-1 (11), and human protein kinase C ␣-isoform (12), respectively, were engineered into the HindIII/ClaI sites of the pLNCx retroviral vector using standard recombinant DNA techniques (13). The pLNCx vector contains the gene to confer neomycin resistance under the control of the 5Ј-and 3Ј-long terminal repeats of the mouse Moloney virus, and the expression of the antisense RNA is under the control of the cytomegalovirus promoter. Antisense RNA to protein kinase A targets both ␣and ␤-isoform of the catalytic subunit of protein kinase A (10,14). RNA antisense to ␤-adrenergic receptor kinase targets both ␤-adrenergic receptor kinase-1 and -2 (11, 15), but not rhodopsin kinase or G-protein-linked receptor kinase-4 and -5 (16). The ␣-, ␤-, and ␥-isoforms of protein kinase C are targeted by RNA antisense to protein kinase C (12,17,18).
Stable Transfection-A431 cells were transfected with the PLNCx plasmid using Lipofectin® (Life Technologies, Inc., Gaithersburg, MD) reagent according to the manufacturer's protocol. Positive transfectants were obtained by selection with the neomycin analog geneticin (0.5 mg/ml). The ability of the antisense RNAs to suppress the expression of protein kinases in the transfectant clones was determined by immunoblotting analysis and assay of residual enzyme activity.
Immunoblotting Analysis-Cells were harvested and homogenized in 10 mM Hepes buffer, pH 7.4, 2 mM MgCl 2 , 2 mM EDTA containing 10 g/ml leupeptin, 10 g/ml aprotinin, and 0.1 mM phenylmethylsulfonyl fluoride. Nuclei were precipitated by low speed centrifugation. Fifty micrograms of post-nuclei fraction protein was subjected to 10% SDSpolyacrylamide gel electrophoresis and the separated proteins were transferred onto a nitrocellulose membrane. Expression of protein kinase C was probed with antibody against the ␣-isoform of protein kinase C (Life Technologies, Inc., Gaithersburg, MD). ␤-Adrenergic receptor kinase levels were probed with polyclonal antibodies raised against the peptide sequence 389 -402 of ␤-adrenergic receptor kinase-1.
Protein Kinase A and C Assays-Protein kinase A and C activities were assessed by using pre-made assay kits purchased from Life Technologies, Inc. The manufacturer's protocol was followed. Protein kinase A activity is defined as the amount of phosphate incorporated into a substrate peptide, Kemptide, in the presence of 10 M cyclic AMP minus that incorporated in the presence of the protein kinase A inhibitor peptide (1 M). The specific activity of protein kinase C is the difference between phosphorylation of an acetylated peptide derived from myelin basic protein in the presence of 10 M phorbol 12-myristate 13-acetate to that in the presence of 20 M protein kinase C inhibitory peptide (19 -36).
Radioligand Binding Studies-The number of ␤ 2 -adrenergic receptors was determined by radioligand binding to intact cells incubated with 0.5 nM [ 125 I]iodocyanopindolol (ICYP) (DuPont NEN, Boston, MA) in the presence or absence of 10 M propranolol at 23°C for 90 min. The incubation buffer contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , and 150 mM NaCl. The affinity constants for ICYP and isoproterenol (Sigma) binding were determined using crude membrane fractions incubated with or without 0.1 mM GTP␥S. The dissociation constant for ICYP was calculated by Scatchard plot analysis with ICYP concentrations ranging from 0 to 1 nM. The high (K h ) and low (K l ) affinities for isoproterenol binding to ␤ 2 AR were determined by isoproterenol (0 -1 M) displacement of ICYP (0.25 nM). The K h values were from a two-site curve fitting (GraphPad Software, San Diego, CA) to the data obtained in the absence of GTP␥S. The K l values were from a single-site curve fitting to the data obtained in the presence of 100 M GTP␥S.
Suppression via Antisense Oligodeoxynucleotides-Specific antisense and sense oligodeoxynucleotides were synthesized and purified to cell culture grade (Operon Tech., Alameda, CA), as described (2). Wild-type A431 cells were treated with 30 M oligodeoxynucleotides for 2 days prior to the analysis of desensitization.
Desensitization and Resensitization of ␤ 2 AR-Two days prior to the desensitization analysis, cells were seeded in 96-well plates at the density of 25,000 -50,000 cells/well. Cells were washed and challenged with or without 1 M isoproterenol for 30 min at 37°C. At the end of the first challenge, cells were washed three times, and incubated in Hepes buffer containing Ro-20 -1724 (0.1 mM, Calbiochem, San Diego, CA) and adenosine deaminase (0.5 unit/ml) for 5 min prior to (and included in) the second challenge with 1 M agonist. For resensitization, isoproterenol-treated cells were washed free of agonist and maintained in buffer containing no phosphodiesterase inhibitor for 60 min after the first challenge. Five minutes before the second challenge of the agonist, cells were incubated again in the presence of Ro-20 -1724 and adenosine deaminase. The agonist-induced cAMP production within 5 min incubation in the second challenge of agonist was measured. cAMP accumulation was determined as described (19). The data are calculated as "% desensitization" in which "100% desensitization" reflects no cAMP accumulation in response to a second challenge with the agonist. "0% desensitization" refers to a cAMP response to a second challenge is equivalent to that obtained in response to the first challenge.
Effect of Protein Kinase C or Protein Phosphatase Inhibitors on Receptor Resensitization-Cells were preincubated in the presence or absence of protein kinase C inhibitors, such as bisindolylmaleimide and calphostin C (Calbiochem), or protein phosphatase inhibitors, such as FK506 (Fujisawa USA, Deerfield, IL), cyclosporin A (Signal Transduction, San Diego, CA), fenvalerate (LC Laboratories, Woburn, MA), okadaic acid, and calyculin A (Boehringer Mannheim, Indianapolis, IN) for 15 min before experiments. Each inhibitor was included throughout the incubation for desensitization and resensitization as described before.

Stable Suppression of Protein Kinases by Antisense RNA-
Antisense sequences of 29 -33 bases in length were engineered into the retroviral expression vector pLNCx (20) targeting protein kinase A, protein kinase C, and ␤-adrenergic receptor kinase, and used to stably transfect A431 cells. After transfection, neomycin-resistant clones were selected and tested for expression of kinase by immunoblotting ( Fig. 1) and by measurement of enzyme activity (not shown). As shown by immunoblotting, the expression of ␤-adrenergic receptor kinase (Fig.  1A, molecular mass ϳ 79 kDa) and protein kinase C (Fig. 1B, molecular mass ϳ 80 kDa) was suppressed effectively in the clones stably expressing the RNA antisense to each. Assay of total protein kinase C activity agreed well with the immunoblotting, displaying Ͼ80% reduction. The expression of PKC␣ and PKC␥ was suppressed by the antisense, whereas PKC␤ and PKC⑀ isoforms were not detected (not shown). As previously reported (21), the assay for ␤-adrenergic receptor kinase activity was of insufficient sensitivity to accurately detect endogenous ␤-adrenergic receptor kinase levels in the extracts of wild-type cells. Likewise, immunoblotting of protein kinase A in wild-type cells was not sensitive enough to detect endogenous levels of the enzyme in whole cell extracts. Total protein kinase A activity, however, was directly assayed and found to be reduced 70 -85% in clones at early passages.
Suppression of Agonist-induced Desensitization: Comparison of Oligodeoxynucleotide Treatment to Stable Expression of Antisense RNA-The initial observations of unique roles of protein kinase A, protein kinase C, and ␤-adrenergic receptor kinase in agonist-induced desensitization were obtained using treatment with oligodeoxynucleotides antisense and sense to mRNA of each kinase (2). Creating stably transfected clones expressing RNA antisense to each protein kinase permitted studies precluded by the small-scale required for oligodeoxynucleotideinduced suppression. Stable transfectants expressing RNA antisense to protein kinase A, protein kinase C, and ␤-adrenergic receptor kinase were analyzed for agonist-induced desensitization and the results were compared to those obtained by oligodeoxynucleotide-induced suppression (Fig. 2). The data demonstrate a sharp reduction in agonist-induced desensitization in the A431 clones deficient in either protein kinase A or ␤-adrenergic receptor kinase. In the protein kinase C-deficient clones, in contrast, agonist-induced desensitization was markedly potentiated. Stable transfection with the empty expression vector itself has no effect. The extent of isoproterenol-induced desensitization was nearly doubled in cells made deficient in protein kinase C. Both wild-type and empty vector transfected cells reached the maximal desensitization (ϳ30% reduction in V max ) at 10 min following challenge with the agonist. The extent of desensitization plateaued from 10 to 40 min. In contrast, protein kinase C-deficient cells displayed a progressive increase in desensitization (30 -50%) from 10 to 40 min following the challenge (not shown). These data agreed well with those obtained with oligodeoxynucleotide-treated cells. The unexpected potentiation of desensitization by protein kinase Cdeficiency (2) was confirmed by these studies. The availability of stable transfectants lacking protein kinase C permitted further analysis of the role of this kinase in desensitization.
␤-Adrenergic Receptor Properties in Protein Kinase C-deficient Cells-The protein kinase C-deficiency provoked a small, but not significant, reduction in the level of ␤ 2 AR expression (Table I) fected with empty vector alone (not shown). The dissociation constant, K d , for radiolabeled ICYP was unaffected and the distribution of receptors in the high-and low-affinity forms were equivalent in wild-type and protein kinase C-deficient cells. The binding constants for the high-and low-affinity sites also were not altered in the protein kinase C-deficient clones. Basal level of cAMP accumulation in protein kinase C-deficient cells was equivalent to that in wild-type cells, whereas the maximal accumulation of cAMP in response to 1 M isoproterenol in protein kinase C-deficient cells was slightly, but not significantly, reduced (Table II). The EC 50 values of isoproterenol-stimulated cAMP accumulation were 2.5 and 5.9 nM for wild-type and protein kinase C-deficient cells, respectively.
Recovery from Agonist-induced Desensitization Is Lost in Protein Kinase C-deficient Cells-The data from the stimulatory responses provided no clue as to the basis for potentiation of desensitization by protein kinase C-deficiency. The ability of the cells to recover from agonist-induced desensitization was explored next. Cells were stimulated with 1 M isoproterenol for 30 min, then washed free of agonist and examined for the ability to re-establish agonist-stimulated cAMP accumulation (Fig. 3). For clones stably transfected with empty vector, washout of agonist promoted a gradual loss of the desensitization, i.e. resensitization. The t 1 ⁄2 for resensitization in the control clones was ϳ25 min. Within 50 min of washout of agonist, resensitization was completed in these clones. The protein kinase C-deficient cells, in sharp contrast, displayed a rather static level of desensitization. Desensitization persisted for more than 60 min following the washout of the agonist in the protein kinase C-deficient cells.
A second approach, treating cells with inhibitors of protein kinase C was explored. Wild-type cells treated with either bisindolylmaleimide (0.3 M) (22) or calphostin C (0.3 M) (23) displayed a blunted resensitization, retaining ϳ70% of agonistinduced desensitization at 60 min following washout of the agonist (not shown). Taken together, the data from the loss-offunction mutant clones and from wild-type clones treated with either inhibitor of protein kinase C provide an explanation for the potentiation of desensitization observed in the protein kinase C-deficient cells, i.e. protein kinase C activity is critical to the recovery phase of agonist-induced desensitization.
Role of Protein Phosphatase Activities in Receptor Resensitization-The loss of receptor resensitization in protein kinase C-deficient cells and the central role of protein phosphorylation in agonist-induced desensitization prompted consideration of protein phosphatases. A series of well characterized protein phosphatase inhibitors were screened for effects on resensitization of ␤ 2 AR, following a 30-min agonist-induced desensitization (Table III). Inhibition of protein phosphatase 1 and 2A by okadaic acid (100 nM) and calyculin A (10 nM) failed to alter significantly receptor resensitization, when measured at 60 and protein kinase C-deficient A431 cells ␤ 2 -Adrenergic receptor (␤ 2 AR) number was determined by radioligand binding to intact cells incubated with the high-affinity, ␤-adrenergic antagonist ligand [ 125 I]iodocyanopindolol (ICYP, 0.5 nM) in the presence or absence of 10 M propranolol. The results are expressed as mean values Ϯ S.E. (n ϭ 3). Ligand binding affinities were determined using crude membrane fractions (instead of cells) incubated with or without GTP␥S. The dissociation constant, K d , for ICYP binding was calculated from Scatchard plots analysis. K h and K l represent the affinity constants for the high-and low-affinity binding sites for isoproterenol determined in the presence and absence of 100 M GTP␥S. The percentages of high-and low-affinity receptor populations are shown in parentheses.
clones Binding properties ICYP Isoproterenol WT 73,000 Ϯ 4000 146 Ϯ 24 4.9 (24%) 0.64 (76%) PKC-deficient 56,000 Ϯ 9000 a 164 Ϯ 34 a 7.9 (21%) a 0.82 (79%) a a Statistically, not different from WT (p Ͻ 0.05). min following agonist washout. In the presence of okadaic acid, the desensitized receptor activity was completely recovered within 60 min. Although calyculin A at 10 nM has been reported to block substantially receptor dephosphorylation (24), it did not affect resensitization in our experiments (data not shown). A minimal, residual degree of desensitization was detected in cells treated with 100 nM calyculin A at 60 min after resensitization. Cells did not tolerate higher concentrations of either okadaic acid or calyculin A, precluding analysis of resensitization at higher concentrations of either inhibitor (not shown). Treating cells with the protein phosphotyrosine phosphatase inhibitor vanadate (100 M), also failed to alter the resensitization. Noticeably, neither okadaic acid nor calyculin A enhanced desensitization, measured at 5 min post the first challenge of agonist.

TABLE II Isoproterenol stimulated responses in wild-type and protein kinase C-deficient A431 cells
Most striking were the effects obtained with the inhibitor of calcium/calmodulin-dependent protein phosphatase (protein phosphatase 2B), FK506. FK506 associates with immunophilin FKBP12 and specifically inhibits protein phosphatase 2B activity (25,26). Treatment with FK506 potentiated agonistinduced desensitization by more than 2-fold (27 versus 75% desensitization) measured at 5 min postwashout. FK506treated cells displayed a persistent desensitization at 60 min postchallenge of agonist, much like deficiency in protein kinase C (Fig. 3). Cells treated with inhibitor of protein phosphatase 2B, either cyclosporin A (100 ng/ml) or fenvalerate (10 nM) (25,27), also displayed blunted resensitization (not shown). Protein phosphatase 2B expression was readily detected in these cells via immunoblotting (not shown).
The effects of FK506 treatment on resensitization were dosedependent (Table IV). Since FK506 and protein kinase C deficiency both suppressed the ability of the cells to recover from agonist-induced desensitization, it was of interest to explore whether their effects were additive. Although both FK506 and protein kinase C-deficiency abolished recovery of cells from agonist-induced desensitization, the effects of the two, in combination, were not additive. Treatment of protein kinase Cdeficient cells with FK506 did not suppress resensitization further than protein kinase C-deficiency alone. As expected, treatment of protein kinase C-deficient cells with phorbol 12myristate 13-acetate failed to change either the extent of desensitization or resensitization (not shown).

DISCUSSION
The attenuation that accompanies chronic stimulation is a fundamental feature of cell signaling. For GPLR, agonist-induced desensitization (1) and down-regulation (28) are nearly universally observed. Protein phosphorylation is central to agonist-induced desensitization, with roles implicated for several protein kinases including protein kinase A (7,29), protein kinase C (9,30), and G-protein-linked receptor kinases (31). In our earlier studies of protein kinases and agonist-induced desensitization, we created loss-of-function mutants by treating cells with oligodeoxynucleotides antisense to specific kinases (2). Elimination of protein kinase A and ␤-adrenergic receptor kinase resulted in a profound loss of attenuation in response to ␤-adrenergic stimulation of cAMP accumulation in a variety of cell types. Loss of protein kinase C, in contrast, potentiated rather than blunted agonist-induced desensitization.
In the current work, use of stably transfected clones permitted further analysis of the effects of protein kinase C-deficiency on desensitization. The hypothesis that protein kinase C acts to suppress desensitization and/or enhance resensitization was FIG. 3. Receptor re-sensitization: time course for control and protein kinase C-deficient cells. Clones stably transfected with empty vector or vector expressing RNA antisense to protein kinase C were challenged with or without 1 M isoproterenol for 30 min, followed by extensive washes. After agnoist washout, cells were incubated in assay buffer for 5-65 min before the second challenge of isoproterenol. The degrees of desensitization in clones expressing empty vector or RNA antisense to protein kinase C measured at 5 min postchallenge were 32 and 44%, respectively. The difference in degree of desensitization measured at 5 min and a designated time postchallenge relatives to the degree of desensitization measured at 5 min postchallenge is calculated as "% resensitization." The results are expressed as mean Ϯ S.E. (n ϭ 4).

TABLE III
Effect of protein phosphatase inhibitors on receptor resensitization A431 cells were pretreated with the inhibitors indicated below for 15 min prior to the first challenge with 1 M isoproterenol for 30 min. After desensitization, the ␤-adrenergic agonist was washed out and cells were resensitized for 5 or 60 min before the second challenge of isoproterenol. The degrees of desensitization were calculated as described under "Experimental Procedures" and are presented as mean Ϯ S.E. (n ϭ 3-5).  tested in loss-of-function mutants, deficient in protein kinase C. Elimination of protein kinase C potentiated desensitization, but was shown not to influence the onset of the process, prompting analysis of the resensitization process. Protein kinase C-deficiency was found not only to influence the recovery of cells from agonist-induced desensitization, but also to block the recovery phase entirely. Loss-of-function mutant cells failed to recover from desensitization for more than an hour following agonist washout. Empty vector transfected cells displayed a recovery process with a t 1 ⁄2 ϳ25 min. This critical observation was confirmed using an independent approach, treatment of wild-type cells with either bisindolylmaleimide or calphostin C, inhibitors of protein kinase C. The central role of protein phosphorylation in agonist-induced desensitization for GPLR prompted an analysis of protein phosphatase activity in the recovery phase. The role and character of phosphatases in the resensitization process were established using well known phosphatase inhibitors, specific for protein phosphatase 1, protein phosphatase 2A, protein phosphatase 2B, and protein phosphotyrosine phosphatase activities. Although protein phosphatase 2A has been shown to dephosphorylate ␤-adrenergic receptor kinase-phosphorylated GPLR in the reconstituted system (32), neither okadaic acid nor calyculin A, inhibitors of protein phosphatase 2A altered significantly the recovery of agonist-induced desensitization. These observations indicate no major role of protein phosphatase 2A in resensitization in these cells, and further suggest that the reversal of ␤-adrenergic receptor kinase-mediated phosphorylation is not essential for the recovery of receptor activity. Likewise, the inability of vanadate ions to alter agonist-induced desensitization argues against a role of protein phosphotyrosine phosphatase in resensitization of GPLR. Although shown recently to be substrates for tyrosine kinases both in vivo (3) and in vitro (4), ␤ 2 AR are cross-regulated by hormones like insulin and insulin-like growth factor-I, whereas activation of tyrosine kinases is not known to occur in homologous desensitization.
In association with immunophilin FKBP12, FK506 is known to inhibit protein phosphatase 2B activity (25,26). Treating cells with FK506 potentiated agonist-induced desensitization of ␤ 2 AR-mediated cAMP accumulation and attenuated sharply the recovery from desensitization that typically follows washout of the agonist. These effects were dose-dependent and suggest a central role of protein phosphatase 2B in the recovery phase from agonist-induced desensitization of GPLRs. The action of FK506 observed in wild-type cells was absent in the protein kinase C-deficient mutants. Taken together these data provide a compelling case for a central role of protein phosphatase 2B in protein kinase C-dependent recovery of cells from agonist-induced desensitization (Fig. 4). In view of the data obtained in the loss-of-function mutant clones lacking protein kinase C, activation of protein kinase C with phorbol esters might be expected to reduce desensitization overall, by accelerating the recovery phase. In the loss-of-function mutants treatment with phorbol 12-myristate 13-acetate had no effect, as expected. Paradoxically, treating wild-type cells with phorbol 12-myristate 13-acetate enhanced desensitization (not shown). The basis for this effect was revealed recently in studies reported by Chuang et al. (33) demonstrating that protein kinase C potentiates the activity of ␤-adrenergic receptor kinase (Fig. 4). Thus, protein kinase C appears to play a pivotal role in regulating GPLR desensitization. In the forward direction, activation of protein kinase C leads to increased ␤-adrenergic receptor kinase activity promoting greater desensitization. In the reverse direction, protein kinase C is obligate and central to the recovery from desensitization. The action of protein kinase C on the recovery phase appears to require protein phosphatase 2B. As determined through metabolic labeling of the cells with 32 P, protein phosphatase 2B was phosphorylated in response to phorbol 12-myristate 13-acetate treatment of the cells (not shown). This novel role for protein kinase C highlights a new dimension in our understanding of cross-talk between GPLR-mediated responses. In view of the ability of catecholamines to activate both ␣-adrenergic receptors (linked to phospholipase C and protein kinase C activation) and ␤-adrenergic (linked to adenylylcyclase activation) receptors coexpressed in many cells, such cross-talk would seem integral in cell signaling.