The 15-Amino Acid Motif of the C Terminus of the ?2-Adrenergic Receptor Is Sufficient to Confer Insulin-Stimulated Counterregulation to the
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内分泌学杂志 2005年第1期
Department of Molecular Pharmacology (S.G., D.Y., E.S., C.C.M.), University Medical Center, State University of New York/Stony Brook, Stony Brook, New York 11794-8651; and Department of Physiology and Biophysics (H.-y.W.), Diabetes and Metabolic Diseases Research Program, University Medical Center, State University of New York/Stony Brook, Stony Brook, New York 11794-8661
Address all correspondence and requests for reprints to: Craig C. Malbon, Department of Pharmacology, Health Sciences Center, State University of New York/Stony Brook, Stony Brook, New York 11794-8651. E-mail craig@pharm.sunysb.edu.
Abstract
Insulin counterregulates catecholamine action in part by inducing the sequestration of ?2-adrenergic receptors. Although similar to agonist-induced sequestration, insulin-induced internalization of ?2-adrenergic receptors operates through a distinct and better-understood cellular pathway. The effects of insulin treatment on the function and trafficking of both ?1- and ?2-adrenergic receptors were tested. The ?2-adrenergic receptors were counterregulated and internalized in response to insulin. The ?1-adrenergic receptors, in sharp contrast, are shown to be resistant to the ability of insulin to counterregulate function and induce receptor internalization. Using chimeric receptors composed of ?1-/?2-adrenergic receptors in tandem with mutagenesis, we explored the role of the C-terminal cytoplasmic tail of the ?2-adrenergic receptors for insulin-induced counterregulation. Substitution of the C-terminal cytoplasmic tail of the ?2-adrenergic receptor on the ?1-adrenergic receptor enabled the chimeric G protein-coupled receptor to be functionally and spatially regulated by insulin. Truncation of the ?2-adrenergic receptor C-terminal cytoplasmic tail to a 15-amino acid motif harboring a potential Src homology 2-binding domain at Y350 and an Akt phosphorylation site at S345,346 was sufficient to enable receptor regulation by insulin.
Introduction
G PROTEIN-COUPLED RECEPTORS (GPCRs) and growth factor receptors with intrinsic tyrosine kinase activity represent two dominant pathways controlling cellular signaling (1, 2, 3, 4). Integration of signaling between GPCR and tyrosine kinase pathways has been investigated and includes the existence of cross-regulation at the most proximal point, receptor-to-receptor interaction with GPCRs acting as substrates for tyrosine kinases (5, 6, 7). Insulin cross-talk with ?2-adrenergic receptor (?2AR) serves an important counterregulation of glucose homeostasis. Treating cells with insulin stimulates the phosphorylation of the ?2AR on tyrosyl residues Y350/354 and Y364 for in vivo studies (5, 6) as well as in vitro (7) studies using recombinant, purified ?2AR and insulin receptors. The Y350 residue, a prominent residue for insulin receptor-catalyzed phosphorylation of the cytoplasmic tail of the ?2AR, is embedded in a sequence motif (Tyr-Gly-Asn-Gly) that is similar to the motifs known to interact with Caenorhabditis elegans sem5 Src homology 2 (SH2) domains when phosphorylated (8). Phosphorylation of the ?2AR by the insulin receptor and the IGF-I receptor includes a motif for tyrosine kinases at Y364 (9), the SH2/Grb2 binding site at Y350 (6, 10), and a potential SHC binding site at Y132 (6, 7, 9). For insulin action, activation of 1-phosphatidylinositol 3-kinase (PI3-kinase) is an early event, following temporally the phosphorylation of the insulin receptor and insulin receptor substrate-1 (11, 12). In response to insulin stimulation, the p85 regulatory subunit of PI3-kinase binds the insulin receptor substrate-1 via SH2 domain(s), activating the catalytic p110 subunit that in turn phosphorylates various phosphoinositides at the 3'-position of the inositol ring (13). Ample reports support the premise that PI 3-kinase and its 3'-phosphoinositide products are critical to intracellular trafficking both of membrane-bound elements in general (14) and downstream elements of tyrosine kinase-mediated cell signaling, particularly that of insulin (15).
We have shown that insulin, much like ?-adrenergic agonists, provokes rapid sequestration of ?2AR (16, 17). In addition, downstream activation of Erk1/2 by insulin in mammalian cells was shown to be amplified by ?2AR expression, i.e. the higher the level of cellular complement of ?2AR, the greater was the amplification of insulin activation of Erk1/2, identifying the ability of the ?2AR to act as a scaffold for some aspect of insulin action. The ability of the ?2AR to amplify the insulin response was dependent on the integrity of the Y350 residue, which is phosphorylated by the insulin receptor and constitutes a binding site for an SH2 domain to which Grb2, and other proteins can bind (18). The later identification of an Akt phosphorylation sites in the C-terminal tail of the ?2AR (19) and the central role of Akt in insulin action (20, 21, 22, 23) brought into focus the question of what domain(s) in the ?2AR confer to this GPCR its ability to be regulated by insulin. In the current work, we demonstrate that unlike the ?2AR, the ?1-adrenergic receptor (?1AR) does not internalize in response to insulin. We make use of this marked difference in receptor trafficking to define the motifs in the ?2AR through which insulin catalyzes sequestration, demonstrating that the donation of the Y350 and Akt phosphorylation sites to the ?1AR enable insulin to internalize the chimeric ?1AR.
Materials and Methods
Plasmids
The human ?1AR gene including the FLAG epitope on the 5' end was generated by PCR using plasmid pUC18-h?1AR (kindly provided by Dr. Stephen Liggett, Department of Medicine, University of Cincinnati, Cincinnati, OH) as the template and a pair of primers designed with HindIII or BamHI linkers. The PCR product was digested with HindIII and BamHI and cloned into the unique HindIII/BamHI sites of peGFP-N1 expression vector (Clontech, Palo Alto, CA) so that the human ?1AR gene was fused to the amino terminus of the GFP. The plasmid encoding the enhanced-green fluorescent protein (GFP)-tagged human ?2AR (in pCDNA3), and including the FLAG epitope at the amino terminus, was a generous gift from Dr. Jeffrey Benovic (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA). A ?1AR/?2AR (?1/?2CTAR) chimera containing the N-terminal FLAG epitope and the C terminus eGFP epitope was made by replacement of the C terminus of the ?1AR with the C terminus of the ?2AR (?1AR from Met1-Ser380 and ?2AR from Pro329-Leu413) by using overlap extension PCR as previously described (24) and cloned into the unique HindIII/BamHI sites of peGFP-N1 expression vector. Another ?1/?2 AR chimera (?1/?2CT342–356) containing the N-terminal FLAG epitope and the C-terminal eGFP was generated by replacement of 15 amino acids in the C terminus of the ?1AR (from Cys393 to Thr407) with 15 amino acids of the ?2AR (from Lys342-Ser356) using overlap extension PCR and cloned into the HindIII/BamHI sites of peGFP-N1 expression vector. The sequence of constructs was confirmed by DNA sequencing.
Cell culture
Chinese hamster ovary (CHO)-K1 cells and human carcinoma HeLa cells were maintained in DMEM supplemented with 5% fetal bovine serum, plus penicillin (60 μg/ml) and streptomycin (100 μg/ml), grown in humidified atmosphere chamber supplied with 5% CO2 and 95% air at 37 C.
Assay of intracellular accumulation of cAMP
CHO-K1 cells transiently transfected with the human ?2AR, ?1AR, ?1/?2CTAR, and ?1/?2CT342–356AR were seeded in a 96-well plate for 24 h. On the day of experiment, cell culture medium was aspirated, the cells washed and replenished with Krebs-Ringer phosphate (KRP) buffer containing 10 μM RO-201724 (a cAMP phosphodiesterase inhibitor), and then treated with the indicated hormones in a total assay volume of 50 μl. The reaction was terminated by the addition of 50 μl of 100% ethanol, and the AMP content was measured by the competitive binding assay as described (25). To assay insulin counterregulation, cells were treated for 30 min with insulin (100 nM) dissolved in a KRP buffer and challenged with the ?-adrenergic agonist isoproterenol (10 μM) for 15 min to enable the accumulation of intracellular cAMP. Each experiment was performed with triplicate determinations.
?-Adrenergic antagonist binding and receptor sequestration assays
The expression of ?2AR, ?1AR, and the chimera was quantified by assay of the binding of the high-affinity, ?-adrenergic antagonist iodocyanolpindolol [125I]CYP to whole-cell preparations of the transiently transfected cells, as detailed elsewhere (24). Nonspecific binding was determined using ?-adrenergic antagonist propranolol (10 μM). The expression and sequestration of receptors on plasma membrane in CHO-K1 cells transiently transfected with the human ?2AR, ?1AR, and chimera were quantified using the hydrophilic, cell-impermeable radiolabeled ?-adrenergic antagonist [3H]CGP-12177 (26). Cells were treated for 30 min with insulin (100 nM) or isoproterenol (10 μM) for at 37 C and then placed at 4 C for 6 h with 70 nM [3H] CGP-12177. The cells were rapidly washed free of unbound ligand and collected on GF/C membranes by vacuum filtration. The radioligand bound to the washed cell mass retained on the filter was quantified by liquid scintillation spectrometry. Binding affinity (Kd) of ?1AR, ?2AR, and chimera was determined using concentrations of [3H]CGP-12177 from 0.023–2.3 nM as previously described (26). Confocal microscopy of eGFP-tagged receptors was performed as described earlier (24).
Membrane preparations
Cells were harvested with ice-cold PBS containing 1 mM EDTA and collected by centrifugation (1000 x g). The cell pellets then were resuspended in 5 ml of a hypotonic buffer composed of 20 mM HEPES (pH 7.4); 2 mM MgCl2; and 1 mM EDTA (HME buffer) plus the protease inhibitors, leupeptin (5 μg/ml), aprotinin (5 μg/ml), and phenylmethylsulfonyl fluoride (200 nM). After incubation on ice for 15 min, the cells were homogenized with five to eight strokes of a Dounce homogenizer operating with a tight-fitting Teflon pestle. Membrane fractions were collected from 1,000 x g postnuclear supernatants by centrifugation at 10,000 x g. The crude membrane pellets were resuspended in 200 μl HME buffer, and protein concentration was determined. The Lowry method was used to determine the amount of protein in the samples before gel loading. The samples were subject to SDS-PAGE using 10% acrylamide gels. The resolved proteins were transferred electrophoretically onto nitrocellulose blots, probed with the anti-GFP antibody, and stained with a secondary antibody to which horseradish peroxidase was coupled. The chemiluminescence reagent was used to detect immunocomplexes.
Site-directed mutagenesis
Double mutation of serine residues 345 and 346 to alanine and tyrosyl residues 350 and 354 to phenylalanine in the ?1/?2CTAR was performed using the Quickchange site directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s suggested protocols. The sequences of the mutagenic primers were as follows: S345/346A, GCTTCTGTGCCTGCGCAGGGCCGCCTTGAAGGCCTATGGC; and Y350/354F, GGTCTTCTTTGAAGGCCTTCGGCAATGGCTTCTCCAGCAACGGCAACACAGGGG. The DNA sequence of the mutant receptors was confirmed by use of direct DNA sequencing of the mutant plasmid DNA.
Results
To investigate the motif(s) that confer the ability of insulin to sequester ?2ARs, we needed to satisfy several criteria. First, ?2ARs are expressed virtually ubiquitously in cells in culture, and a cell line essentially deficient of ?2ARs is optimal for such studies. The CHO-K1 cells were used because these cells express vanishing few, if any, ?2AR (27). To investigate the domains/protein motif(s) through which insulin exerts its effects on sequestration of ?2AR, a recipient GPCR was required that was similar to the ?2AR but one that failed to undergo insulin-stimulated counterregulation. Earlier observations suggested that the action of the ?1AR, which shares many properties in common with the ?2AR, does not display counterregulation by insulin (2). Finally, the studies required that a functional assay of ?AR activity be coupled with the ability to study the sequestration by radioligand binding and independently by confocal microscopy.
Expression of ?1AR, ?2AR, and chimera in CHO-K1 cells
The ability to express the GFP-tagged ?1AR and ?2AR was investigated in CHO-K1 cells transiently transfected with mammalian expression vectors harboring the cDNAs of each. Crude cell membranes were prepared from the cells transiently transfected with the appropriate expression vector and harvested 30 h later. Using [125I]ICYP to assess the expression of total ?AR, we observed the following values (femtomoles per 106 cells): the ?1AR-expressing cells, 225.5 ± 49.5, and ?2AR-expressing cells, 186.0 ± 23 (27). Binding affinity to their antagonist [3H]CGP-12177 was determined using Scatchard plot. The results revealed Kd values (nanomoles) for ?1AR of 0.52 ± 0.02 and ?2AR of 0.66 ± 0.14. Thus, CHO-K1 cells transiently transfected with the same expression vector, but harboring different ?AR cDNAs, yielded comparable amounts of receptor expression. Aliquots of the crude cell membranes were subjected to SDS-PAGE and the separated proteins blotted with antibodies specific for the GFP-moiety (Fig. 1). The results of the immunoblotting demonstrate the expression of the receptors, ?1AR and ?2AR [mobility (Mr) 98 kDa], both demonstrating relative mobilities that agree well with the expected Mr for their GFP-tagged versions of their ?AR subtypes (28). Some lower Mr forms likely to be products of differential glycosylation were apparent for the blots of either receptor subtype (29, 30).
FIG. 1. Immunoblots (IBs) from membrane preparations of wild-type CHO-K1 cells and CHO-K1 cells with transient transfection of ?1AR-GFP, ?2AR-GFP, and ?1/?2CTAR-GFP. CHO-K1 cells were transiently transfected with expression vector harboring the human ?2AR, ?1AR, or ?1/?2CTAR chimera. Cells were then homogenized with a Dounce homogenizer and subjected to centrifugation at 10,000 x g. Samples were subject to SDS-PAGE using 10% acrylamide gels and probed with an antibody against GFP. Immunoblotting demonstrates expression of ?2AR, ?1AR, or ?1/?2CTAR chimera with relative mobilities consistent with the expected Mr for their GFP-tagged versions of their ?AR subtypes. Some lower Mr forms of the either receptor subtype are likely to be products of differential glycosylation.
We also constructed a ?1/?2CTAR chimera (human ?1AR from Met1-Ser380 and human ?2AR from Pro329-Leu413) to ascertain what effect, if any, the substitution of the ?2AR C terminus in the ?1AR would have on the ability of the chimera to respond to insulin. In transiently transfected CHO-K1 cells, the ?1/?2CTAR chimeric receptor was expressed at levels (265.0 ± 21.9 fmol/106 cells) comparable with ?1AR and ?2AR, displaying the same approximated relative Mr on SDS-PAGE as the ?1AR (Fig. 1). Kd value (nanomoles) for binding of [3H]CGP-12177 to the ?1/?2CTAR chimera was 0.51 ± 0.11, similar to ?1AR and ?2AR subtypes.
?2AR and the ?1/?2CTAR chimera, but not ?1AR, demonstrate insulin-sensitive counterregulation of isoproterenol-stimulated cAMP response
The ability of the ?-adrenergic agonist isoproterenol (10 μM) to stimulate cAMP accumulation in CHO cells transiently transfected with expression vectors harboring the cDNA for the ?1AR, ?2AR, and ?1/?2CTAR was probed in cells expressing comparable amounts of receptors (Fig. 2). For the ?1AR-expressing cells, isoproterenol (10 μM) stimulated a robust increase in intracellular cAMP accumulation, and this response was not sensitive to counterregulation by insulin (100 nM). In cells expressing the ?2AR, isoproterenol stimulated a nearly equivalent cAMP response as observed in the cells expressing the ?1AR. When stimulated by both isoproterenol and insulin together, cells expressing the ?2AR, in contrast to those expressing the ?1AR, displayed an insulin-sensitive counterregulation of the isoproterenol-stimulated cAMP response, as reported earlier in other cell lines (6, 16, 31, 32, 33).
FIG. 2. Insulin counterregulates the isoproterenol-stimulated cAMP response mediated by the ?2AR and the ?1/?2CTAR chimera but not the ?1AR. CHO-K1 cells transiently transfected with expression vector harboring the human ?2AR, ?1AR, or ?1/?2CTAR chimera were treated with or without 100 nM insulin (Ins) for 30 min and then challenged with 10 μM isoproterenol (Iso) for 15 min. Insulin suppressed isoproterenol-stimulated cAMP response in the cell transfected with the vectors harboring the ?2AR and ?1/?2CTAR chimera but not the ?1AR. These data are mean values ± SEM representative of three separate experiments. Asterisks denote P 0.05 for the difference between insulin and KRP buffer treatments.
To ascertain whether the C-terminal region of the ?2AR is the dominant element of the receptor that confers the ability to be counterregulated by insulin, we studied a chimeric receptor composed of the core of the ?1AR (residues Met1-Ser380, which lacks the entire C-terminal cytoplasmic tail) to which the cytoplasmic C-terminal tail of the ?2AR (residues Pro329-Leu413) has been spliced (?1/?2CTAR, Fig. 2). The ?1/?2CTAR was expressed at levels comparable with those observed for either the ?1AR or ?2AR and was able to stimulate cAMP accumulation in response to isoproterenol. Unlike the ?1AR that failed to display counterregulation by insulin, the ?1/?2CTAR chimera was found to display an insulin-stimulated counterregulation of the cAMP response to isoproterenol. These data reveal that by substitution of the C terminus of the ?2AR to the ?1AR, an insulin-stimulated counterregulation was conferred to the ?1AR.
?2AR and ?1/?2CTAR chimera, but not ?1AR, demonstrate insulin-stimulated internalization
We sought to quantify the level of cell surface ?ARs to test further the observations made by functional assay of insulin action on ?-adrenergic agonist-stimulated cAMP accumulation. The high-affinity, radiolabeled, and hydrophilic antagonist CGP-12177 is unique as a radioligand binding reagent because it is restricted to the extracellular aqueous environment and binds only to those ?ARs localized in the cell membrane. Using [3H]CGP-12177 and intact cells, the surface complement of ?1AR, ?2AR, and ?1/?2CTAR chimera were quantified in cells after a challenge with either isoproterenol (10 μM) or insulin (100 nM, Fig. 3). For either ?1AR or ?2AR, treatment with isoproterenol provoked a 60% reduction in the cell surface complement. The ?2AR displayed internalization of more than 40% in response to100 nM insulin. The ?1AR, in contrast, displayed no internalization in response to treatment of the cells with100 nM insulin.
FIG. 3. Insulin internalizes the ?2AR and the ?1/?2CTAR chimera but not the ?1AR. CHO-K1 cells transiently transfected with expression vectors harboring the human ?1AR, ?2AR, or ?1/?2CTAR chimera were treated with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso) for 30 min at 37 C. The internalization of the receptor was measured by use of a hydrophilic, cell-impermeable, radiolabeled ?-adrenergic antagonist [3H]CGP-12177. Insulin treatment promotes internalization of the ?2AR and ?1/?2CTAR but not of the ?1AR. All three receptors displayed internalization in response to stimulation with the ?-adrenergic agonist isoproterenol. These data are mean values ± SEM of three separate experiments. The surface receptor expression in unstimulated cells is set at 100%. Asterisks denote P 0.05 for the difference between insulin treatment and control.
We also tested the ability of the ?1/?2CTAR chimeric receptor to respond to agonist-induced internalization by 10 μM isoproterenol (Fig. 3). The ?1/?2CTAR chimera displayed a robust approximately 80% reduction in cell surface localization in response to stimulation with the ?-adrenergic agonist. Unlike the parent ?1AR, which displays no insulin-sensitive internalization, the ?1/?2CTAR chimera displayed counterregulation by insulin. More than 25% of the chimeric receptor was internalized within 30 min of challenge with insulin.
Insulin internalizes the ?2AR and the ?1/?2CTAR chimera, but not the ?1AR: analysis by confocal microscopy
Confocal microscopy of the eGFP-tagged version of these receptors would allow us to detail the localization of the receptors in response to treatment with ?-adrenergic agonist and insulin. In human carcinoma cells (HeLa), most of the eGFP-tagged receptors are localized to the cell membrane under basal conditions and therefore serve as a good model for visualizing receptor trafficking. As such, we explored the localization of the eGFP-tagged ?1AR, ?2AR, and ?1/?2CTAR chimera in HeLa cells. Isoproterenol readily stimulated the internalization of ?2AR-GFP and ?1AR-GFP expressed in these HeLa cells (Fig. 4). Both the ?2AR and ?1AR localized to the cell membrane (see white arrows) in the absence of treatment with hormones, appearing as punctate structures decorating the lipid bilayer of the plasma membrane. Treatment with isoproterenol provoked marked sequestration of the ?2AR-GFP within 30 min (see yellow arrowheads). This agonist-induced sequestration is a hallmark of GPCRs (34). The ?1AR-GFP, like the ?2AR-GFP, displayed a robust internalization in response to ?-adrenergic agonist-induced stimulation. In response to the counterregulatory effects of insulin, the ?2AR-GFP displayed internalization. This confirmed the imaging data obtained with the hydrophilic radioligand CGP-12177. In sharp contrast to the data obtained with the ?2AR-GFP, confocal images of the ?1AR-GFP reveal no internalization in response to insulin. The failure to detect internalization of ?1AR in response to insulin also confirmed the [3H]CGP-12177 radioligand binding data, providing a molecular explanation for the inability of insulin to counterregulate isoproterenol-stimulated cAMP accumulation in the ?1AR-bearing cells.
FIG. 4. Insulin internalizes the ?2AR and the ?1/?2CTAR chimera but not the ?1AR: analysis by confocal microscopy. HeLa cells transiently transfected with expression vectors harboring the human ?1AR-GFP, ?2AR-GFP, or ?1/?2CTAR-GFP chimera were treated for 30 min with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso) for 30 min at 37 C. The cells were fixed and the internalization of these GFP-tagged receptors was examined by confocal microscopy. The results displayed are from a single experiment and represent more than three independent experiments. White arrows indicate receptor localized to the cell membrane, appearing as punctate structures decorating the lipid bilayer. Yellow arrowheads indicate sequestered receptors that were no longer on the plasma membrane.
The GFP-tagged ?1/?2CTAR chimera also was expressed in cells and examined by confocal microscopy. Like the GPCRs from which it was constructed, the ?1/?2CTAR chimera-GFP displays ?-agonist-induced sequestration from the plasma membrane to intracellular, perinuclear regions of the cell (Fig. 4). Unlike the parent ?1AR from which the core of the chimera is composed, however, the ?1/?2CTAR chimera now clearly displays the ability to be sequestered to the intracellular, perinuclear region of the cell in response to insulin. The ?1/?2CTAR chimera by virtue of the substitution of the C-terminal tail from the ?2AR, has acquired the ability to be functionally and spatially regulated by a prominent member of the receptor tyrosine kinase family.
Mutation of the SH2 domain and Akt phosphorylation sites in the C terminus of the ?2AR abolishes insulin action on ?1/?2CTAR chimera
It has been shown that stimulating cells with insulin catalyzes phosphorylation of an SH2 domain (Y350) and Akt phosphorylation sites (S345/346) in the cytoplasmic C-terminal domain of the ?2AR, both in vivo and in vitro (6, 7, 19, 35). We explored whether a Y350/354F double mutation that eliminates the SH2-binding domain in the C-terminal tail of the ?2AR would impact on the ability of the ?1/?2CTAR chimera to signal and be counterregulated by insulin (Fig. 5). The Y350F mutation in the ?1/?2CTAR chimera abolished the ability of insulin to counterregulate isoproterenol-stimulated cAMP accumulation. Likewise, mutation of an Akt phosphorylation site (S345/346A) in the ?1/?2CTAR chimera abolished insulin-stimulated counterregulation of the cAMP response to isoproterenol. Elimination of either the Y350 or S345/346 residues can abolish the ability of insulin to counterregulate ?-adrenergic action on the chimera.
FIG. 5. Mutation of either Y350/354F or S345/346A of the ?1/?2CTAR abolishes counterregulation of ?-adrenergic agonist-stimulated cAMP accumulation by insulin. CHO-K1 cells transiently transfected with the human ?1/?2CTAR, Y350/354F ?1/?2CTAR (Y350/354F), and S345/346A ?1/?2CTAR (S345/346A) were treated with or without 100 nM insulin (Ins) for 30 min and then with 10 μM isoproterenol (Iso) for 15 min. Insulin suppresses the isoproterenol-stimulated cAMP response in the ?1/?2CTAR transfected cells but not in the Y350/354F or S345/346A mutants. The data are mean values ± SEM representative of three separate experiments. Asterisks denote P 0.05 for the difference between treatment insulin and KRP buffer treatments.
The ability of insulin to stimulate the sequestration of the ?1/?2CTAR chimera harboring either the Y350F mutation or S345/346A mutation was also explored, using the CGP-12177 hydrophilic radioligand (Fig. 6). The ?1/?2CTAR chimera displayed the ability to be sequestered to intracellular, perinuclear areas of the cell in response to insulin, a property inherent in the C-terminal domain of the ?2AR present in the chimera. Mutation of either the SH2 domain or the Akt phosphorylation site abolished the ability of these mutated chimeras to be sequestered from the cell surface in response to insulin, although their abilities to be sequestered in response to ?-adrenergic agonist was unaffected. Confocal microscopy of the mutant chimeras expressed in HeLa cells (data not shown) only confirmed the inability of either the Y350/354F or S345/346A mutant forms of the chimera from undergoing insulin-stimulated internalization. Both of the mutant chimeras retained the ability to undergo ?-adrenergic agonist-induced internalization (data not shown).
FIG. 6. Mutation of either Y350/354F or S345/346A of the ?1/?2CTAR abolishes sequestration of the ?AR chimera in response to insulin. CHO-K1 cells transiently transfected with the human ?1/?2CTAR, Y350/354F ?1/?2CTAR (Y350/354F), and S345/346A ?1/?2CTAR (S345/346A) were treated with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso) for 30 min at 37 C. The internalization of the receptor was measured using a hydrophilic, impermeant, radiolabeled ?-adrenergic antagonist [3H]CGP-12177 (70 nM). Insulin treatment promotes internalization of the ?1/?2CTAR in the transfected cells but not the Y350/354F or S345/346A mutants. All three chimers internalized in response to isoproterenol. The data are mean values ± SEM representative of three separate experiments. The surface receptor expression in unstimulated cells set at 100%. Asterisks denote P 0.05 for the difference between insulin treatment and control.
Elucidation of a 15-amino acid motif of the ?2AR that confers insulin-stimulated counterregulation when substituted in the ?1AR
To further define the sites on the ?2AR that are necessary for insulin mediated counterregulation, the C-terminal cytoplasmic tail was truncated to a short 15-amino acid motif from Lys342-Ser356. The motif included the Y350/354 (potential SH2-binding domain) and the S345/346 (potential Akt phosphorylation site). This 15-amino acid motif extended two to three amino acids on either side of these binding sites to maintain recognition sites flanking the sites of phosphorylation. We tested the ability of the ?1/?2CT342–356AR chimeric receptor to respond to counterregulation of cAMP accumulation by 100 nM insulin. The ?1/?2CT342–356AR was able to stimulate cAMP accumulation in response to isoproterenol. This response was sensitive to counterregulation by 100 nM insulin (Fig. 7A).
FIG. 7. Elucidation of a 15-amino acid motif (Lys342-Ser356) of the ?2AR C-terminal cytoplasmic tail is sufficient to confer insulin-stimulated counterregulation of the ?1AR. CHO-K1 cells transiently transfected with the human ?1/?2CT342–356AR were treated with or without 100 nM insulin (Ins) for 30 min and then challenged with 10 μM isoproterenol (Iso) for 15 min. Insulin suppressed isoproterenol-stimulated cAMP response in the cell transfected with the vectors harboring the ?1/?2CT342–356AR chimera (A). The internalization of the receptor was measured by use of a hydrophilic, cell-impermeable, radiolabeled ?-adrenergic antagonist [3H]CGP-12177 (70 nM). Insulin treatment promotes internalization of the ?1/?2CT342–356AR (B). The data are mean values ± SEM representative of three separate experiments. Asterisks denote P 0.05 for the difference between insulin and KRP buffer treatments. HeLa cells transiently transfected with expression vectors harboring the human ?1/?2CT342–356AR-GFP chimera were treated for 30 min at 37 C with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso). The cells were fixed and the internalization of these GFP-tagged receptors was examined by confocal microscopy (C). The results displayed are from a single experiment and representative of more than three independent experiments. White arrows indicate receptor localized to the cell membrane. Yellow arrowheads indicate sequestered receptors that were no longer on the plasma membrane.
We further evaluated the ability of insulin, compared with isoproterenol, to stimulate the internalization of the ?1/?2CT342–356AR. The ?1/?2CT342–356AR chimera displayed a robust approximately 60% reduction in cell surface localization in response to ?-adrenergic agonist. Unlike the parent ?1AR, it displayed approximately 15% internalization within 30 min of challenge with insulin (Fig. 7B). The tagged ?1/?2CT342–356AR chimera was also expressed in cells and examined by confocal microscopy. As demonstrated with the hydrophilic radioligand [3H]CGP-12177 binding data, the ?1/?2CT342–356AR displays the ability to be sequestered intracellularly, in response to ?-adrenergic agonist, and, more importantly, to insulin (Fig. 7C). Thus, the 15-amino acid motif of the C-terminal tail of the ?2AR containing the Y350 and Akt phosphorylation sites inserted into the ?1AR conferred response to insulin-mediated counterregulation of cAMP accumulation and receptor sequestration.
Discussion
Insulin counterregulates catecholamine action at many loci, including directly at the ?2AR, receptor to receptor. Insulin exerts its counterregulation of ?2AR by interfering with signal transduction from the ?2AR to the heterotrimer G protein Gs by creating a functional SH2 binding domain to which molecules such as Grb2, dynamin, and the p85 regulatory subunit of PI3-kinase can bind the ?2AR (phosphoY350) and trafficking the ?2AR away from the cell membrane. These insulin-induced changes in ?2AR function and sequestration are essential for the ability of insulin to suppress ?-adrenergic action in normal physiology. The actions of agonist-induced desensitization and insulin-stimulated counterregulation of ?2AR appear to achieve the same goals of uncoupling the ?2AR from its cognate G protein and sequestering the receptor to the intracellular compartment, yet the mechanisms by which these two regulatory pathways exert their influence is quite different (36).
Although ?AR-agonist and insulin each catalyze phosphorylation of the ?2AR, the sites phosphorylated on the ?2AR and the protein kinases involved in the regulation are distinctly different (36). Insulin stimulates its receptor to catalyze the phosphorylation of several specific tyrosyl residues in the C terminus of the ?2AR, especially Y350/354. Phosphorylation of Y350 in vivo or in vitro creates an SH2 binding domain capable of binding many well-known signaling and adaptor molecules, including the p85 regulatory subunit of PI3-kinase, dynamin, and Grb2 (7). ?AR-agonist, in contrast, stimulates protein kinase A and/or members of the GPCR kinase family to phosphorylate the ?2AR, events also largely confined to the C-terminal cytoplasmic tail of this prototypic GPCR (4). The phosphorylation event itself and subsequent binding of other molecules to the phosphorylated ?2AR provide a likely explanation of the loss of the ability of the receptor to signal properly to Gs, essentially disrupting the functional capacity of the receptor. Somewhat later, but within 30 min of treatment with either ?-adrenergic agonist or insulin, the phosphorylated ?2AR undergoes sequestration to an intracellular, perinuclear compartment of the cell not accessible to catecholamine stimulation. Although the kinetics of the intracellular trafficking of the ?2AR by ?-adrenergic agonist, compared with insulin, appear similar, detailed analyses of the pathways with chemical inhibitors and dominant-negative mutant versions of key signaling in the pathway molecules clearly demonstrate that these pathways for receptor trafficking events are different (36).
The current work explored to what extent the C-terminal tail of the ?2AR provides the actual basis for the ability of insulin to counterregulate functionally and spatially the ?2AR. The ?1AR, like the ?2AR, binds and is activated by catecholamines, signals to the same heterotrimeric G protein Gs, and provokes the activation of adenylyl cyclase and accumulation of intracellular cAMP. Both the ?1AR and ?2AR are expressed ubiquitously but display unique aspects of regulation (4, 37, 38). ?2ARs are subject to insulin-stimulated counterregulation but not its ?1AR counterpart (31). In the current work, we tested this premise directly in several cell lines, observing that the ?1AR is largely resistant to insulin action on signaling to cAMP accumulation and receptor sequestration. To test whether the C-terminal tail of the ?2AR contains the motif(s) essential for insulin counterregulation, we compared the effects of insulin on ?1AR, ?2AR, and an interesting ?1/?2CTAR chimera in which the C-terminal tail is ?2AR in origin, whereas the core of the chimera is ?1AR in origin.
The results clearly demonstrate that donation of the C-terminal tail of the ?2AR confers to the chimera the ability to be regulated by insulin. Insulin was able to counterregulate ?-adrenergic agonist stimulation of cAMP accumulation in the ?1/?2CTAR chimera but not the parent ?1AR. The presence of the ?2AR C terminus on the chimera likewise enabled insulin to exert its ability to sequester the ?1/?2CTAR chimera to intracellular, perinuclear areas of the cells, much like that observed for the ?2AR but not ?1AR.
More detailed analysis was performed by targeted mutagenesis of two important domains of the cytoplasmic C-terminal tail of the ?2AR for the action of insulin, i.e. the tyrosine phosphorylation site (Y350) that creates an SH2 binding domain when phosphorylated and the sites of Akt-catalyzed phosphorylation (S345/346). Mutation of either of these insulin-targeted sites impaired the ability of the ?2AR C terminus to confer insulin-stimulated counterregulation of function and intracellular trafficking to the ?1/?2CTAR chimera.
To further define the sites necessary for counterregulation of insulin on the ?2AR, we constructed a shortened version of the C-terminal tail of the ?2AR to include only the putative sites of SH2 domain and Akt phosphorylation site. This motif of 15 amino acids from the C-terminal tail of the ?2AR extends from Lys342 to Ser356. Reducing the C-terminal tail of the ?2AR from 84 amino acids to 15 amino acids maintained insulin-mediated counterregulation of cAMP accumulation and sequestration of the ?1AR. Thus, the SH2 domain and the Akt phosphorylation sites are obligate for the ability of the ?2AR to be counterregulated by insulin and represent, as a unit, a transferable motif that links insulin counterregulation to GPCRs.
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Address all correspondence and requests for reprints to: Craig C. Malbon, Department of Pharmacology, Health Sciences Center, State University of New York/Stony Brook, Stony Brook, New York 11794-8651. E-mail craig@pharm.sunysb.edu.
Abstract
Insulin counterregulates catecholamine action in part by inducing the sequestration of ?2-adrenergic receptors. Although similar to agonist-induced sequestration, insulin-induced internalization of ?2-adrenergic receptors operates through a distinct and better-understood cellular pathway. The effects of insulin treatment on the function and trafficking of both ?1- and ?2-adrenergic receptors were tested. The ?2-adrenergic receptors were counterregulated and internalized in response to insulin. The ?1-adrenergic receptors, in sharp contrast, are shown to be resistant to the ability of insulin to counterregulate function and induce receptor internalization. Using chimeric receptors composed of ?1-/?2-adrenergic receptors in tandem with mutagenesis, we explored the role of the C-terminal cytoplasmic tail of the ?2-adrenergic receptors for insulin-induced counterregulation. Substitution of the C-terminal cytoplasmic tail of the ?2-adrenergic receptor on the ?1-adrenergic receptor enabled the chimeric G protein-coupled receptor to be functionally and spatially regulated by insulin. Truncation of the ?2-adrenergic receptor C-terminal cytoplasmic tail to a 15-amino acid motif harboring a potential Src homology 2-binding domain at Y350 and an Akt phosphorylation site at S345,346 was sufficient to enable receptor regulation by insulin.
Introduction
G PROTEIN-COUPLED RECEPTORS (GPCRs) and growth factor receptors with intrinsic tyrosine kinase activity represent two dominant pathways controlling cellular signaling (1, 2, 3, 4). Integration of signaling between GPCR and tyrosine kinase pathways has been investigated and includes the existence of cross-regulation at the most proximal point, receptor-to-receptor interaction with GPCRs acting as substrates for tyrosine kinases (5, 6, 7). Insulin cross-talk with ?2-adrenergic receptor (?2AR) serves an important counterregulation of glucose homeostasis. Treating cells with insulin stimulates the phosphorylation of the ?2AR on tyrosyl residues Y350/354 and Y364 for in vivo studies (5, 6) as well as in vitro (7) studies using recombinant, purified ?2AR and insulin receptors. The Y350 residue, a prominent residue for insulin receptor-catalyzed phosphorylation of the cytoplasmic tail of the ?2AR, is embedded in a sequence motif (Tyr-Gly-Asn-Gly) that is similar to the motifs known to interact with Caenorhabditis elegans sem5 Src homology 2 (SH2) domains when phosphorylated (8). Phosphorylation of the ?2AR by the insulin receptor and the IGF-I receptor includes a motif for tyrosine kinases at Y364 (9), the SH2/Grb2 binding site at Y350 (6, 10), and a potential SHC binding site at Y132 (6, 7, 9). For insulin action, activation of 1-phosphatidylinositol 3-kinase (PI3-kinase) is an early event, following temporally the phosphorylation of the insulin receptor and insulin receptor substrate-1 (11, 12). In response to insulin stimulation, the p85 regulatory subunit of PI3-kinase binds the insulin receptor substrate-1 via SH2 domain(s), activating the catalytic p110 subunit that in turn phosphorylates various phosphoinositides at the 3'-position of the inositol ring (13). Ample reports support the premise that PI 3-kinase and its 3'-phosphoinositide products are critical to intracellular trafficking both of membrane-bound elements in general (14) and downstream elements of tyrosine kinase-mediated cell signaling, particularly that of insulin (15).
We have shown that insulin, much like ?-adrenergic agonists, provokes rapid sequestration of ?2AR (16, 17). In addition, downstream activation of Erk1/2 by insulin in mammalian cells was shown to be amplified by ?2AR expression, i.e. the higher the level of cellular complement of ?2AR, the greater was the amplification of insulin activation of Erk1/2, identifying the ability of the ?2AR to act as a scaffold for some aspect of insulin action. The ability of the ?2AR to amplify the insulin response was dependent on the integrity of the Y350 residue, which is phosphorylated by the insulin receptor and constitutes a binding site for an SH2 domain to which Grb2, and other proteins can bind (18). The later identification of an Akt phosphorylation sites in the C-terminal tail of the ?2AR (19) and the central role of Akt in insulin action (20, 21, 22, 23) brought into focus the question of what domain(s) in the ?2AR confer to this GPCR its ability to be regulated by insulin. In the current work, we demonstrate that unlike the ?2AR, the ?1-adrenergic receptor (?1AR) does not internalize in response to insulin. We make use of this marked difference in receptor trafficking to define the motifs in the ?2AR through which insulin catalyzes sequestration, demonstrating that the donation of the Y350 and Akt phosphorylation sites to the ?1AR enable insulin to internalize the chimeric ?1AR.
Materials and Methods
Plasmids
The human ?1AR gene including the FLAG epitope on the 5' end was generated by PCR using plasmid pUC18-h?1AR (kindly provided by Dr. Stephen Liggett, Department of Medicine, University of Cincinnati, Cincinnati, OH) as the template and a pair of primers designed with HindIII or BamHI linkers. The PCR product was digested with HindIII and BamHI and cloned into the unique HindIII/BamHI sites of peGFP-N1 expression vector (Clontech, Palo Alto, CA) so that the human ?1AR gene was fused to the amino terminus of the GFP. The plasmid encoding the enhanced-green fluorescent protein (GFP)-tagged human ?2AR (in pCDNA3), and including the FLAG epitope at the amino terminus, was a generous gift from Dr. Jeffrey Benovic (Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA). A ?1AR/?2AR (?1/?2CTAR) chimera containing the N-terminal FLAG epitope and the C terminus eGFP epitope was made by replacement of the C terminus of the ?1AR with the C terminus of the ?2AR (?1AR from Met1-Ser380 and ?2AR from Pro329-Leu413) by using overlap extension PCR as previously described (24) and cloned into the unique HindIII/BamHI sites of peGFP-N1 expression vector. Another ?1/?2 AR chimera (?1/?2CT342–356) containing the N-terminal FLAG epitope and the C-terminal eGFP was generated by replacement of 15 amino acids in the C terminus of the ?1AR (from Cys393 to Thr407) with 15 amino acids of the ?2AR (from Lys342-Ser356) using overlap extension PCR and cloned into the HindIII/BamHI sites of peGFP-N1 expression vector. The sequence of constructs was confirmed by DNA sequencing.
Cell culture
Chinese hamster ovary (CHO)-K1 cells and human carcinoma HeLa cells were maintained in DMEM supplemented with 5% fetal bovine serum, plus penicillin (60 μg/ml) and streptomycin (100 μg/ml), grown in humidified atmosphere chamber supplied with 5% CO2 and 95% air at 37 C.
Assay of intracellular accumulation of cAMP
CHO-K1 cells transiently transfected with the human ?2AR, ?1AR, ?1/?2CTAR, and ?1/?2CT342–356AR were seeded in a 96-well plate for 24 h. On the day of experiment, cell culture medium was aspirated, the cells washed and replenished with Krebs-Ringer phosphate (KRP) buffer containing 10 μM RO-201724 (a cAMP phosphodiesterase inhibitor), and then treated with the indicated hormones in a total assay volume of 50 μl. The reaction was terminated by the addition of 50 μl of 100% ethanol, and the AMP content was measured by the competitive binding assay as described (25). To assay insulin counterregulation, cells were treated for 30 min with insulin (100 nM) dissolved in a KRP buffer and challenged with the ?-adrenergic agonist isoproterenol (10 μM) for 15 min to enable the accumulation of intracellular cAMP. Each experiment was performed with triplicate determinations.
?-Adrenergic antagonist binding and receptor sequestration assays
The expression of ?2AR, ?1AR, and the chimera was quantified by assay of the binding of the high-affinity, ?-adrenergic antagonist iodocyanolpindolol [125I]CYP to whole-cell preparations of the transiently transfected cells, as detailed elsewhere (24). Nonspecific binding was determined using ?-adrenergic antagonist propranolol (10 μM). The expression and sequestration of receptors on plasma membrane in CHO-K1 cells transiently transfected with the human ?2AR, ?1AR, and chimera were quantified using the hydrophilic, cell-impermeable radiolabeled ?-adrenergic antagonist [3H]CGP-12177 (26). Cells were treated for 30 min with insulin (100 nM) or isoproterenol (10 μM) for at 37 C and then placed at 4 C for 6 h with 70 nM [3H] CGP-12177. The cells were rapidly washed free of unbound ligand and collected on GF/C membranes by vacuum filtration. The radioligand bound to the washed cell mass retained on the filter was quantified by liquid scintillation spectrometry. Binding affinity (Kd) of ?1AR, ?2AR, and chimera was determined using concentrations of [3H]CGP-12177 from 0.023–2.3 nM as previously described (26). Confocal microscopy of eGFP-tagged receptors was performed as described earlier (24).
Membrane preparations
Cells were harvested with ice-cold PBS containing 1 mM EDTA and collected by centrifugation (1000 x g). The cell pellets then were resuspended in 5 ml of a hypotonic buffer composed of 20 mM HEPES (pH 7.4); 2 mM MgCl2; and 1 mM EDTA (HME buffer) plus the protease inhibitors, leupeptin (5 μg/ml), aprotinin (5 μg/ml), and phenylmethylsulfonyl fluoride (200 nM). After incubation on ice for 15 min, the cells were homogenized with five to eight strokes of a Dounce homogenizer operating with a tight-fitting Teflon pestle. Membrane fractions were collected from 1,000 x g postnuclear supernatants by centrifugation at 10,000 x g. The crude membrane pellets were resuspended in 200 μl HME buffer, and protein concentration was determined. The Lowry method was used to determine the amount of protein in the samples before gel loading. The samples were subject to SDS-PAGE using 10% acrylamide gels. The resolved proteins were transferred electrophoretically onto nitrocellulose blots, probed with the anti-GFP antibody, and stained with a secondary antibody to which horseradish peroxidase was coupled. The chemiluminescence reagent was used to detect immunocomplexes.
Site-directed mutagenesis
Double mutation of serine residues 345 and 346 to alanine and tyrosyl residues 350 and 354 to phenylalanine in the ?1/?2CTAR was performed using the Quickchange site directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer’s suggested protocols. The sequences of the mutagenic primers were as follows: S345/346A, GCTTCTGTGCCTGCGCAGGGCCGCCTTGAAGGCCTATGGC; and Y350/354F, GGTCTTCTTTGAAGGCCTTCGGCAATGGCTTCTCCAGCAACGGCAACACAGGGG. The DNA sequence of the mutant receptors was confirmed by use of direct DNA sequencing of the mutant plasmid DNA.
Results
To investigate the motif(s) that confer the ability of insulin to sequester ?2ARs, we needed to satisfy several criteria. First, ?2ARs are expressed virtually ubiquitously in cells in culture, and a cell line essentially deficient of ?2ARs is optimal for such studies. The CHO-K1 cells were used because these cells express vanishing few, if any, ?2AR (27). To investigate the domains/protein motif(s) through which insulin exerts its effects on sequestration of ?2AR, a recipient GPCR was required that was similar to the ?2AR but one that failed to undergo insulin-stimulated counterregulation. Earlier observations suggested that the action of the ?1AR, which shares many properties in common with the ?2AR, does not display counterregulation by insulin (2). Finally, the studies required that a functional assay of ?AR activity be coupled with the ability to study the sequestration by radioligand binding and independently by confocal microscopy.
Expression of ?1AR, ?2AR, and chimera in CHO-K1 cells
The ability to express the GFP-tagged ?1AR and ?2AR was investigated in CHO-K1 cells transiently transfected with mammalian expression vectors harboring the cDNAs of each. Crude cell membranes were prepared from the cells transiently transfected with the appropriate expression vector and harvested 30 h later. Using [125I]ICYP to assess the expression of total ?AR, we observed the following values (femtomoles per 106 cells): the ?1AR-expressing cells, 225.5 ± 49.5, and ?2AR-expressing cells, 186.0 ± 23 (27). Binding affinity to their antagonist [3H]CGP-12177 was determined using Scatchard plot. The results revealed Kd values (nanomoles) for ?1AR of 0.52 ± 0.02 and ?2AR of 0.66 ± 0.14. Thus, CHO-K1 cells transiently transfected with the same expression vector, but harboring different ?AR cDNAs, yielded comparable amounts of receptor expression. Aliquots of the crude cell membranes were subjected to SDS-PAGE and the separated proteins blotted with antibodies specific for the GFP-moiety (Fig. 1). The results of the immunoblotting demonstrate the expression of the receptors, ?1AR and ?2AR [mobility (Mr) 98 kDa], both demonstrating relative mobilities that agree well with the expected Mr for their GFP-tagged versions of their ?AR subtypes (28). Some lower Mr forms likely to be products of differential glycosylation were apparent for the blots of either receptor subtype (29, 30).
FIG. 1. Immunoblots (IBs) from membrane preparations of wild-type CHO-K1 cells and CHO-K1 cells with transient transfection of ?1AR-GFP, ?2AR-GFP, and ?1/?2CTAR-GFP. CHO-K1 cells were transiently transfected with expression vector harboring the human ?2AR, ?1AR, or ?1/?2CTAR chimera. Cells were then homogenized with a Dounce homogenizer and subjected to centrifugation at 10,000 x g. Samples were subject to SDS-PAGE using 10% acrylamide gels and probed with an antibody against GFP. Immunoblotting demonstrates expression of ?2AR, ?1AR, or ?1/?2CTAR chimera with relative mobilities consistent with the expected Mr for their GFP-tagged versions of their ?AR subtypes. Some lower Mr forms of the either receptor subtype are likely to be products of differential glycosylation.
We also constructed a ?1/?2CTAR chimera (human ?1AR from Met1-Ser380 and human ?2AR from Pro329-Leu413) to ascertain what effect, if any, the substitution of the ?2AR C terminus in the ?1AR would have on the ability of the chimera to respond to insulin. In transiently transfected CHO-K1 cells, the ?1/?2CTAR chimeric receptor was expressed at levels (265.0 ± 21.9 fmol/106 cells) comparable with ?1AR and ?2AR, displaying the same approximated relative Mr on SDS-PAGE as the ?1AR (Fig. 1). Kd value (nanomoles) for binding of [3H]CGP-12177 to the ?1/?2CTAR chimera was 0.51 ± 0.11, similar to ?1AR and ?2AR subtypes.
?2AR and the ?1/?2CTAR chimera, but not ?1AR, demonstrate insulin-sensitive counterregulation of isoproterenol-stimulated cAMP response
The ability of the ?-adrenergic agonist isoproterenol (10 μM) to stimulate cAMP accumulation in CHO cells transiently transfected with expression vectors harboring the cDNA for the ?1AR, ?2AR, and ?1/?2CTAR was probed in cells expressing comparable amounts of receptors (Fig. 2). For the ?1AR-expressing cells, isoproterenol (10 μM) stimulated a robust increase in intracellular cAMP accumulation, and this response was not sensitive to counterregulation by insulin (100 nM). In cells expressing the ?2AR, isoproterenol stimulated a nearly equivalent cAMP response as observed in the cells expressing the ?1AR. When stimulated by both isoproterenol and insulin together, cells expressing the ?2AR, in contrast to those expressing the ?1AR, displayed an insulin-sensitive counterregulation of the isoproterenol-stimulated cAMP response, as reported earlier in other cell lines (6, 16, 31, 32, 33).
FIG. 2. Insulin counterregulates the isoproterenol-stimulated cAMP response mediated by the ?2AR and the ?1/?2CTAR chimera but not the ?1AR. CHO-K1 cells transiently transfected with expression vector harboring the human ?2AR, ?1AR, or ?1/?2CTAR chimera were treated with or without 100 nM insulin (Ins) for 30 min and then challenged with 10 μM isoproterenol (Iso) for 15 min. Insulin suppressed isoproterenol-stimulated cAMP response in the cell transfected with the vectors harboring the ?2AR and ?1/?2CTAR chimera but not the ?1AR. These data are mean values ± SEM representative of three separate experiments. Asterisks denote P 0.05 for the difference between insulin and KRP buffer treatments.
To ascertain whether the C-terminal region of the ?2AR is the dominant element of the receptor that confers the ability to be counterregulated by insulin, we studied a chimeric receptor composed of the core of the ?1AR (residues Met1-Ser380, which lacks the entire C-terminal cytoplasmic tail) to which the cytoplasmic C-terminal tail of the ?2AR (residues Pro329-Leu413) has been spliced (?1/?2CTAR, Fig. 2). The ?1/?2CTAR was expressed at levels comparable with those observed for either the ?1AR or ?2AR and was able to stimulate cAMP accumulation in response to isoproterenol. Unlike the ?1AR that failed to display counterregulation by insulin, the ?1/?2CTAR chimera was found to display an insulin-stimulated counterregulation of the cAMP response to isoproterenol. These data reveal that by substitution of the C terminus of the ?2AR to the ?1AR, an insulin-stimulated counterregulation was conferred to the ?1AR.
?2AR and ?1/?2CTAR chimera, but not ?1AR, demonstrate insulin-stimulated internalization
We sought to quantify the level of cell surface ?ARs to test further the observations made by functional assay of insulin action on ?-adrenergic agonist-stimulated cAMP accumulation. The high-affinity, radiolabeled, and hydrophilic antagonist CGP-12177 is unique as a radioligand binding reagent because it is restricted to the extracellular aqueous environment and binds only to those ?ARs localized in the cell membrane. Using [3H]CGP-12177 and intact cells, the surface complement of ?1AR, ?2AR, and ?1/?2CTAR chimera were quantified in cells after a challenge with either isoproterenol (10 μM) or insulin (100 nM, Fig. 3). For either ?1AR or ?2AR, treatment with isoproterenol provoked a 60% reduction in the cell surface complement. The ?2AR displayed internalization of more than 40% in response to100 nM insulin. The ?1AR, in contrast, displayed no internalization in response to treatment of the cells with100 nM insulin.
FIG. 3. Insulin internalizes the ?2AR and the ?1/?2CTAR chimera but not the ?1AR. CHO-K1 cells transiently transfected with expression vectors harboring the human ?1AR, ?2AR, or ?1/?2CTAR chimera were treated with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso) for 30 min at 37 C. The internalization of the receptor was measured by use of a hydrophilic, cell-impermeable, radiolabeled ?-adrenergic antagonist [3H]CGP-12177. Insulin treatment promotes internalization of the ?2AR and ?1/?2CTAR but not of the ?1AR. All three receptors displayed internalization in response to stimulation with the ?-adrenergic agonist isoproterenol. These data are mean values ± SEM of three separate experiments. The surface receptor expression in unstimulated cells is set at 100%. Asterisks denote P 0.05 for the difference between insulin treatment and control.
We also tested the ability of the ?1/?2CTAR chimeric receptor to respond to agonist-induced internalization by 10 μM isoproterenol (Fig. 3). The ?1/?2CTAR chimera displayed a robust approximately 80% reduction in cell surface localization in response to stimulation with the ?-adrenergic agonist. Unlike the parent ?1AR, which displays no insulin-sensitive internalization, the ?1/?2CTAR chimera displayed counterregulation by insulin. More than 25% of the chimeric receptor was internalized within 30 min of challenge with insulin.
Insulin internalizes the ?2AR and the ?1/?2CTAR chimera, but not the ?1AR: analysis by confocal microscopy
Confocal microscopy of the eGFP-tagged version of these receptors would allow us to detail the localization of the receptors in response to treatment with ?-adrenergic agonist and insulin. In human carcinoma cells (HeLa), most of the eGFP-tagged receptors are localized to the cell membrane under basal conditions and therefore serve as a good model for visualizing receptor trafficking. As such, we explored the localization of the eGFP-tagged ?1AR, ?2AR, and ?1/?2CTAR chimera in HeLa cells. Isoproterenol readily stimulated the internalization of ?2AR-GFP and ?1AR-GFP expressed in these HeLa cells (Fig. 4). Both the ?2AR and ?1AR localized to the cell membrane (see white arrows) in the absence of treatment with hormones, appearing as punctate structures decorating the lipid bilayer of the plasma membrane. Treatment with isoproterenol provoked marked sequestration of the ?2AR-GFP within 30 min (see yellow arrowheads). This agonist-induced sequestration is a hallmark of GPCRs (34). The ?1AR-GFP, like the ?2AR-GFP, displayed a robust internalization in response to ?-adrenergic agonist-induced stimulation. In response to the counterregulatory effects of insulin, the ?2AR-GFP displayed internalization. This confirmed the imaging data obtained with the hydrophilic radioligand CGP-12177. In sharp contrast to the data obtained with the ?2AR-GFP, confocal images of the ?1AR-GFP reveal no internalization in response to insulin. The failure to detect internalization of ?1AR in response to insulin also confirmed the [3H]CGP-12177 radioligand binding data, providing a molecular explanation for the inability of insulin to counterregulate isoproterenol-stimulated cAMP accumulation in the ?1AR-bearing cells.
FIG. 4. Insulin internalizes the ?2AR and the ?1/?2CTAR chimera but not the ?1AR: analysis by confocal microscopy. HeLa cells transiently transfected with expression vectors harboring the human ?1AR-GFP, ?2AR-GFP, or ?1/?2CTAR-GFP chimera were treated for 30 min with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso) for 30 min at 37 C. The cells were fixed and the internalization of these GFP-tagged receptors was examined by confocal microscopy. The results displayed are from a single experiment and represent more than three independent experiments. White arrows indicate receptor localized to the cell membrane, appearing as punctate structures decorating the lipid bilayer. Yellow arrowheads indicate sequestered receptors that were no longer on the plasma membrane.
The GFP-tagged ?1/?2CTAR chimera also was expressed in cells and examined by confocal microscopy. Like the GPCRs from which it was constructed, the ?1/?2CTAR chimera-GFP displays ?-agonist-induced sequestration from the plasma membrane to intracellular, perinuclear regions of the cell (Fig. 4). Unlike the parent ?1AR from which the core of the chimera is composed, however, the ?1/?2CTAR chimera now clearly displays the ability to be sequestered to the intracellular, perinuclear region of the cell in response to insulin. The ?1/?2CTAR chimera by virtue of the substitution of the C-terminal tail from the ?2AR, has acquired the ability to be functionally and spatially regulated by a prominent member of the receptor tyrosine kinase family.
Mutation of the SH2 domain and Akt phosphorylation sites in the C terminus of the ?2AR abolishes insulin action on ?1/?2CTAR chimera
It has been shown that stimulating cells with insulin catalyzes phosphorylation of an SH2 domain (Y350) and Akt phosphorylation sites (S345/346) in the cytoplasmic C-terminal domain of the ?2AR, both in vivo and in vitro (6, 7, 19, 35). We explored whether a Y350/354F double mutation that eliminates the SH2-binding domain in the C-terminal tail of the ?2AR would impact on the ability of the ?1/?2CTAR chimera to signal and be counterregulated by insulin (Fig. 5). The Y350F mutation in the ?1/?2CTAR chimera abolished the ability of insulin to counterregulate isoproterenol-stimulated cAMP accumulation. Likewise, mutation of an Akt phosphorylation site (S345/346A) in the ?1/?2CTAR chimera abolished insulin-stimulated counterregulation of the cAMP response to isoproterenol. Elimination of either the Y350 or S345/346 residues can abolish the ability of insulin to counterregulate ?-adrenergic action on the chimera.
FIG. 5. Mutation of either Y350/354F or S345/346A of the ?1/?2CTAR abolishes counterregulation of ?-adrenergic agonist-stimulated cAMP accumulation by insulin. CHO-K1 cells transiently transfected with the human ?1/?2CTAR, Y350/354F ?1/?2CTAR (Y350/354F), and S345/346A ?1/?2CTAR (S345/346A) were treated with or without 100 nM insulin (Ins) for 30 min and then with 10 μM isoproterenol (Iso) for 15 min. Insulin suppresses the isoproterenol-stimulated cAMP response in the ?1/?2CTAR transfected cells but not in the Y350/354F or S345/346A mutants. The data are mean values ± SEM representative of three separate experiments. Asterisks denote P 0.05 for the difference between treatment insulin and KRP buffer treatments.
The ability of insulin to stimulate the sequestration of the ?1/?2CTAR chimera harboring either the Y350F mutation or S345/346A mutation was also explored, using the CGP-12177 hydrophilic radioligand (Fig. 6). The ?1/?2CTAR chimera displayed the ability to be sequestered to intracellular, perinuclear areas of the cell in response to insulin, a property inherent in the C-terminal domain of the ?2AR present in the chimera. Mutation of either the SH2 domain or the Akt phosphorylation site abolished the ability of these mutated chimeras to be sequestered from the cell surface in response to insulin, although their abilities to be sequestered in response to ?-adrenergic agonist was unaffected. Confocal microscopy of the mutant chimeras expressed in HeLa cells (data not shown) only confirmed the inability of either the Y350/354F or S345/346A mutant forms of the chimera from undergoing insulin-stimulated internalization. Both of the mutant chimeras retained the ability to undergo ?-adrenergic agonist-induced internalization (data not shown).
FIG. 6. Mutation of either Y350/354F or S345/346A of the ?1/?2CTAR abolishes sequestration of the ?AR chimera in response to insulin. CHO-K1 cells transiently transfected with the human ?1/?2CTAR, Y350/354F ?1/?2CTAR (Y350/354F), and S345/346A ?1/?2CTAR (S345/346A) were treated with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso) for 30 min at 37 C. The internalization of the receptor was measured using a hydrophilic, impermeant, radiolabeled ?-adrenergic antagonist [3H]CGP-12177 (70 nM). Insulin treatment promotes internalization of the ?1/?2CTAR in the transfected cells but not the Y350/354F or S345/346A mutants. All three chimers internalized in response to isoproterenol. The data are mean values ± SEM representative of three separate experiments. The surface receptor expression in unstimulated cells set at 100%. Asterisks denote P 0.05 for the difference between insulin treatment and control.
Elucidation of a 15-amino acid motif of the ?2AR that confers insulin-stimulated counterregulation when substituted in the ?1AR
To further define the sites on the ?2AR that are necessary for insulin mediated counterregulation, the C-terminal cytoplasmic tail was truncated to a short 15-amino acid motif from Lys342-Ser356. The motif included the Y350/354 (potential SH2-binding domain) and the S345/346 (potential Akt phosphorylation site). This 15-amino acid motif extended two to three amino acids on either side of these binding sites to maintain recognition sites flanking the sites of phosphorylation. We tested the ability of the ?1/?2CT342–356AR chimeric receptor to respond to counterregulation of cAMP accumulation by 100 nM insulin. The ?1/?2CT342–356AR was able to stimulate cAMP accumulation in response to isoproterenol. This response was sensitive to counterregulation by 100 nM insulin (Fig. 7A).
FIG. 7. Elucidation of a 15-amino acid motif (Lys342-Ser356) of the ?2AR C-terminal cytoplasmic tail is sufficient to confer insulin-stimulated counterregulation of the ?1AR. CHO-K1 cells transiently transfected with the human ?1/?2CT342–356AR were treated with or without 100 nM insulin (Ins) for 30 min and then challenged with 10 μM isoproterenol (Iso) for 15 min. Insulin suppressed isoproterenol-stimulated cAMP response in the cell transfected with the vectors harboring the ?1/?2CT342–356AR chimera (A). The internalization of the receptor was measured by use of a hydrophilic, cell-impermeable, radiolabeled ?-adrenergic antagonist [3H]CGP-12177 (70 nM). Insulin treatment promotes internalization of the ?1/?2CT342–356AR (B). The data are mean values ± SEM representative of three separate experiments. Asterisks denote P 0.05 for the difference between insulin and KRP buffer treatments. HeLa cells transiently transfected with expression vectors harboring the human ?1/?2CT342–356AR-GFP chimera were treated for 30 min at 37 C with either 100 nM insulin (Ins) or 10 μM isoproterenol (Iso). The cells were fixed and the internalization of these GFP-tagged receptors was examined by confocal microscopy (C). The results displayed are from a single experiment and representative of more than three independent experiments. White arrows indicate receptor localized to the cell membrane. Yellow arrowheads indicate sequestered receptors that were no longer on the plasma membrane.
We further evaluated the ability of insulin, compared with isoproterenol, to stimulate the internalization of the ?1/?2CT342–356AR. The ?1/?2CT342–356AR chimera displayed a robust approximately 60% reduction in cell surface localization in response to ?-adrenergic agonist. Unlike the parent ?1AR, it displayed approximately 15% internalization within 30 min of challenge with insulin (Fig. 7B). The tagged ?1/?2CT342–356AR chimera was also expressed in cells and examined by confocal microscopy. As demonstrated with the hydrophilic radioligand [3H]CGP-12177 binding data, the ?1/?2CT342–356AR displays the ability to be sequestered intracellularly, in response to ?-adrenergic agonist, and, more importantly, to insulin (Fig. 7C). Thus, the 15-amino acid motif of the C-terminal tail of the ?2AR containing the Y350 and Akt phosphorylation sites inserted into the ?1AR conferred response to insulin-mediated counterregulation of cAMP accumulation and receptor sequestration.
Discussion
Insulin counterregulates catecholamine action at many loci, including directly at the ?2AR, receptor to receptor. Insulin exerts its counterregulation of ?2AR by interfering with signal transduction from the ?2AR to the heterotrimer G protein Gs by creating a functional SH2 binding domain to which molecules such as Grb2, dynamin, and the p85 regulatory subunit of PI3-kinase can bind the ?2AR (phosphoY350) and trafficking the ?2AR away from the cell membrane. These insulin-induced changes in ?2AR function and sequestration are essential for the ability of insulin to suppress ?-adrenergic action in normal physiology. The actions of agonist-induced desensitization and insulin-stimulated counterregulation of ?2AR appear to achieve the same goals of uncoupling the ?2AR from its cognate G protein and sequestering the receptor to the intracellular compartment, yet the mechanisms by which these two regulatory pathways exert their influence is quite different (36).
Although ?AR-agonist and insulin each catalyze phosphorylation of the ?2AR, the sites phosphorylated on the ?2AR and the protein kinases involved in the regulation are distinctly different (36). Insulin stimulates its receptor to catalyze the phosphorylation of several specific tyrosyl residues in the C terminus of the ?2AR, especially Y350/354. Phosphorylation of Y350 in vivo or in vitro creates an SH2 binding domain capable of binding many well-known signaling and adaptor molecules, including the p85 regulatory subunit of PI3-kinase, dynamin, and Grb2 (7). ?AR-agonist, in contrast, stimulates protein kinase A and/or members of the GPCR kinase family to phosphorylate the ?2AR, events also largely confined to the C-terminal cytoplasmic tail of this prototypic GPCR (4). The phosphorylation event itself and subsequent binding of other molecules to the phosphorylated ?2AR provide a likely explanation of the loss of the ability of the receptor to signal properly to Gs, essentially disrupting the functional capacity of the receptor. Somewhat later, but within 30 min of treatment with either ?-adrenergic agonist or insulin, the phosphorylated ?2AR undergoes sequestration to an intracellular, perinuclear compartment of the cell not accessible to catecholamine stimulation. Although the kinetics of the intracellular trafficking of the ?2AR by ?-adrenergic agonist, compared with insulin, appear similar, detailed analyses of the pathways with chemical inhibitors and dominant-negative mutant versions of key signaling in the pathway molecules clearly demonstrate that these pathways for receptor trafficking events are different (36).
The current work explored to what extent the C-terminal tail of the ?2AR provides the actual basis for the ability of insulin to counterregulate functionally and spatially the ?2AR. The ?1AR, like the ?2AR, binds and is activated by catecholamines, signals to the same heterotrimeric G protein Gs, and provokes the activation of adenylyl cyclase and accumulation of intracellular cAMP. Both the ?1AR and ?2AR are expressed ubiquitously but display unique aspects of regulation (4, 37, 38). ?2ARs are subject to insulin-stimulated counterregulation but not its ?1AR counterpart (31). In the current work, we tested this premise directly in several cell lines, observing that the ?1AR is largely resistant to insulin action on signaling to cAMP accumulation and receptor sequestration. To test whether the C-terminal tail of the ?2AR contains the motif(s) essential for insulin counterregulation, we compared the effects of insulin on ?1AR, ?2AR, and an interesting ?1/?2CTAR chimera in which the C-terminal tail is ?2AR in origin, whereas the core of the chimera is ?1AR in origin.
The results clearly demonstrate that donation of the C-terminal tail of the ?2AR confers to the chimera the ability to be regulated by insulin. Insulin was able to counterregulate ?-adrenergic agonist stimulation of cAMP accumulation in the ?1/?2CTAR chimera but not the parent ?1AR. The presence of the ?2AR C terminus on the chimera likewise enabled insulin to exert its ability to sequester the ?1/?2CTAR chimera to intracellular, perinuclear areas of the cells, much like that observed for the ?2AR but not ?1AR.
More detailed analysis was performed by targeted mutagenesis of two important domains of the cytoplasmic C-terminal tail of the ?2AR for the action of insulin, i.e. the tyrosine phosphorylation site (Y350) that creates an SH2 binding domain when phosphorylated and the sites of Akt-catalyzed phosphorylation (S345/346). Mutation of either of these insulin-targeted sites impaired the ability of the ?2AR C terminus to confer insulin-stimulated counterregulation of function and intracellular trafficking to the ?1/?2CTAR chimera.
To further define the sites necessary for counterregulation of insulin on the ?2AR, we constructed a shortened version of the C-terminal tail of the ?2AR to include only the putative sites of SH2 domain and Akt phosphorylation site. This motif of 15 amino acids from the C-terminal tail of the ?2AR extends from Lys342 to Ser356. Reducing the C-terminal tail of the ?2AR from 84 amino acids to 15 amino acids maintained insulin-mediated counterregulation of cAMP accumulation and sequestration of the ?1AR. Thus, the SH2 domain and the Akt phosphorylation sites are obligate for the ability of the ?2AR to be counterregulated by insulin and represent, as a unit, a transferable motif that links insulin counterregulation to GPCRs.
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