Cascade of autophosphorylation in the beta-subunit of the insulin receptor

Insulin stimulated autophosphorylation of the beta-subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild-type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the beta-subunit: the regulatory region containing Tyr-1146, Tyr-1150, and Tyr-1151, and the C-terminus containing Tyr-1316 and Tyr-1322. In the presence of antiphosphotyrosine antibody (alpha-PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that alpha-PY inhibited autophosphorylation of both tyrosyl residues in the C-terminus and one tyrosyl residue in the regulatory region, either Tyr-1150 or Tyr-1151. Thus, a bis-phosphorylated form of the regulatory region accumulated in the presence of alpha-PY, which contained Tyr(P)-1146 and either Tyr(P)-1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the beta-subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the beta-subunit was mainly (greater than 80%) bis-phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris-phosphorylation but not bis-phosphorylation of the regulatory region of the beta-subunit (White et al.: Journal of Biological Chemistry 263:2969-2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.     

Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins

Effects of aluminum, magnesium, and fluoride (AMF) on members of both the trimeric G protein and smaller (20-25 kDa) monomeric GTP-binding protein families were examined. The dissociation of GDP from G proteins was blocked by AMF but was unchanged with the addition of AMF to any of six of the monomeric GTP-binding proteins. Biochemical activities and properties of one of the smaller GTP-binding proteins, ADP-ribosylation factor, were also found to be unaffected by AMF. It is concluded that the ability of AMF to activate the trimeric G proteins is not shared by the smaller GTP-binding proteins and thus should prove to be a useful discriminator between cellular activities regulated by these two families of regulatory proteins.     

Healing modes correlate with visuotectal pattern formation in regenerating embryonic Xenopus retina

After removal of the nasal or the temporal two-thirds of the embryonic (stage 32) eye, the remaining one-third sized fragment undergoes wound healing and then, in most cases, regenerates to form a new eye. Using gross anatomy and histology techniques, we categorized eye fragments into three healing mode categories over the first 24 hr after surgery (stage 37-38). Representative animals were reared through metamorphosis and their visuotectal projections were assayed using standard electrophysiology techniques. In the "rounded-up" healing mode, the cut edges of the fragment pinch to close the wound; retinal cell type layers (pigmented retinal epithelium (pre), photoreceptors, interneurons, ganglion cells) and a lens are present by 24 hr postsurgery. No extraneous or disorganized cells are present either internal or external to the fragments. These fragments regenerated to form normal projections 83% of the time and pattern duplicated projections only 17% of the time. In the "intermediate" healing mode, wound closure is not complete by 24 hr post surgery and groups of disorganized cells are present in the fragment and amassed between the healing cut edges. These fragments formed pattern duplicated projections 72% of the time. In the tongue healing mode, an ectopic mass of cells, contiguous with the main body of the fragment, forms a supernumerary retina in the region of the ablation. At 24 hr post surgery, the cells of the main body fragment form retinal layers; the cells of the tongue, excluding the presence of differentiated pre cells, remain undifferentiated, resembling ciliary margin. The cut edges of the main body fragment eventually fuse with the tongue to form a single eyeball. Tongue fragments formed pattern duplicated projections 100% of the time. In addition, pattern duplicated points derived from nasal fragments appeared most often in the posterior region of the tectum, the normal site of innervation of the nasal retina. This differed significantly from temporal fragment derived duplicated points which appeared more often in the front of the tectum, the normal site of innervation by temporal retina. Thus, the specificity of pattern duplicated innervation is related to the positional values remaining in the fragment after partial retinal ablation. The data indicate that cell movements during healing, whether overt as in the tongue healing mode, or remaining internal to the fragment as in the intermediate healing mode, are intimately correlated with pattern forming mechanisms which underlie pathological visuotectal duplication.     

Mechanism for markedly hyperresponsive insulin-stimulated glucose transport activity in adipose cells from insulin-treated streptozotocin diabetic rats. Evidence for increased glucose transporter intrinsic activity

The effects of insulin therapy in streptozotocin diabetic rats on the glucose transport response to insulin in adipose cells have been examined. At sequential intervals during subcutaneous insulin infusion, isolated cells were prepared and incubated with or without insulin, and 3-O-methylglucose transport was measured. Insulin treatment not only reversed the insulin-resistant glucose transport associated with diabetes, but resulted in a progressive hyperresponsiveness, peaking with a 3-fold overshoot at 7-8 days (12.1 +/- 0.3 versus 3.4 +/- 0.1 fmol/cell/min, mean +/- S.E.) and remaining elevated for more than 3 weeks. During the peak overshoot, glucose transporters in subcellular membrane fractions were assessed by cytochalasin B binding. Insulin therapy restored glucose transporter concentration in the plasma membranes of insulin-stimulated cells from a 40% depleted level previously reported in the diabetic state to approximately 35% greater than control (38 +/- 4 versus 28 +/- 2 pmol/mg of membrane protein). Glucose transporter concentration in the low-density microsomes from basal cells was also restored from an approximately 45% depleted level back to normal (50 +/- 4 versus 50 +/- 6 pmol/mg of membrane protein), whereas total intracellular glucose transporters were further increased due to an approximately 2-fold increase in low-density microsomal membrane protein. However, these increases remained markedly less than the enhancement of insulin-stimulated glucose transport activity in the intact cell. Thus, insulin treatment of diabetic rats produces a marked and sustained hyperresponsive insulin-stimulated glucose transport activity in the adipose cell with little more than a restoration to the non-diabetic control level of glucose transporter translocation. Because this enhanced glucose transport activity occurs through an increase in Vmax, insulin therapy appears to be associated with a marked increase in glucose transporter intrinsic activity.     

Characterization of an endogenous substrate of the insulin receptor in cultured cells

Using antiphosphotyrosine antibodies, we have characterized the tyrosine phosphorylation of an endogenous substrate of the insulin receptor in Fao hepatoma cells and in Chinese hamster ovary cells transfected with a eukaryotic expression vector containing the human insulin receptor cDNA. In Fao cells, besides the beta-subunit of the insulin receptor, a protein with a molecular mass between 170 and 210 kDa designated pp185, undergoes tyrosine phosphorylation immediately after insulin stimulation reaching a maximum level within 30 s. After 4 h of continuous insulin stimulation, the labeling of pp185 decreased to less than half of its original intensity, whereas the insulin receptor was unchanged. After 24 h of insulin stimulation, the phosphotyrosine-containing insulin receptor decreased by 75% owing to down-regulation, whereas the pp185 was completely undetectable. By several biochemical and physiological criteria, the pp185 is distinct from the insulin receptor. The pp185 and the beta-subunit of the insulin receptor were strongly labeled with [32P]orthophosphate, but in contrast to the insulin receptor, the pp185 was not labeled by cross-linking with 125I-insulin or surface 125I iodination. Unlike the insulin receptor, the pp185 was extracted from Fao cells without detergent, and tryptic phosphopeptide mapping of the pp185 and the insulin receptor yielded distinct patterns. Thus, the pp185 is not located at the external face of the plasma membrane and does not bind insulin. Treatment of Fao cells with the phorbol ester, phorbol 12-myristate 13-acetate, stimulated the phosphorylation of two proteins with molecular weights of 170 and 210 kDa which were immunoprecipitated with the anti-phosphotyrosine antibody. Subsequent insulin stimulation increased the phosphorylation of the 210 kDa protein, but the pp185 was not detected. Increasing the concentration of the human insulin receptor in the Chinese hamster ovary cells by transfection with a plasmid containing the human insulin receptor cDNA caused a higher level of tyrosine phosphorylation of the beta-subunit and the pp185. These data support the notion that the insulin signal may be transmitted to a cellular substrate (pp185) which may initiate insulin action at intracellular sites.