The beta-adrenergic receptor is a substrate for the insulin receptor tyrosine kinase.

G-protein-linked receptors and intrinsic tyrosine-kinase growth receptors represent two prominent modalities in cell signaling. Cross-regulation among members of both receptor superfamilies has been reported, including the counter-regulatory effects of insulin on β-adrenergic catecholamine action. Cells stimulated by insulin show loss of function and increased phosphotyrosine content of β2-adrenergic receptors. Phosphorylation of tyrosyl residues 350/354 of β2-adrenergic receptors is obligatory for counter-regulation by insulin (Karoor, V., Baltensperger, K., Paul, H., Czech, M., and Malbon, C. C.(1995) J. Biol. Chem. 270, 25305-25308), suggesting the hypothesis that G-protein-linked receptors themselves may act as substrates for the insulin receptor and other growth factor receptors. This hypothesis was evaluated directly using recombinant human insulin receptor, hamster β2-adrenergic receptor, and an in vitro reconstitution and phosphorylation assay. Insulin is shown to stimulate insulin receptor-catalyzed phosphorylation of the β2-adrenergic receptor. Phosphoamino acid analysis establishes that insulin receptor-catalyzed phosphorylation of the β2-adrenergic receptor in vitro is confined to phosphotyrosine. High pressure liquid chromatography and two-dimensional mapping reveal insulin receptor-catalyzed phosphorylation of the β2-adrenergic receptor at residues Tyr132/Tyr141, Tyr350/Tyr354, and Tyr364, known sites of phosphorylation in response to insulin in vivo. Insulin-like growth factor-I receptor as well as the insulin receptor displays the capacity to phosphorylate the β2-adrenergic receptor in vitro, establishing a new paradigm, i.e. G-protein-linked receptors acting as substrates for intrinsic tyrosine kinase growth factor receptors.

Protein phosphorylation plays a prominent role in the regulation of cell signaling. A populous family of G-protein-linked receptors mediate activation of a diverse class of effectors, such as adenylyl cyclase, phospholipase C, and various ion channels, via a less populous class of G-proteins (1). These G-protein-linked receptors share many features, including regulation via protein phosphorylation (2,3). Insulin counter-regulates the action of ␤-adrenergic catecholamine stimulation, at a point proximal to the ␤-adrenergic receptor (4). Cells stimulated by insulin show increased phosphotyrosine content and loss of function of ␤ 2 -adrenergic receptors (4). The insulin receptor, upon ligand binding, expresses intrinsic tyrosine kinase activation (5), raising the intriguing hypothesis that G-proteinlinked receptors and intrinsic tyrosine kinase growth receptors may interact directly, the former a substrate for the latter. Recently, we have deduced structural information on sites of phosphotyrosine labeling in vivo (6). In the current work, we directly test the hypothesis that the ␤ 2 -adrenergic receptor is a substrate for growth factor receptor tyrosine kinase, using recombinant receptors and a defined reconstitution assay in vitro. The results demonstrate that growth factor tyrosine kinase receptors (e.g. insulin receptor and the IGF-I 1 receptor) can directly phosphorylate a G-protein-linked receptor.

Recombinant ␤ 2 AR, Insulin Receptor, and Purified IGF-I Receptor-
Recombinant hamster ␤ 2 -adrenergic receptor (r␤AR) was expressed using the baculovirus-Sf9 insect cell expression system (7) and purified by affinity, HPLC, and lectin chromatography (8). Recombinant human insulin receptor (rIR) was purified by lectin chromatography (9) from Chinese hamster ovary (CHO)-T cells, which stably overexpress the human insulin receptor (10) or from COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (11). The IGF-I receptor (IGF-IR) was prepared from lectin chromatography of cell extracts of human osteogenic sarcoma, a cell line replete in IGF-IR (11).
Phosphorylation of ␤ 2 AR in Vitro-In vitro, phosphorylation of the r␤AR was achieved in a reconstitution assay, whereby 5 l (100 -200 fmol) of rIR and 20 l (10 -20 pmol) of ␤ 2 AR (or 20 l of buffer) were incubated at 22°C in a final volume of 45 l in (final concentrations) 25 mM Tris/HCl (pH 7.4), 50 mM NaCl, 10 mM MgCl 2 , 3 mM MnCl 2 , 100 M Na 3 VO 4 , 1 mM dithiothreitol, 0.1% (w/v) Triton X-100. 5 l of [␥-32 P]ATP (40 Ci/mmol) were then added to give a final concentration of 5 M ATP. The phosphorylation reaction was terminated at 30 min by the addition of 50 l of 2ϫ concentrated Laemmli sample buffer containing 100 mM dithiothreitol. Proteins were denatured for 5 min at 95°C and then separated by SDS-PAGE. Phosphorylated proteins were made visible by exposing the dried gel to X-Omat AR film (Kodak). The amount of label incorporated into r␤ 2 AR by insulin-stimulated rIR in this detergent-dispersed reconstitution system was defined by phosphoroimage analysis (Beta-scan) and compared with the label incorporated in the rIR ␤-subunit. Under the conditions employed, insulin-stimulated phosphorylation of r␤ 2 AR was approximately 15% of that incor-* 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, Universitat Bern, Friedbuhl-Strasse 49, CH-3010 Bern, Switzerland.
Phosphoamino Acid Analysis of ␤ 2 AR-rIR (5 l, 100 -200 fmol) from CHO-T cells or from COS-1 cells was reconstituted with r␤AR (20 l, 10 -20 pmol) in the absence or the presence of insulin (100 nM) and incubated for 30 min at 22°C with [␥-32 P]ATP (5 M) as described above. Phosphorylated proteins were then separated by SDS-PAGE and visualized by autoradiography of the dried gel. The regions of interest were excised from the gel, rehydrated, and subjected to acid hydrolysis in 6 M HCl (7,12). Phosphoamino acids were separated by thin-layer electrophoresis at pH 3.5 and visualized by autoradiography as described (4,9). Proportional amounts of radioactivity as detected in the rehydrated gel pieces were resolved and detected.
Phosphorylation of Synthetic Peptide Substrates-Peptides corresponding to the cytoplasmic domains of the ␤ 2 AR displaying phosphorylation of tyrosyl residues in vivo (6) were synthesized, purified by HPLC, and subjected to in vitro phosphorylation by rIR in the absence or the presence of insulin (100 nM). The peptide sequences employed as internal standards for the HPLC and high voltage electrophoresis after tryptic digestion were as follows: L339, LLCLRRSSSKAYGNGYSS-NSNGKTD; T362, TDYMGEASGCQLGQEK; R62, RLQTVTNYFITS-LACAD; Y132, YIAITSPFKYQSLLTKNKAR; and I135, ITSPFKYQS-LLTKNKAR. Partially purified rIR (wheat germ agglutinin extracts from CHO-T cells) was incubated in the absence or the 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 2ϫ concentrated Laemmli sample buffer. Phosphopeptides were separated by Tricine gel electrophoresis (13).
Reverse-phase HPLC of Tryptic Phosphopeptides-r␤AR radiolabeled in vitro by rIR with [␥-32 P]ATP were separated on SDS-PAGE (6). Synthetic peptides containing tyrosine residues 141, 350, 354, and 364 were labeled in vitro with [␥-32 P]ATP and separated on Tricine gels. The bands corresponding to r␤ 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 (9). 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) was measured for each fraction. The standards for tryptic digests of L339 and T362 reported earlier (6) are included for reference.
Two-dimensional Peptide Mapping-Tryptic digestion of r␤ 2 -adrenergic receptor and of synthetic peptides phosphorylated in vitro by rIR was performed as described above. Two-dimensional mapping of the tryptic fragments was performed as described recently (6). Briefly, 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, 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, 15:10:3:12). The plates were dried, and the peptides were identified by autoradiography. The standards for tryptic digests of L339 and T362 reported earlier (6) are included for reference.

RESULTS AND DISCUSSION
In cultured smooth muscle cells, insulin impairs the function and increases the phosphotyrosine content of ␤ 2 AR (4). To address the possibility that a G-protein-linked receptor might serve as a substrate for an intrinsic tyrosine kinase growth receptor, we reconstituted the two receptors in vitro. rIR was isolated and partially purified from a Chinese hamster ovary cell line (CHO-T) that stably overexpresses the human insulin receptor (10). This rIR preparation reduced levels of uncleaved proreceptor and showed marked increase of rIR ␤-subunit phosphorylation upon insulin stimulation (Fig. 1, lanes 1 and  2). Marked insulin-stimulated phosphorylation of the r␤AR (M r , 45,000) as well as of the rIR ␤-subunit (M r , 86,000) was observed following reconstitution of purified r␤ 2 AR expressed in baculovirus-infected Sf9 cells and rIR purified from CHO-T cells in the presence of detergent (Fig. 1, lanes 3 and 4). No phosphorylation of r␤AR was evident in reconstitutions performed in the absence of rIR. Phosphorylation of some incompletely processed proreceptor (M r , 180,000) present in the IR preparations was observed occasionally as shown here. Insulinstimulated phosphorylation of r␤AR was observed in reconstitutions with lectin-purified IR from either CHO-T cells or a second source, SV40-transformed African green monkey kidney COS-1 cells, which were transiently transfected with the human insulin receptor cDNA (not shown).
Phosphoamino acid analysis of the r␤AR and rIR products from an in vitro phosphorylation reaction revealed phosphotyrosine labeling of the r␤AR, whether performed with rIR expressed in CHO-T (Fig. 2, lanes 1 and 2) or COS-1 (lanes 3 and  4) cells. The rIR ␤-subunit was predominantly phosphorylated on tyrosine residues (Fig. 2, lanes 5 and 6), providing an internal control. Some phosphoserine in the rIR ␤-subunit was detected, which is due to low level serine kinase activity of the IR tyrosine kinase (9). Similarly, upon overexposure of the autoradiography, some phosphoserine could be detected in phosphorylated r␤AR (not shown), which is consistent with the hypothesis that the insulin receptor directly interacts with and phosphorylates the r␤AR.
We explored whether the ␤ 2 -adrenergic receptor was a substrate for a second growth factor-activated tyrosine kinase receptor, the IGF-I receptor, which is structurally closely related to the IR (14). We assayed the ability of insulin to stimulate phosphorylation of r␤AR in vitro in reconstitution studies with IGF-IR purifed from human osteogenic sarcoma cell extracts (Fig. 3, lanes 2-5) and compared it with the phosphorylation of r␤AR by rIR (Fig. 3, lane 1, and Fig. 1). The IGF-1R phosphorylated r␤AR in response to stimulation by high concentrations (1 M) of insulin (lane 3). IGF-1R-dependent phosphorylation of the 45,000 M r protein was absent in the reconstitutions devoid of r␤AR (lanes 4 and 5). A small amount of phosphorylation was observed in the lectin-purified extract rich in IGF-IR in the absence of hormonal stimulation (lane 3), which may reflect phosphorylation catalyzed by platelet-derived growth factor receptor, which was activated (autophosphorylated) in this fraction (see *, Fig. 3, lanes 2-5). These observations demonstrate that tyrosine kinase growth factor receptors, in addition to the IR, can catalyze specific phosphorylation of r␤AR in response to insulin.
In an effort to explore the site(s) for insulin-stimulated, rIR-catalyzed phosphorylation, recently we prepared synthetic peptides corresponding to cytoplasmic regions of the ␤ 2 AR that 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 (6). Maps of tryptic digests might permit analysis of the sites phosphorylated on the ␤ 2 AR in response to insulin in vitro (Fig. 4). Tryptic digests of peptides phosphorylated in vitro by rIR in response to insulin provided markers for HPLC analysis (Fig. 4, A-C). The retention times for the tryptic fragments subjected to HPLC separation agreed well with the retention times calculated from the sequence information (not shown). For tryptic digests of phosphorylated I135 peptide, uncleaved peptide was detected routinely (*, Fig. 4A). Tryptic digests of phosphopeptide Y132 display the same mobility as the fragments of I135 (not shown), i.e. fraction 4 (Fig. 4A). The Y132 peptide was shown previously to be the preferred substrate for insulin-stimulated, rIR-catalyzed phosphorylation (6). Using these labeled standards, we established that ␤ 2 AR reconsti-  2-5), which is highly enriched in IGF-1 receptor were reconstituted as described above (Fig. 1) in the presence of [␥-32 P]ATP with (lanes 1-3) or without (lanes 4 and 5) r␤AR in the absence (lanes 2 and 4) or the presence of insulin (INS; lane 1, 100 nM insulin; lanes 3 and 5, 1 M insulin to stimulate the IGF-1R tyrosine kinase activity). Phosphoproteins were separated by SDS-PAGE and visualized by autoradiography of the dried gel. The extent of the phosphorylation of r␤AR catalyzed by rIR and IGF-IR was quantified by excising gel pieces containing the tyrosine kinase receptors and r␤AR. *, represents the phosphorylated/activated form of the platelet-derived growth factor receptor, as established by immunoblotting of the gels with anti-platelet-derived growth factor antibody (not shown). harboring Tyr 364 following in vitro insulin-stimulated rIR-catalyzed phosphorylation. D, HPLC analysis of tryptic fragments of r␤ 2 AR following reconstitution with and insulin-stimulated phosphorylation in vitro by rIR. *, represents phosphopeptide I135 resistant to tryptic digestion. **, represents a small and variable amount of disulfidebridged tryptic peptide of Tyr 362 , reflecting the presence of a cysteinyl residue in the fragment. The standards for tryptic digests of L339 and T362 reported earlier (6) are included for reference. tuted in vitro with rIR in the presence of insulin was phosphorylated predominantly on peptides harboring residues Tyr 131 / Tyr 142 , Tyr 350 /Tyr 354 , and Tyr 364 (Fig. 4D). Phosphorylation of the r␤ 2 AR by rIR in vitro was not detected in the absence of insulin.

FIG. 3. Direct phosphorylation of the r␤AR in vitro by the IGF-I receptor as well as insulin receptor. Recombinant IR from CHO-T cells (lane 1) or the glycoprotein fraction from human osteogenic sarcoma cells (lanes
High voltage electrophoresis followed by thin-layer chromatography of the tryptic fragments (Fig. 5, A-C) provides additional markers for analysis of phosphopeptides derived from the ␤ 2 AR phosphorylated by insulin-stimulated rIR using the in vitro, reconstitution assay (Fig. 5D). The two-dimensional analysis extends the results of reverse-phase HPLC, establishing that the predominant sites of insulin-stimulated phosphorylation catalyzed by the rIR in vitro are Tyr 132 /Tyr 141 , Tyr 350 / Tyr 354 , and Tyr 364 (Fig. 5D). Peptides harboring Tyr 132 and/or Tyr 141 are substrates for rIR-catalyzed phosphorylation in response to insulin stimulation, the peptide harboring both Tyr 132 and Tyr 141 being the preferred substrate (6). The analysis for I135 only is displayed (Fig. 5A), being representative of tryptic fragments from Y132 also, which behave identically with those of I135 in both the HPLC and two-dimensional analyses (not shown). Two-dimensional analysis of tryptic fragments of ␤ 2 AR phosphorylated by rIR in vitro revealed labeling of Tyr 132 /Tyr 141 , as shown here. Thus, in vitro phosphorylation of the ␤ 2 AR by the IR tyrosine kinase is confined to Tyr 132 / Tyr 141 , Tyr 350 /Tyr 354 , and Tyr 364 .
In the current work we exploited the ability to prepare rIR, rbAR, and IGF-IR-enriched fractions from human osteogenic sarcoma cells to directly test the hypothesis that a G-proteinlinked receptor can act as a substrate for an intrinsic tyrosinekinase growth factor receptor. The following observations provide compelling evidence in support of this hypothesis: (i) insulin stimulates rIR-catalyzed phosphorylation of the r␤ 2 AR in a reconstituted, in vitro assay; (ii) insulin-stimulated phosphorylation of the r␤ 2 AR in vitro is confined to tyrosyl residues; (iii) the sites phosphorylated in vitro in response to insulin are those phosphorylated in vivo, as determined by structural analysis of ␤ 2 AR isolated from metabolically labeled cells; (iv) insulin at high concentrations stimulates in vitro phosphorylation of r␤ 2 AR catalyzed by purified IGF-IR; and (v) mutagenesis of these tyrosyl residues of the ␤ 2 AR in vivo results in loss of insulin-stimulated counter-regulation of ␤ 2 AR function (6). In addition, the bradykinin receptor has been shown to be phosphorylated on tyrosyl residues in response to serum (15), and the angiotensin II AT1 receptor has been reported to be a substrate for phosphorylation by the src family of tyrosine kinases (16). Based upon these observations we propose a new paradigm in which two prominent pathways in cell signaling cross-talk to each other at the most proximal point, receptor to receptor.
Interestingly, the sites on the ␤ 2 AR phosphorylated by the IR and IGF-IR include a well known motif for tyrsoine kinase growth factor receptors at Tyr 364 (17), a prominent GRB2 site at Tyr 350 (6,18), and a potential SHC binding site at Tyr 132 (19). The co-migration of the tryptic fragments harboring Tyr 132 and Tyr 141 preclude definition of the contribution of each to phosphotyrosine labeling by the IR, either in vitro (present study) or in vivo (6). Insulin-stimulated, IR-catalyzed phosphorylation of peptide Y132, which harbors Tyr 132 /Tyr 141 , however, was prominent, whereas that for peptide I135, which lacks Tyr 132 , was decidely poor (6). Further analysis of this site will be required, because phosphorylation of Tyr 132 creates a Shc binding site, and this site is conserved among many Gprotein-linked receptors, including receptors for neuropeptide Y, tachykinin, A2-adenosine, thyrotropin releasing factor, and serotonin (Genebank). Although speculation, the full range of adaptor molecules known to play a prominent role in tyrosine kinase receptor signaling may be made available to G-proteinlinked receptors, once phosphorylated by a tyrosine kinase activated in response to a growth factor.
FIG. 5. Two-dimensional phosphopeptide mapping of the ␤-adrenergic receptor demonstrates insulin-stimulated rIR-catalyzed phosphorylation of tyrosyl residues 350, 354, and 364 in vitro. A, high voltage electrophoresis and thin-layer chromatography two-dimensional analysis of tryptic fragment I135 (ITSPFKYQSLLT-KNKAR) harboring Tyr 141 following in vitro insulin-stimulated rIRcatalyzed phosphorylation. B, high voltage electrophoresis and thinlayer chromatography two-dimensional analysis of tryptic fragment of L339 (AYGNGYSSNSNGK) harboring Tyr 350 /Tyr 354 following in vitro insulin-stimulated rIR-catalyzed phosphorylation. C, high voltage electrophoresis and thin-layer chromatography two-dimensional analysis of tryptic fragment T362 (TDYMGEASGCQLGQEK) harboring Tyr 364 following in vitro insulin-stimulated rIR-catalyzed phosphorylation. D, high voltage electrophoresis and thin-layer chromatography two-dimensional analysis of tryptic fragments of r␤ 2 AR following reconstitution with and insulin-stimulated phosphorylation by rIR in vitro. The standards for tryptic digests of L339 and T362 reported earlier (6) are included for reference.