©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation of Tyrosyl Residues 350/354 of the -Adrenergic Receptor Is Obligatory for Counterregulatory Effects of Insulin (*)

(Received for publication, July 26, 1995; and in revised form, August 23, 1995)

Vijaya Karoor (1) Kurt Baltensperger (2)(§) Hyacinth Paul (1) Michael P. Czech (2) Craig C. Malbon (1)(¶)

From the  (1)Department of Molecular Pharmacology, Diabetes and Metabolic Diseases Research Program, State University of New York, Stony Brook, New York 11794-8651 and the (2)Program in Molecular Medicine and the Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01605

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Insulin stimulates a loss of function and increased phosphotyrosine content of the beta(2)-adrenergic receptor in intact cells, raising the possibility that the beta(2)-receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the beta(2)-adrenergic receptor by the insulin receptor in vitro and peptide mapping of the beta(2)-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 beta(2)-adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate beta-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes beta-adrenergic stimulation itself.


INTRODUCTION

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 beta(2)-adrenergic receptor, beta(2)AR),^1 in contrast, activate adenylyl cyclase 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 beta(2)-adrenergic receptor(4) . In the current study the structural basis for these counterregulatory effects of insulin exerted on the beta(2)-adrenergic receptor is explored.


MATERIALS AND METHODS

Preparation of Recombinant beta(2)AR and Insulin Receptor

Recombinant hamster beta(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) .

Phosphorylation of beta(2)AR in Vivo

In vivo, DDT(1)MF-2 hamster vas deferens smooth muscle cells were cultured in Dulbecco's modified Eagle's medium (DMEM), metabolically labeled in phosphate-free DMEM containing 0.5% fetal bovine serum and [P]orthophosphate (1 mCi/ml) for 4 h at 37 °C(4) . At the end of the 4-h incubation, insulin or vehicle was added as indicated in the figure legends. To terminate phosphorylation, cells were washed and then lysed. The lysis buffer was composed of Triton X-100 (1%), sodium dodecyl sulfate (0.1%), dithiothreitol (6.0 µM), aprotinin (5 µg/ml), leupeptin (5 µg/ml), bacitracin (100 µg/ml), benzamidine (100 µg/ml), sodium orthovanadate (2 mM), NaCl (150 mM), EDTA (5 mM), NaF (50 mM), sodium pyrophosphate (40 mM), KH(2)PO(4) (50 mM), sodium molybdate (10 mM), and Tris-HCl (20 mM, pH 7.4). Immunoprecipitation was performed in the lysis buffer(10) . Each sample was precleared with nonimmune serum-protein A-agarose complex for 2 h prior to immunoprecititation with anti-receptor antibody CM-4(4, 10) . The immunoprecipitated proteins were denatured for 5 min at 95 °C and then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% (w/v) acrylamide gels(4) . Phosphorylated proteins were made visible by exposing the dried gel to X-Omat AR film (Kodak).

Stoichiometry of Phosphorylation

In vivo, duplicate cultures of DDT(1)MF-2 smooth muscle cells were labeled metabolically with [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 beta(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 [P]ATP in the supernatant was determined as described by England and Walsh(11) . With the determination of the specific activity of the cellular [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 beta(2)AR.

Phosphorylation of Synthetic Peptide Substrates

Peptides corresponding to each cytoplasmic domain of the beta(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, TDYMGEASGCQLGQEK; R62, RLQTVTNYFITSLACAD; Y132, AITSPFKYQSLLTKNKAR; I135, ITSPFKYQSLLTKNKAR; E121,ETLSVIAVDRYIAITSPFK. Partially purified rIR (wheat germ agglutinin extracts from CHO-T cells) was incubated in the absence or presence of insulin (100 nM), 10 µM [-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 times 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 (P window) in the gel piece. The data shown are from a single experiment, replicated once with similar results.

Reverse-phase HPLC of Tryptic Phosphopeptides

P-Labeled beta(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 [-P]ATP and rIR, and then separated on Tricine gels. The bands corresponding to beta(2)AR or the synthetic peptides were excised from the gels and treated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (40 µg/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 µl/min. Fractions were collected at 1-min intervals and Cerenkov radiation (P window) measured for each fraction.

Two-dimensional Peptide Mapping

Tryptic digestion of beta(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 µl) 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 beta(2)AR cDNA was performed using the Transformer Mutagenesis® kit (Clontech), according to the manufacturer's suggested protocols. The sequences of the mutagenic primers were as follows: Y350F/Y354F, CTTCAAAAGCCTTTGGGAACGGCTTCTCCAGCA; 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 µg/ml). Expression of beta(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 beta-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 beta(2)AR, we prepared synthetic peptides to corresponding to each cytoplasmic regions of the beta(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 beta(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. Insulin-stimulated phosphorylation of the peptides by the rIR was 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).


Figure 1: Topological model of beta(2)-adrenergic receptor, highlighting candidate tyrosyl residues for insulin-stimulated phosphorylation. A, model of beta(2)AR organization and location of synthetic sequences used as substrates for insulin-stimulated rIR-catalyzed phosphorylation of the beta(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.




Figure 2: Synthetic peptides corresponding to cytoplasmic sequences of the C terminus of the beta(2)AR that possess Tyr-350/Tyr-354 and Tyr-364 (L339 and T362, respectively) are preferred substrates for phosphorylation by rIR. A, insulin-dependent phosphorylation of beta(2)AR-peptides by the rIR. rIRbeta, beta-subunit of the recombinant IR. B, dose-response-relationship of betaAR peptide phosphorylation by the rIR. The synthetic peptides were incubated with rIR in the presence of insulin (100 nM) at concentrations indicated. Phosphorylation of the peptides was quantified and computed as picomoles of phosphate/tyrosyl residue.



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 for insulin-stimulated phosphorylation of L339 peptide was 100 µM. At 3 mM concentrations of peptide, saturation of rIR-catalyzed phosphorylation for the other peptides was not achieved, precluding the calculation of ED 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) > T362 (Tyr-364) > Y132 (Tyr-132) 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 beta(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).


Figure 3: Reverse-phase HPLC phosphopeptide mapping of the beta-adrenergic receptor demonstrates insulin-stimulated rIR-catalyzed phosphorylation of tyrosyl residues 350, 354 and 364 in vivo. C-F, insulin promotes phosphorylation of the beta(2)-adrenergic receptor in metabolically labeled DDT(1) MF-2 smooth muscle cells. Tryptic peptides of in vitro labeled L339 (harboring Tyr-350 and Tyr-354) and of T362 (harboring Tyr-364) were employed as standards (panels A and B, respectively). Cells metabolically labeled with [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 beta(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.



In vivo, metabolic labeling of DDT(1)MF-2 smooth muscle cells in culture with [P]orthophosphate revealed the phosphotyrosine content of beta(2)AR to increase from 0.86 ± 0.10 (basal) to 1.76 ± 0.39 (n = 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 beta(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 beta(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 profiles (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.


Figure 4: Two-dimensional phosphopeptide mapping of the beta-adrenergic receptor demonstrates insulin-stimulated phosphorylation of tyrosyl residues 350, 354, and 364 in vivo. A-D, insulin promotes phosphorylation of the beta(2)-adrenergic receptor in metabolically labeled DDT(1) MF-2 smooth muscle cells. Tryptic peptides of in vitro-labeled L339 (harboring Tyr-350 and Tyr-354) and of T362 (harboring Tyr-364) were employed as standards (panels A and B, respectively). Cells metabolically labeled with [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 beta(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.



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 beta(2)AR. Tyr-350/Tyr-354 (double substitution) or Tyr-364 were mutated to phenylalanine and the mutant receptor expressed in CHO cells. The beta-adrenergic agonist isoproterenol (1 µM) stimulated cyclic AMP accumulation in CHO cells expressing wild-type and Y350F/Y354F mutant receptors, but not the Y364F mutant beta(2)AR (Table 1). Tyrosine to phenylalanine substitution of residues 350 and 354 abolishes the ability of insulin to counterregulate beta-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 beta(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 cross-talk from tyrosine kinase to G-protein-linked receptors (4) . By study of rIR-catalyzed phosphorylation of beta(2)AR peptides in vitro, by peptide mapping of beta(2)AR phosphorylated in cells stimulated by insulin in vivo, and by site-directed mutagenesis studies, beta(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 +2 from the tyrosine residue, while residues +1 and +3 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.

beta(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 beta(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 beta(2)AR function. Mutation of Tyr-350/Tyr-354 to alanine has been shown to alter beta(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 ( (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 beta(2)AR involved in G-protein coupling and that it is subject to covalent modification by counterregulatory, tyrosine kinase receptors.


FOOTNOTES

*
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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Pharmakologisches Institut, Universität Bern, Friedbuhl-Strasse 49, CH-3010 Bern, Switzerland.

To whom all correspondence and proofs are to be addressed. Tel.: 516-444-7873; Fax: 516-444-7696.

(^1)
The abbreviations used are: beta(2)AR, beta(2)-adrenergic receptor; HPLC, high performance liquid chromatography; rIR, recombinant human insulin receptor; CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1,bis(hydroxymethyl)ethyl]glycine; SH2, Src homology 2.


ACKNOWLEDGEMENTS

We thank Drs. T. Fischer, R. Carraway, and J. Hoogasian for peptide synthesis and purification.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.