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The amyloid precursor protein (APP) is the source of Abeta fragments implicated in the formation of senile plaques in Alzheimer's disease (AD). APP-related proteins are also expressed at high levels in the embryonic nervous system and may serve a variety of developmental functions, including the regulation of neuronal migration. To investigate this issue, we have cloned an orthologue of APP (msAPPL) from the moth, Manduca sexta, a preparation that permits in vivo manipulations of an identified set of migratory neurons (EP cells) within the developing enteric nervous system. Previously, we found that EP cell migration is regulated by the heterotrimeric G protein Goalpha: when activated by unknown receptors, Goalpha induces the onset of Ca2+ spiking in these neurons, which in turn down-regulates neuronal motility. We have now shown that msAPPL is first expressed by the EP cells shortly before the onset of migration and that this protein undergoes a sequence of trafficking, processing, and glycosylation events that correspond to discrete phases of neuronal migration and differentiation. We also show that msAPPL interacts with Goalpha in the EP cells, suggesting that msAPPL may serve as a novel G-protein-coupled receptor capable of modulating specific aspects of migration via Goalpha-dependent signal transduction.

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp6Ov-rc. In this mutant, lysine-295 is replaced with methionine. SF2 pp6ov-src was found to have a half-life similar to that of wild-type pp6Ov-c and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp6Ov-src were morphologically untransformed and do not form tumors. The SF2 pp6Ov-S isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp6Ov-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp6OVS-was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp6Ov-sr itself. By contrast, SF2 pp6Ov-srC could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp6Ov-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp6O-src elicits an allosteric change required for phosphorylation of serine in the protein.The tumorigenicity and in vitro transforming ability of Rous sarcoma virus (RSV) reside in pp60v-src (4), the protein encoded by the viral oncogene v-src (3, 33, 48). pp60v-src has tyrosine-specific protein kinase activity (10,11,21,26,27,36) and is itself phosphorylated, both on serine-17 (and, to a lesser degree, on at least one other serine) in the aminoterminal half of the protein (9, 16) and on tyrosine-416 (32, 41). Recent evidence has also demonstrated the existence of phosphotyrosines in the amino-terminal half of the protein that may play a role in regulation of enzymatic activity (7,12,34).Evidence from studies on mutants of v-src suggests that pp60v-src may have as-yet-undiscovered functions independent of the kinase activity which are necessary for full expression of transformation (5, 13, 45). To examine this possibility more closely, it would be desirable to identify residues involved in kinase activity and then mutate them to eliminate this activity while preserving other possible functions of pp6Ov-src. Both indirect (2) and direct (23) evidence now exists that lysine-295 is intimately involved with the ATP binding of pp60v-src. We wanted to obtain a mutation at this site that would eliminate tyrosine kinase activity without altering overall structure or other active functions. In this paper, we report on the construction and expression of such a mutant, which we call RSV-SF2, and the effect of this mutation on the structure and activity of pp6Ov-src and on the expression of some parameters of transformation. Site-specific mutagenesis. Procedures for site-specific mutagenesis on M13 and selection of mutated DNA have been described earlier (43). The 19-base oligonucleotide used for converting lysine-295 of v-src to me...

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Evaluation and optimization of drug metabolism and pharmacokinetic data plays an important role in drug discovery and development and several reliable in vitro ADME models are available. Recently higher throughput in vitro ADME screening facilities have been established in order to be able to evaluate an appreciable fraction of synthesized compounds. The ADME screening process can be dissected in five distinct steps: (1) plate management of compounds in need of in vitro ADME data, (2) optimization of the MS/MS method for the compounds, (3) in vitro ADME experiments and sample clean up, (4) collection and reduction of the raw LC-MS/MS data and (5) archival of the processed ADME data. All steps will be described in detail and the value of the data on drug discovery projects will be discussed as well. Finally, in vitro ADME screening can generate large quantities of data obtained under identical conditions to allow building of reliable in silico models.