The regular arrangement of leaves around a plant's stem, called phyllotaxis, has for centuries attracted the attention of philosophers, mathematicians and natural scientists; however, to date, studies of phyllotaxis have been largely theoretical. Leaves and flowers are formed from the shoot apical meristem, triggered by the plant hormone auxin. Auxin is transported through plant tissues by specific cellular influx and efflux carrier proteins. Here we show that proteins involved in auxin transport regulate phyllotaxis. Our data indicate that auxin is transported upwards into the meristem through the epidermis and the outermost meristem cell layer. Existing leaf primordia act as sinks, redistributing auxin and creating its heterogeneous distribution in the meristem. Auxin accumulation occurs only at certain minimal distances from existing primordia, defining the position of future primordia. This model for phyllotaxis accounts for its reiterative nature, as well as its regularity and stability.

Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

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 ...

Recent observations suggest that insulin increases cellular levels of activated, GTP-bound Ras protein.We tested whether the acute actions of insulin on hexose uptake and glucose-transporter redistribution to the cell surface are mimicked by activated Ras. 3T3-L1 fibroblasts expressing an activated mutant (Lys-61) N-Ras protein exhibited a 3-fold increase in 2-deoxyglucose uptake rates compared with nontransfected cells. Insulin stimulated hexose uptake by -2-fold in parental fibroblasts but did not stimulate hexose uptake in the N-Ras6lK-expressing fibroblasts. Overexpression of N-Ras6lK also mimicked the large effect of insulin on 2-deoxyglucose transport in 3T3-L1 adipocytes, and again the effects of the two agents were not additive. Total glucose transporter protein (GLUT) 1 was similar between parental and N-Ras6lK_ expressing 3T3-L1 fibroblasts or adipocytes, whereas total GLUT-4 protein was actually lower in the N-Ras6lK-expressing compared with parental adipocytes. However, expression of N-Ras6lK in 3T3-L1 adipocytes markedly elevated both GLUT-1 and GLUT-4 in plasma membranes relative to intracellular membranes, and insulin had no further effect. These modulations of glucose transporters by N-Ras6lK expression are not due to upstream regulation of insulin receptors because receptor tyrosine phosphorylation and association of phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins were unaffected. These results show that activated Ras mimics the actions of insulin on membrane trafficking of glucose transporters, consistent with the concept that Ras proteins function as intermediates in this insulin signaling pathway.Insulin is a major physiological regulator of many metabolic pathways, including those directing protein synthesis, lipogenesis, and glucose utilization. These biological actions of insulin are initiated by the activation of a specific, heterotetrameric receptor (1) with intrinsic tyrosine kinase activity (2), leading to rapid autophosphorylation of receptor tyrosine residues (3), as well as the tyrosine phosphorylation of other cellular substrates (4). Early cellular events preceding insulin regulation of target metabolic enzymes include the association of phosphatidylinositol 3-kinase with tyrosinephosphorylated proteins (5-8), an increase in the amounts of the protooncogene product Ras present in the active, GTPbound state (9-11), and the activation of numerous protein serine/threonine kinases (12, 13). Some of the insulinactivated protein kinases appear to operate in cascades (14, 15), and recent evidence indicates that phosphatase I modulation by insulin is a result of protein kinase activations (16). It is thought that insulin-regulated protein kinase and phosphatase activities catalyze downstream phosphorylation/ dephosphorylation reactions that acutely regulate insulinsensitive proteins and enzymes. Thus, conversion of the elevated insulin-receptor tyrosine kinase activity to serine/ threonine phosphorylation activity is a key element of signal transmission by insu...