Molecular cloning and characterization of the 5' region of the mouse trkA proto-oncogene.

The trkA proto-oncogene encodes a high-affinity NGF receptor that is essential for the survival, differentiation and maintenance of many neural and non-neural cell types. Altered expression of the trkA gene or trkA receptor malfunction have been implicated in neurodegeneration, tumor progression and oncogenesis. We have cloned and characterized the 5' region of the mouse trkA gene and have identified its promoter. trkA promoter sequences are GC-rich, lack genuine TATA or CAAT boxes, and are contained within a CpG island which extends over the entire first coding exon. The mouse trkA transcription start site is located 70/71 bp upstream to the AUG translation initiation codon. Sequence analysis showed that the gene encoding the insulin receptor-related receptor, IRR, is located just 1.6 kbp upstream to the trkA gene and is transcribed in the opposite direction. We have used trkA-CAT transcriptional fusions to study trkA promoter function in transient transfection experiments. RNase protection assays and CAT protein ELISA analyses showed that a 150 bp long DNA segment, immediately upstream to the start site, is sufficient to direct accurate transcription in trkA-expressing cells. Dissection of this fragment allowed us to identify a 13 bp cis-regulatory element essential for both promoter activity and cell-type specific expression. Deletion of this 13 bp segment as well as modification of its sequence by site-directed mutagenesis led to a dramatic decline in promoter activity. Gel mobility shift assays carried out with double-stranded oligonucleotides containing the 13 bp element revealed several specific DNA-protein complexes when nuclear extracts from trkA-expressing cells were used. Supershift experiments showed that the Sp1 transcription factor was a component of one of these complexes. Our results identify a minimal trkA gene promoter, located very close to the transcription start site, and define a 13 bp enhancer within this promoter sequence.

Proximal promoter sequences mediate cell-specific and elevated expression of the favorable prognosis marker TrkA in human neuroblastoma cells.

The nerve growth factor receptor, TrkA, has a critical role in the survival, differentiation, and function of neurons in the peripheral and central nervous systems. Recent studies have demonstrated a strong correlation between abundant expression of TrkA and a favorable prognosis of the pediatric tumor, neuroblastoma. This correlation suggests that TrkA may actively promote growth arrest and differentiation of neuroblastoma tumor cells and may be an important therapeutic target in the treatment of this disease. In the present study, we have examined the mechanistic basis for TrkA gene expression in human neuroblastoma cells. Northern blotting and nuclear run-on analyses demonstrated that transcription is a primary determinant of both cell-specific and variable expression of the TrkA gene in neuroblastoma cell lines that express it to different degrees. Cell-specific and variable transcription in neuroblastoma cells was recapitulated by transient transfection of TrkA promoter-luciferase reporter constructs, and regulatory sequences mediating these processes were localized to a 138-base pair region lying just upstream of the transcription initiation region. This neuroblastoma regulatory region formed multiple DNA-protein complexes in gel shift assays that were highly enriched in neuroblastoma cells exhibiting abundant TrkA expression. Thus, TrkA-positive neuroblastoma cells are distinguished by differential expression of putative transcription factors that ultimately may serve as targets for up-regulating TrkA expression in tumors with poor prognosis.

Epidermal growth factor secretion by submandibular glands is not perturbed in the early phase of streptozotocin-induced diabetes in mice.

In rodents, diabetes mellitus is accompanied by a decreased concentration of epidermal growth factor (EGF) in plasma and urine and by a reduced number of EGF receptors on the surface of target cells. A combination of these two abnormalities may reduce the effects of EGF and could be implicated in some complications of diabetes. The aim of the present work was to investigate the possible implication of the major source of EGF in the organism, the submandibular glands, upon the reduced EGF concentration in blood and urine. Firstly, we measured the content of mice submandibular gland EGF by radioimmunoassay and by morphometric determinations of the relative volume occupied by granular convoluted tubules in the gland and by the EGF-containing granules in the cells at the light and electron microscopical levels respectively. We found no differences in the EGF content of submandibular glands of streptozotocin-treated diabetic animals compared to control ones. Secondly, we estimated stimulus-secretion coupling by EGF-secreting cells, present in an enriched preparation of granular convoluted tubules (GCT) perifused in thermoregulated chambers. Phenylephrine was the only agent tested that was capable of stimulating EGF secretion. The stimulation was dose-dependent and similar in streptozotocin-diabetic and control mice. Thus, the EGF deficiency observed in diabetic mice is related neither to a defect of EGF content in the submandibular glands nor to an abnormal EGF secretion by the glands.

Epidermal growth factor receptor internalization and biosynthesis in the diabetic rat.

The number of surface EGF receptors as well as their internalization rate and biosynthesis were analyzed in hepatocytes freshly isolated from control, streptozotocin-diabetic, and insulin-treated diabetic rats. All three parameters were decreased in diabetic animals and values were corrected by insulin treatment. Moreover, the inhibition of synthesis was specific for the EGF receptor since the other biosynthetically labeled proteins were not affected. These data demonstrate that the reduced number of hepatocyte surface EGF receptors results from an inhibition of EGF-receptor synthesis which is not compensated by a reduced internalization rate.

The relationship of ligand receptor mobility to internalization of polypeptide hormones and growth factors.

Polypeptide hormones and growth factors bind to cell surface receptors and are internalized by receptor-mediated endocytosis. Both [125I]insulin and [125I]epidermal growth factor (EGF) are internalized to a much greater extent than [125I]glucagon in freshly isolated rat hepatocytes. All three ligands bind initially and preferentially to the microvillous surface of the hepatocyte, but only [125I]insulin and [125I]EGF undergo significant redistribution to the nonvillous surface of the cell. Thus, the degree of lateral mobility of the ligand receptor complex is strongly correlated with the extent of internalization of the ligand. Since the beta-subunit of the insulin and the EGF receptors span the plasma membrane only once and both receptors are autophosphorylated, it is possible that these are important determinants of the receptor mobility.

Effect of pinealectomy on liver insulin and glucagon receptor concentrations in the rat.

The studies described here were undertaken to characterize the hepatic insulin and glucagon receptors of control (C), pinealectomized (Pn), and melatonin-treated pinealectomized (Pn + Mel) rats. Compared with C rats, an increase in plasma glucose and glucagon levels and a reduction in circulating concentrations of insulin in Pn animals were observed. Melatonin treatment of Pn rats reverses all three parameters toward the normal values. In liver membranes, insulin binding was lower in Pn than in C rats, and glucagon binding was greater in Pn than in C animals; in Pn + Mel rats both insulin and glucagon binding reverse toward the normal values that were observed in C rats. The modifications in hormone binding reflect changes in the number of receptors but not in the affinity constants. The time courses of hormone association and dissociation from liver membranes were similar in all three experimental groups. The degradation of both hormones by liver membranes was similar in all three groups. Insulin receptor degradation also was similar in the three groups, while glucagon receptor degradation was similar in the liver membranes of C and Pn rats but smaller in Pn + Mel animals. These findings suggest that the pineal gland may modulate the circulating levels and liver receptor concentrations of insulin and glucagon. In addition, our results indicate that insulin and glucagon did not induce a down-regulation of liver receptors in Pn rats.

Effect of pinealectomy and of diabetes on liver insulin and glucagon receptor concentrations in the rat.

The studies described in this paper were undertaken to characterize the circulating and hepatic insulin and glucagon receptor concentrations of control (C), diabetic (Db), and pinealectomized-diabetic (Pn + Db) rats. Compared with C rats, an increase in plasma glucose and glucagon levels and a reduction in circulating insulin concentrations in Db animals was observed; these differences were greater in Pn + Db rats. In liver membranes, insulin binding was lower in Db and in Pn + Db than in C rats, and glucagon binding was greater in Db and in Pn + Db than in C rats. The modifications in hormone binding did not reflect changes in the affinity constants. The time courses of hormone association and dissociation from liver membranes were similar in all three experimental groups. The degradation of both hormones and their receptors was similar in all three groups. These findings indicate that in either pinealectomized-diabetic or diabetic rats there were significant changes in the circulating and liver insulin and glucagon receptor concentrations and that the changes in the circulating levels of both pancreatic hormones were more pronounced in pinealectomized-diabetic animals. In addition, the absence in Db and in Pn + Db rats of the insulin and glucagon down-regulation of their own receptors could further modify metabolic activities in these animals.

Ligand-induced changes in insulin receptors in cell surface and Golgi fractions of fetal rat liver.

The studies described in this paper were undertaken to characterize the insulin receptors present in the plasma membranes and Golgi fractions of fetal rat liver and to determine their subcellular distribution after the administration of exogenous insulin. Purification patterns of both types of liver membranes from fetal and adult rats were similar, as verified by morphological and biochemical approaches. In both groups insulin binding was significantly greater in plasma membranes than in Golgi fractions. However, in plasma membranes insulin binding was similar in both groups, while in Golgi fractions it was greater in fetal than in adult rats, although the difference was not statistically significant. The modifications in insulin binding reflect changes in the number of receptors, but not in the affinity constants. The time courses of insulin association and dissociation from liver membranes were unaffected by development. Degradation of insulin by liver membranes was lower in fetal than in adult rats, although this does not seem to be responsible for the differences observed in binding. No significant differences in the degradation of insulin receptors between different groups of liver membranes were found. The effects of a single injection of insulin on the subcellular distribution of insulin receptors in liver were examined. Insulin administration to adult rats resulted in a marked decrease in insulin binding in liver plasma membranes but a significant increase in Golgi fractions, occurring within 1.5 min. By contrast, in 21-day-old fetuses insulin injection slightly increased insulin binding to liver plasma membranes at 15 min, while in Golgi fractions an increase in insulin binding was only observed 30 min after insulin injection. These findings suggest that the slow ligand-induced translocation of the insulin receptor from the cell surface to Golgi fractions in the fetus might explain the absence of insulin receptor down-regulation in fetal hepatocytes.

Characterization of glucagon receptors in Golgi fractions of fetal rat liver.

The present study was designed to determine if Golgi fractions from fetal rat liver contain glucagon receptors and to characterize the properties of such receptors. Purification patterns of liver plasma membranes and Golgi fractions from fetal and adult rats were similar, as verified by morphological and biochemical approaches. Glucagon binding was greater in plasma membranes of adult than fetal rats, while in Golgi fractions glucagon binding was similar in both groups. The modifications in in glucagon binding reflect changes in glucagon receptors. Glucagon association and glucagon receptor inactivation by liver membranes were similar in the two groups of animals, while glucagon degradation was lower in fetal than in adult rats.

Characterization of glucagon receptors in liver membranes and isolated hepatocytes during rat ontogenic development.

We studied the functional properties of hepatic glucagon receptors during rat development. Glucagon binding to liver membranes and isolated hepatocytes was significantly less in foetuses and weaning rats than in adult animals, reflecting changes in the number of receptors rather than any change in receptor affinity for the hormone. After correcting for the smaller surface area of foetal hepatocytes, glucagon receptor concentrations were still lower in foetuses than in adult rats. The time courses of glucagon association to liver membranes and of glucagon receptor degradation of prenatal and postnatal rats were similar, while inactivation of glucagon by liver membranes was significantly lower in foetal than in adult rats. Also, glucagon-stimulated production of cAMP was smaller in younger rats. These findings suggest that, according to several criteria, glucagon receptors in the foetus are functionally normal and their delayed development may be important for the metabolic processes occurring during the perinatal age.

Glucagon-like peptide-1 does not have a role in hepatic carbohydrate metabolism.

Glucagon-like peptide-1 does not have specific, high-affinity receptors on rat liver membranes, does not displace glucagon from glucagon receptors on these membranes and does not stimulate the production of cyclic AMP by isolated rat hepatocytes. In the presence of glucagon, high concentrations of glucagon-like peptide-1 do not significantly alter the production of cyclic AMP. Thus, glucagon-like peptide-1 appears unlikely to have a direct action on hepatic carbohydrate metabolism.