guanosine-diphosphate and Pheochromocytoma

Two major isoforms of angiotensin II receptors, AT1 and AT2, have been defined on the basis of their ligand selectivity. While AT1 is known to mediate typical biological actions of angiotensin II as a cardiovascular regulator, the biological function of AT2 has not yet been established. In the present study using a rat pheochromocytoma cell line, which expresses AT2 exclusively, we found that angiotensin II inhibits phosphotyrosine phosphatase activity in vivo as measured by the inhibition of hydrolysis of [32P]-phosphate from the 32P-labeled synthetic peptide substrate, Raytide. This phosphotyrosine phosphatase inhibition was completely reversed by pertussis toxin, which indicates a G-protein coupled mechanism. In SDS-polyacrylamide gel electrophoresis we found that the phosphotyrosine group of an 85 kDa protein was a substrate mainly preserved, presumably as a consequence of the plausible intracellular phosphotyrosine phosphatase inhibition by angiotensin II.

Topics: Adrenal Gland Neoplasms; Angiotensin II; Angiotensin Receptor Antagonists; Animals; Biphenyl Compounds; Cell Membrane; Chromatography, Affinity; Guanosine Diphosphate; Imidazoles; Losartan; Oligopeptides; PC12 Cells; Pertussis Toxin; Pheochromocytoma; Phosphates; Phosphoproteins; Phosphorus Radioisotopes; Phosphotyrosine; Protein Tyrosine Phosphatases; Pyridines; Rats; Receptors, Angiotensin; Tetrazoles; Thionucleotides; Tyrosine; Virulence Factors, Bordetella

To determine the amino acid residues required for the signal-transducing activity of the human c-Ha-Ras protein, we introduced point mutations at residues 45-54 near the 'effector region' (residues 32-40). We transfected PC12 cells with these mutant genes and also micro-injected the mutant proteins, bound with an unhydrolyzable GTP analog, into PC12 cells. Both procedures showed that Val45----Glu and Gly48----Cys mutations impaired the ability of the Ras protein to induce morphological change of PC12 cells. These mutations did not affect the guanine nucleotide-binding activity or GTPase activity in the absence or presence of bovine GTPase-activating protein (GAP). Therefore, the Val45 and Gly48 residues should be included by definition in the effector region responsible for the signal transduction, while only a subset of the effector-region residues is required for enhancement of the GTPase activity by GAP.

Topics: Adrenal Gland Neoplasms; Amino Acids; Genes, ras; GTP Phosphohydrolases; GTPase-Activating Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Mutagenesis, Site-Directed; Pheochromocytoma; Proteins; Proto-Oncogene Proteins p21(ras); ras GTPase-Activating Proteins; Signal Transduction; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured

The ras gene product (p21) specifically binds GDP or GTP. In analogy with the reaction mechanism of other GTP-binding proteins, only the GTP-bound conformation is believed to be the biologically active one. Previously, we reported that not only oncogenic p21(Val-12) but also proto-oncogenic p21(Gly-12) could induce morphological differentiation in rat pheochromocytoma PC12 cells when microinjected in the complexed form with GTP gamma S [(1987) Mol. Cell. Biol. 7, 4553-4556]. In the present report we transformed PC12 cells with the oncogenic ras gene placed under the metallothionein I promoter. It was found that the transformed cells, when induced with Cd2+, differentiated in the absence of NGF. Then we analyzed the guanine nucleotide bound to p21 in the intact PC12 cells. It was found that conditionally induced p21(Val-12) was mostly present in the GTP-bound form, whereas the endogenous p21(Gly-12) was in the GDP-bound form. These results indicate again that p21.GTP induces the morphological differentiation of PC12 cells.

Topics: Animals; Cell Line, Transformed; Guanine Nucleotides; Guanosine Diphosphate; Guanosine Triphosphate; Immunoassay; Pheochromocytoma; Plasmids; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; Transfection; Tumor Cells, Cultured

Rat pheochromocytoma (PC12) cells differentiate to neuronal cells in response to nerve growth factor. It has been shown that microinjection of oncogenic but not proto-oncogenic p21 protein induces morphological differentiation in PC12 cells (D. Bar-Sagi and J. R. Feramisco, Cell 42:841-848, 1985). In this paper we describe a recombinant human proto-oncogenic Ha-ras protein which can effectively induce neurite extension of PC12 cells when microinjected as a complex with guanosine-5'-O-(3-thiotriphosphate). The protein was found to be less effective when complexed with GTP. On the other hand, an oncogenic ras protein coinjected with guanosine-5'-O-(2-thiodiphosphate) was entirely inactive. These results indicate that the binary p21-GTP complex, but not the p21-GDP complex, is effective in inducing differentiation in PC12 cells, irrespective of the oncogenic or the proto-oncogenic protein.

Topics: Adrenal Gland Neoplasms; Animals; Axons; Cell Differentiation; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Microinjections; Pheochromocytoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins p21(ras); Rats; Recombinant Proteins; Thionucleotides; Tumor Cells, Cultured

The amount and distribution of brown adipose tissue (BAT) in healthy adult human subjects remain uncertain. BAT has been described in patients with pheochromocytoma. This pathological situation was chosen to try to characterize human BAT using both morphological and biochemical criteria. Typical multilocular brown adipocytes were observed; the cytoplasm of these adipocytes was filled with mitochondria with numerous tightly packed cristae, as in rat or hamster BAT. Isolated mitochondria exhibited a loose respiratory coupling sensitive to guanosine diphosphate; the chloride conductance of these mitochondria was reduced by the addition of this nucleotide. It is concluded that in patients with pheochromocytoma, there is a large amount of a tissue around the adrenal glands and kidneys which fullfills the ultrastructural and biochemical criteria for a BAT.

Topics: Adipose Tissue, Brown; Adrenal Gland Neoplasms; Adult; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Female; Guanosine Diphosphate; Humans; Middle Aged; Mitochondria; Oxygen Consumption; Pheochromocytoma