No abstract

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-proteinlinked 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. MATERIALS AND METHODSRecombinant ␤ 2 AR, Insulin Receptor, and Purified IGF-I ReceptorRecombinant 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 syst...

Insulin stimulates a loss of function and increased phosphotyrosine content of the ␤ 2 -adrenergic receptor in intact cells, raising the possibility that the ␤ 2 -receptor itself is a substrate for the insulin receptor tyrosine kinase. Phosphorylation of synthetic peptides corresponding to cytoplasmic domains of the ␤ 2 -adrenergic receptor by the insulin receptor in vitro and peptide mapping of the ␤ 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 ␤ 2 -adrenergic receptor as primary targets. Mutation of tyrosyl residues 350, 354 (double mutation) to phenylalanine abolishes the ability of insulin to counterregulate ␤-agonist stimulation of cyclic AMP accumulation. Phenylalanine substitution of tyrosyl reside 364, in contrast, abolishes ␤-adrenergic stimulation itself.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 ␤ 2 -adrenergic receptor, ␤ 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 ␤ 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 METHODSPreparation 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).Phosphorylation of ␤ 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 [ 32 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 M), aprotinin (5 g/ml), leupeptin (5 g/ml), bacitracin (100 g/ml), benzamidine (100 g/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 ...