Pulling strings below the surface: hormone receptor signaling through inhibition of protein tyrosine phosphatases.

Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptor's tyrosine residues. At any given moment, the receptor's phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.

Identification of tyrosine phosphatases that dephosphorylate the insulin receptor. A brute force approach based on "substrate-trapping" mutants.

Many pharmacologically important receptors, including all cytokine receptors, signal via tyrosine (auto)phosphorylation, followed by resetting to their original state through the action of protein tyrosine phosphatases (PTPs). Establishing the specificity of PTPs for receptor substrates is critical both for understanding how signaling is regulated and for the development of specific PTP inhibitors that act as ligand mimetics. We have set up a systematic approach for finding PTPs that are specific for a receptor and have validated this approach with the insulin receptor kinase. We have tested nearly all known human PTPs (45) in a membrane binding assay, using "substrate-trapping" PTP mutants. These results, combined with secondary dephosphorylation tests, confirm and extend earlier findings that PTP-1b and T-cell PTP are physiological enzymes for the insulin receptor kinase. We demonstrate that this approach can rapidly reduce the number of PTPs that have a particular receptor or other phosphoprotein as their substrate.

Highly controlled gene expression using combinations of a tissue-specific promoter, recombinant adenovirus and a tetracycline-regulatable transcription factor.

Controllable gene expression is a desirable feature both in gene therapy protocols and for the study of gene function in animals and plants. We have exploited the modular character of the tetracycline (tc)-regulatable genetic switch to show that its components can be encoded by any combination of recombinant adenovirus and/or transgenic mice. Transgenic mice were constructed that express the tc-regulatable trans-activator tTA muscle specifically. These were injected with recombinant adenovirus expressing a luciferase reporter controlled by the tTA-regulatable promoter. Virus injected into muscle, but not into a control organ (brain) resulted in luciferase activity. Conversely, injection of tTA producing adenovirus into mice that were transgenic for a trkB/Fc fusion protein gene under tc promoter control resulted in swift expression of serum trkB/Fc receptor-body. Both modes of gene induction were fully inhibited by administration of tc. We demonstrate that a careful choice of these tools allows exquisite in vivo control over transgene expression in a temporal, tc-regulatable, topical and tissue-specific manner.