Protein tyrosine phosphorylation is a potential mechanism for initial sinaling in PC12 cells during dfferentiation in response to nerve growth factor (NGF). NGF-induced tyrosine phosphorylation has been found to be initiated by the trk protooncogene, which participates in the formation of highaffinity NGF binding sites. In contrast to transfection of wildtype low-affinity p75 NGF receptors, transfection of p75NGm with mutations in the cytoplasmic domain resulted in an inability of NGF to elicit tyrosine phosphorylation of intracellular substrates, indicating that p75NG is involved in initiating pbosphorylation events by NGF. Even though the p75NGFR receptor does not possess any inherent tyrosne kinase activity, these experiments demonstrate that the p75NGFR has a potential role in NGF-induced tyrosine phosphorylation.Protein tyrosine phosphorylation is initiated by growth factor binding to receptors for insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), all of which possess intrinsic tyrosine kinase activity (1). Nerve growth factor (NGF) interacts with a 75-kDa receptor (2) that lacks a cytoplasmic tyrosine kinase domain (3,4), yet rapid and transient protein tyrosine phosphorylation has been observed upon treatment of PC12 cells with NGF (5-8). The kinase responsible for this activity has been identified as the trk protooncogene, a receptor tyrosine kinase that binds NGF and is activated in PC12 cells, neuroblastoma cells, and embryonic sensory neurons (8,9,38 Constructs. Construction of the deletion mutants of the NGF receptor has been described (15). All cDNAs were subcdoned into the EcoRI site of the pMV7 expression vector and recombinant retroviruses produced as described (18). Retroviral Gene Transfer. Cell lines expressing the wildtype and mutant NGF receptor cDNAs were generated after retroviral infection of helper-free virus stocks. NR18 transfectants were isolated after neomycin (0.5 mg of G418 per ml; GIBCO) selection and purification by rosetting with the monoclonal antibody ME20.4 and rabbit anti-mouse IgGcoupled human erythrocytes (2,3,15).Immunoblotting. Cells were grown to near confluence on 60-mm tissue culture dishes and were serum starved for 1 hr by incubation in serum-free DMEM. The medium was then replaced with DMEM containing 50 ng of NGF per ml or 20 ng of EGF per ml (Bioproducts for Science, Indianapolis). At the indicated times the cells were rapidly rinsed with ice-cold Tris-buffered saline, solubilized in 120 ,l of sol buffer [10 mM Tris hydrochloride, pH 7.4/1% sodium dodecyl sulfate (SDS)/1 mM sodium orthovanadate/0.1 mM sodium molybdate/1 mM phenylmethylsulfonyl fluoride/5 ,ug of aprotinin per ml/5 ,ug of leupeptin per ml], and immediately frozen in dry ice and ethanol. The samples were then boiled for 5 min. Protein concentrations were determined with a protein assay kit (Bio-Rad). Proteins (150 ,ug) were separated by SDS/ PAGE on 6-13% linear gradient polyacrylamide gels (unless otherwise noted) and electro...

Evidence is presented for hormone-controlled adenosine 3',5'-cyclic monophosphate (cAMP)-mediated NaCl diuresis in Malpighian tubules of the blood-feeding yellow-fever mosquito Aedes aegypti. Studies in isolated Malpighian tubules reveal that cAMP added to the peritubular bath selectively stimulates NaCl secretion and not KCl secretion by increasing the Na conductance of the basolateral membrane of primary cells. These effects are duplicated by forskolin and theophylline in parallel with increased intracellular concentrations of endogenous cAMP. Two natriuretic peptides that we have isolated by high-pressure liquid chromatography (HPLC) methods from mosquito heads also increase NaCl and fluid secretion in isolated Malpighian tubules together with increased intracellular levels of cAMP. These results are consistent with a mechanism of NaCl diuresis in which the natriuretic peptides and cAMP are respectively the primary and secondary messengers that couple the ingestion of a blood meal to the excretion of the unwanted salt and water fraction of the meal. This hypothesis is supported by in vivo studies that reveal elevated intracellular cAMP levels in Malpighian tubules at the time of maximum NaCl diuresis.

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Beta-amyloid1-42 (Abeta) is a naturally occuring peptide whose accumulation in the brain is putatively coupled to Alzheimer's disease pathogenesis. Deleterious effects of Abeta on neurons have been linked to the inappropriate activation of signaling pathways within the cell (reviewed in Yankner, 1996), including tyrosine phosphorylation of focal adhesion kinase (FAK) (Zhang et al., 1994, 1996a,b). Here we have investigated the effects of Abeta on paxillin in a neural cell line. Paxillin, a substrate for FAK, is thought to act as a signal "integrator," functioning to link other proteins into multi-molecular signaling complexes (reviewed in Turner, 1994). Treatment of the rat central nervous system B103 cell line with aggregates of Abeta was found to induce the tyrosine phosphorylation of paxillin within 30 min, nearly 24 hr prior to significant cell death. Particularly striking was a subsequent "mobilization" of paxillin to the cytoskeleton in Abeta-treated cells. The amount of paxillin associated with the cytoskeleton in Abeta-treated cells was increased 10-fold over controls. The Abeta-induced paxillin accumulation could be visualized immunocytochemically, with an increase in number and size of paxillin-labeled focal contacts upon treatment with Abeta. This effect was specific, in that vinculin, another focal contact protein, was unaffected by Abeta. Disruption of f-actin, which inhibits both Abeta-induced neurotoxicity (Furukawa and Mattson, 1995) and focal contact signaling in B103 cells (Zhang et al., 1996b) was found to block the cytoskeletal paxillin accumulation. The rapid tyrosine phosphorylation and cytoskeletal mobilization of paxillin links Abeta to the activation of focal contact signaling events that may influence neuronal cytoskeletal architecture, gene expression, synaptic plasticity and cell viability.

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