Phosphorylation of tyrosyl residues 350/354 of the beta-adrenergic receptor is obligatory for counterregulatory effects of insulin.

Insulin stimulates a loss of function and increased phosphotyrosine content of the β-adrenergic receptor in intact cells, raising the possibility that the β-receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the β-adrenergic receptor by the insulin receptor in vitro and peptide mapping of the β-adrenergic receptor phosphorylated in vivo in cells stimulated by insulin reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal region of the β-adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate β-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes β-adrenergic stimulation itself.


Insulin stimulates a loss of function and increased phosphotyrosine content of the ␤ 2 -adrenergic receptor in intact cells, raising the possibility that the
The counterregulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well known, whereas the molecular details of insulin regulation of G-protein-linked pathways remain unknown. Upon ligand binding, the insulin receptor displays tyrosine kinase activity which is critical to signal propagation (1). G-protein-linked receptors (like the ␤ 2adrenergic receptor, ␤ 2 AR), 1 in contrast, activate adenylyl cy-clase via G s and are phosphorylated during agonist-induced desensitization (2,3). We demonstrated recently that the well known counterregulatory actions of insulin included loss of function and increased phosphorylation of the ␤ 2 -adrenergic receptor (4). In the current study the structural basis for these counterregulatory effects of insulin exerted on the ␤ 2 -adrenergic receptor is explored.

MATERIALS AND METHODS
Preparation of Recombinant ␤ 2 AR and Insulin Receptor-Recombinant hamster ␤ 2 -adrenergic receptor was expressed using the baculovirus-Sf9 insect cell expression system (5) and purified by affinity, HPLC, and lectin chromatography (6). Recombinant human insulin receptor (rIR) was purified by lectin chromatography (7) from Chinese hamster ovary (CHO) T cells, which stably overexpress the human insulin receptor (8), or from COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (9).
Stoichiometry of Phosphorylation-In vivo, duplicate cultures of DDT 1 MF-2 smooth muscle cells were labeled metabolically with [ 32 P]orthophosphate as described above. After 4 h of labeling, the medium was aspirated. One culture was washed, lysed, and used as the source for determination of receptor number by ICYP binding and isolation of the labeled ␤ 2 AR by immunoprecipitation. The replicate culture was washed and total protein precipitated with 0.5 M perchloric acid. The precipitate was collected by centrifugation and the supernatant neutralized with KOH. The specific activity of the [ 32 P]ATP in the supernatant was determined as described by England and Walsh (11). With the determination of the specific activity of the cellular [ 32 P]ATP pool, the derivative amount of labeled phosphate incorporated into receptor protein, and the amount of receptor, the stoichiometry of the phosphorylation was calculated as moles of phosphate/mol of ␤ 2 AR.
Phosphorylation of Synthetic Peptide Substrates-Peptides corresponding to each cytoplasmic domain of the ␤ 2 AR harboring a tyrosyl residue were synthesized, purified by HPLC, and subjected to in vitro phosphorylation by rIR in the absence or presence of insulin (100 nM), as described above. The peptide sequences are employed were as follows: L339, LLCLRRSSSKAYGNGYSSNSNGKTD; T362, TDYMGEA-SGCQLGQEK; R62, RLQTVTNYFITSLACAD; Y132, AITSPFK-YQSLLTKNKAR; I135, ITSPFKYQSLLTKNKAR; E121,ET LSVIAVDRYIAITSPFK. Partially purified rIR (wheat germ agglutinin extracts from CHO-T cells) was incubated in the absence or presence of insulin (100 nM), 10 M [␥-32 P]ATP, and the synthetic peptides at the concentrations indicated for 30 min at 22°C. The reaction was stopped by adding an equal volume of 2 ϫ concentrated Laemmli sample buffer. Phosphopeptides were separated by Tricine gel electrophoresis (12,13). After fixing for 30 min, the wet gel was subjected to autoradiography for 30 min. For quantitation, the radioactive bands were identified in the gel and then excised. Phosphate incorporation was estimated from quantitation of Cerenkov radiation ( 32 P window) in the gel piece. The data shown are from a single experiment, replicated * This work was supported in part by postdoctoral fellowships from the Juvenile Diabetes Foundation (to K. B. and C. C. M.) and by grants from the National Institutes of Health (to C. C. M., A. R., and M. P. C). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ¶ Present address: Pharmakologisches Institut, Universitä t Bern, Friedbuhl-Strasse 49, CH-3010 Bern, Switzerland.
once with similar results.
Reverse-phase HPLC of Tryptic Phosphopeptides-32 P-Labeled ␤ 2 AR immunoprecipitated from metabolically labeled DDT 1 MF-2 cells were separated on SDS-PAGE as described above. Synthetic peptides containing tyrosine residues 350, 354, and 364 were phosphorylated in vitro with [␥-32 P]ATP and rIR, and then separated on Tricine gels. The bands corresponding to ␤ 2 AR or the synthetic peptides were excised from the gels and treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (40 g/ml) for 18 h at 37°C (7). The tryptic eluate was then separated on a microbore HPLC (Applied Biosystems) using a 220-mm Aquapore OD-300 column and a gradient of acetonitrile (0 -50% in 45 min) in 0.1% trifluoroacetic acid at a flow rate of 200 l/min. Fractions were collected at 1-min intervals and Cerenkov radiation ( 32 P window) measured for each fraction.
Two-dimensional Peptide Mapping-Tryptic digestion of ␤ 2 -adrenergic receptor phosphorylated in vivo as well as of synthetic peptides phosphorylated in vitro was performed as described above. The tryptic eluates from the HPLC peaks were separated in two dimensions on cellulose, thin-layer plates. Aliquots (10 l) of tryptic eluates were spotted onto the TLC plates and electrophoresed at 1000 V for 60 min in pH 1.9 buffer (formic acid/glacial acetic acid/water; ratio 50:156: 1794). Following electrophoresis, the plate was air-dried overnight and subjected to chromatography at a right angle to the direction of electrophoresis in a phosphochromatography buffer (1-butanol/pyridine/ acetic acid/water; ratio 15:10:3:12). The plates were dried and the peptides identified by autoradiography.
Site-directed Mutagenesis-Single mutation of tyrosyl residue 364 and the double mutation of tyrosyl residues 350 and 354 to phenylalanine in the hamster ␤ 2 AR cDNA was performed using the Transformer Mutagenesis® kit (Clontech), according to the manufacturer's sug-gested protocols. The sequences of the mutagenic primers were as follows: Y350F/Y354F, CTTCAAAAGCCTTTGGGAACGGCTTCTC-CAGCA; Y364F, CAAAACAGACTTCATGGGGGAGGC. Suspected mutations were confirmed by direct DNA sequencing of the mutant plasmid DNA. The mutant and wild-type cDNAs were then subcloned into the expression vector pCMV5 for subsequent transfection of CHO cells.
Transfection and Cyclic AMP Determination-CHO wild-type cells were co-transfected with either mutant, wild-type, or empty vector plasmids (all in combination with plasmid pCW1 containing the neomycin resistance gene) using Lipofectin® from Life Technologies, Inc. according to the manufacturer's protocol. Mutant cells were selected for neomycin-resistance in DMEM containing 10% fetal bovine serum and G418 (10 g/ml). Expression of ␤ 2 AR (ICYP binding, fmol/10 5 cells) was 1.8, 2.1, and 1.9 for wild-type, double mutant Y350F/Y354F, and single mutant T364F transfectants, respectively. For assay of ␤-adrenergic stimulation of cyclic AMP accumulation, cells were suspended in Krebs-Ringer phosphate buffer and treated with the indicated hormones for 15 min at 37°C. The reaction was terminated by the addition of HCl (0.1 M final). Cyclic AMP accumulation was measured using a competition binding assay (14).

RESULTS AND DISCUSSION
In an effort to explore the site(s) for insulin-stimulated phosphorylation of the ␤ 2 AR, we prepared synthetic peptides to corresponding to each cytoplasmic regions of the ␤ 2 AR that harbors candidate tyrosyl residues, i.e. Tyr-70, Tyr-132, Tyr-141, Tyr-350, Tyr-354, and Tyr-364, and analyzed their potential as substrates for rIR (Fig. 1). The ␤ 2 AR peptides were reconstituted with rIR in the absence (not shown) or presence of insulin (100 nM). For the in vitro assay, no labeling of peptides by rIR was observed in the absence of insulin. Insulinstimulated phosphorylation of the peptides by the rIR was FIG. 1. Topological model of ␤ 2 -adrenergic receptor, highlighting candidate tyrosyl residues for insulin-stimulated phosphorylation. A, model of ␤ 2 AR organization and location of synthetic sequences used as substrates for insulin-stimulated rIR-catalyzed phosphorylation of the ␤ 2 AR and as precursors for tryptic fragments used as markers for peptide mapping of the labeled tyrosyl residues. B, synthetic peptides used to map cytoplasmic tyrosyl residues for phosphorylation by activated tyrosine kinase growth factor receptors. Six sequences were selected to be used as substrates for insulin-stimulated rIR-phosphorylation. The peptides were designed as substrates for phosphorylation as well as a source of markers for reverse-phase HPLC and two-dimensional mapping of tryptic fragments. The derivative peptide fragments are displayed. compared, after electrophoretic separation of the labeled products from the rIR ( Fig. 2A). Insulin stimulated rIR-catalyzed phosphorylation of peptides L339 (Tyr-350 and Tyr-354), T362 (Tyr-364), and to a lesser extent peptides Y132 (Tyr-132 and Tyr-141) and I135 (Tyr-141).
Since peptide L339 contains both Tyr-350 and Tyr-354, it was important to explore if one site, or the other, or both were phosphorylated in vitro by rIR in the presence of insulin. When L339 peptide analogs that carried phenylalanine substitutions at either Tyr-350 or Tyr-354 were reconstituted with rIR in the in vitro system, both analogs served equally well as substrates for insulin-stimulated phosphorylation (data not shown). Elimination of the YIA sequence of Y132 to create peptide I135 reduced insulin-stimulated, rIR-catalyzed phosphorylation of the residual peptide, suggesting either that Tyr-132 is the site of phosphorylation or that the YIA sequence is required for recognition/phosphorylation of the Tyr-141. The absence of labeling of the E122 peptide containing Tyr-132 and the detection of some label in tryptic fragments containing Tyr-141 supports the latter interpretation. Phosphopeptides from peptides R62 (Tyr-70) and E121 (Tyr-132) could not be detected, although the presence of the unphosphorylated peptides in the gel could be detected by silver staining. Phosphoamino acid analysis of the labeled peptides was performed, and for all phosphopeptides, labeling was confined to phosphotyrosine (not shown). These data demonstrate that residues Tyr-350, Tyr-354, and Tyr-364 (and to a lesser extent Tyr-141) are phosphorylated by the rIR tyrosine kinase in vitro.
The efficacy of peptide phosphorylation was assessed by comparing the amount of phosphate incorporated into each peptide by insulin-stimulated rIR at various concentrations of peptides (Fig. 2B). Peptide L339 was clearly the best substrate for the rIR. The ED 50 for insulin-stimulated phosphorylation of L339 peptide was ϳ100 M. At 3 mM concentrations of peptide, saturation of rIR-catalyzed phosphorylation for the other peptides was not achieved, precluding the calculation of ED 50 values for the other peptides. The rank order of insulin-stimulated phosphorylation for the synthetic peptides employed at 1 mM, from best to worst substrate, was L339 (Tyr-350 and Tyr-354) Ͼ T362 (Tyr-364) Ͼ Y132 (Tyr-132) Ͼ Ͼ I135 (Tyr-141). Phosphorylation of peptides R62 and E122, containing Tyr-70 and Tyr-132, respectively, was not detected.
The synthetic peptides were designed not only to probe all cytosolic tyrosyl residues available for phosphorylation by IR, but also to provide a source of tryptic fragments in which the candidate sites for tyrosine kinase phosphorylation were imbedded (Fig. 1, A and B). Maps of tryptic digests might permit analysis of the sites phosphorylated on the ␤ 2 AR in response to insulin in vivo (Fig. 1). Tryptic digests of peptides phosphorylated in vitro by rIR in response to insulin provided markers for HPLC analysis (Fig. 3, A and B). The retention times for the tryptic fragments subjected to HPLC separation agreed well with the retention times calculated from the sequence information (not shown).
In vivo, metabolic labeling of DDT 1 MF-2 smooth muscle cells in culture with [ 32 P]orthophosphate revealed the phosphotyrosine content of ␤ 2 AR to increase from 0.86 Ϯ 0.10 (basal) to 1.76 Ϯ 0.39 (n ϭ 4) mol/mol receptor in response to insulin (20 min, 100 nM). Some phosphotyrosine was found in tryptic fragments harboring Tyr-350 and Tyr-354 of ␤ 2 AR isolated from cells in the absence of stimulation by insulin (Fig. 3, C and D).
In the presence of insulin, increased phosphorylation of the ␤ 2 AR was observed, confined largely to Tyr-350, Tyr-354, and Tyr-364 (Fig. 3, E and F). Insulin-stimulated phosphorylation displayed two patterns in which labeling occurred either at both Tyr-350/Tyr-354 and Tyr-364 (Fig. 3F) or more prominently at Tyr-350/Tyr-354 with reduced labeling of Tyr-364 (Fig. 3E). Other peaks occasionally observed in the HPLC pro- of T362 (harboring Tyr-364) were employed as standards (panels A and B, respectively). Cells metabolically labeled with [ 32 P]orthophosphate (see "Materials and Methods") were incubated for 20 min without (panels C and D) or with (panels E and F) 100 nM insulin. After lysis of the cells, the ␤ 2 AR was immunoprecipitated, and the phosphorylated receptor isolated, and then digested with trypsin. Reverse-phase HPLC analysis of the tryptic fragments was performed as described under "Materials and Methods." Chromatograms from two separate experiments, representative of five independent experiments, are displayed. The label in fraction 30 of panel F was observed on occasion, but contained no phosphotyrosine. . Cells metabolically labeled with [ 32 P]orthophosphate (see "Materials and Methods") were incubated for 20 min without (data not shown) or with (panels C and D) 100 nM insulin. After lysis of the cells, the ␤ 2 AR was immunoprecipitated, and the phosphorylated receptor isolated, and then digested with trypsin. High voltage electrophoresis and thin layer chromatography of the tryptic fragments was performed as described under "Materials and Methods." Analyses from two separate experiments are displayed.
files (e.g. fraction 30, Fig. 3D) were subjected to phosphoamino acid analysis and found to contain no phosphotyrosine. High voltage electrophoresis followed by thin-layer chromatography of the tryptic fragments confirmed the identity of the HPLC peaks (Fig. 4, A and B) and provided additional markers for analysis of phosphopeptides derived from insulin-stimulated rIR-catalyzed phosphorylation of receptor peptides (Fig. 4, C and D). The two-dimensional analysis confirmed the results of reverse-phase HPLC, establishing that the predominant sites of insulin-stimulated phosphorylation are Tyr-350/Tyr-354, and to a lesser extent Tyr-364.
Site-directed mutagenesis of the tyrosyl residues was performed to test independently the role of Tyr-350/Tyr-354 and Tyr-364 in the counterregulatory effects of insulin on the ␤ 2 AR. Tyr-350/Tyr-354 (double substitution) or Tyr-364 were mutated to phenylalanine and the mutant receptor expressed in CHO cells. The ␤-adrenergic agonist isoproterenol (1 M) stimulated cyclic AMP accumulation in CHO cells expressing wildtype and Y350F/Y354F mutant receptors, but not the Y364F mutant ␤ 2 AR (Table I). Tyrosine to phenylalanine substitution of residues 350 and 354 abolishes the ability of insulin to counterregulate ␤-agonist-stimulated cyclic AMP accumulation in CHO cells. The Y364F mutation, in contrast, abolishes isoproterenol-stimulated cyclic AMP accumulation itself. These data, gathered from an independent approach, clearly highlight a critical role of Tyr-350/Tyr-354 in expression of the counterregulatory actions of insulin upon ␤ 2 AR.
Our results illuminate cross-regulation among two major transmembrane signaling pathways (4). The recent report that bradykinin B2 receptors isolated from WI-38 human lung fibroblasts in culture can be detected by anti-phosphotyrosine antibodies (16) provides additional evidence to support crosstalk from tyrosine kinase to G-protein-linked receptors (4). By study of rIR-catalyzed phosphorylation of ␤ 2 AR peptides in vitro, by peptide mapping of ␤ 2 AR phosphorylated in cells stimulated by insulin in vivo, and by site-directed mutagenesis studies, ␤ 2 AR tyrosyl residues 350, 354, and 364 are shown to be sites of insulin-stimulated phosphorylation. The peptide sequences flanking Tyr-364 suggest a growth factor tyrosine kinase recognition motif (17), which agrees well with our data implicating this residue as a phosphorylation site for the IR tyrosine kinase. Interestingly, the best peptide substrate, peptide L339, harbors tyrosyl residue 350, which lies in a sequence motif (Tyr-Gly-Asn-Gly) with similarity to motifs known to interact with Caenorhabditis elegans sem5 Src homology 2 (SH2) domains when phosphorylated (18). A common feature of these motifs is an Asn residue at position ϩ2 from the tyrosine residue, while residues ϩ1 and ϩ3 contain aliphatic side chains. It is tempting to speculate that residue Tyr-350, once phosphorylated by IR tyrosine kinase, constitutes a potential binding site for SH2-containing proteins such as the mammalian homolog of sem5, GRB2.
␤ 2 AR and IR co-exist in a number of mammalian tissues, including skeletal muscle and liver. How tyrosine kinase receptor-catalyzed phosphorylation of G-protein-linked receptors, like the ␤ 2 AR, contributes to physiological regulation remains an important question, derivative of exciting results presented herein. The data suggest that phosphorylation or mutagenesis of the tyrosyl residues in this domain (350 -364) impairs ␤ 2 AR function. Mutation of Tyr-350/Tyr-354 to alanine has been shown to alter ␤ 2 AR coupling to G s (19), much like the Y364F mutations (this study). Phosphorylation of Tyr-350/Tyr-354 by the IR is shown to impair G s coupling (Ref. 4 and this study), but further analysis of the multi-site phosphorylation of this receptor will be required making use of the mutant cell lines developed herein. Taken together these data provide strong evidence that this region is a regulatory domain of the ␤ 2 AR involved in G-protein coupling and that it is subject to covalent modification by counterregulatory, tyrosine kinase receptors.

TABLE I
Tyrosine to phenylalanine substitution of Tyr-350/Tyr-354 and of Tyr-364 in the ␤ 2 -adrenergic receptor alters the counterregulatory effects of insulin CHO cells were stably transfected with pCMV5 vector expressing wild-type ␤ 2 -adrenergic receptors or receptors with the tyrosine to phenylalanine substitutions indicated below. At least four separate clones for each mutation were analyzed. Clones were selected with a level of ␤ 2 -adrenergic receptor expression between 18 and 21 fmol/10 6 cells. cAMP accumulation was measured in unstimulated cells (basal) and cells challenged with 1 M isoproterenol alone, 1 M isoproterenol plus 100 nM insulin, or 100 nM insulin alone, as described under "Materials and Methods." The values displayed are mean values Ϯ S.E. from at least four independent determinations, each performed in triplicate. The S.E. for the increment of change values were less than 10% of the mean values and were omitted from this table for sake of simplicity.