Mechanism of action of STAMP - a new comodulator of glucocorticoid receptors

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The four aims of this project are 1) to determine whether the modulatory actions of STAMP are limited to GRs and ARs, 2) to define the interaction surfaces that are used in the interactions of GR, TIF2, and STAMP, 3) to identify the domains of each factor that are needed for the observed modulation of EC50, Amax, and percent partial agonist activity (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485), and 4) to search for other activities of STAMP. With regard to the first aim, progesterone receptors (PRs) interact with STAMP in a mammalian two-hybrid assay (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). Therefore, it was not unexpected that PR induction properties (Amax, percent partial agonist activity, and EC50) are similarly modulated by STAMP, albeit to a slightly lower magnitude than GR under the same conditions. However, different STAMP sequences are important for maximal interaction with PR vs. GR., which suggests that more significant differences in the responses of PR and GR to STAMP may be elicited under other conditions. Aims 2 and 3 are being assessed by quantifying the ability of different deletions in GR, TIF2, and STAMP, both separately and in combination, to retain the modulatory activity for EC50, percent partial agonist activity, and Amax. ChiP-reChIP (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485) and co-IP experiments support a model where GR, TIF2, and STAMP act via a complex containing all three factors. Preliminary data indicate that different regions of STAMP are required for the optimal modulation of each induction property with GR. Furthermore, significant portions of GR, TIF2, and STAMP can be deleted without major changes in the final modulatory activities. Finally, Aim 4 seeks to determine whether STAMP mediates any other actions. The only identifiable domain in STAMP is a tyrosine tubulin ligase-like (TTL) domain, which is not required for STAMP modulatory activity (He and Simons Jr., 2007, Mol. Cell. Biol., 27, 1467-1485). However, it was recently reported that an overlapping region in TTLL family proteins has polyglutamylase activity (van Dijk et al., 2007, Mol Cell, 26, 437-448). We have confirmed that STAMP (TTLL5) has polyglutamylase activity and recognizes TIF2 in addition to tubulin. The level of TIF2 polyglutamylation is very low, though, and unrelated to the modulatory activity of STAMP. Preliminary data indicate that STAMP also affects the growth behavior of cells in a cell-selective manner. In summary, the new cofactor (STAMP) that acts in an additive, and sometimes synergistic, manner with the coactivator TIF2 to alter several properties of PR-regulated gene expression. Thus STAMP modulates the transcriptional properties of AR, GR, and PR. As a result of our on-going studies, we have gained new molecular information about the modulation of the dose-response curve and total activity of agonists and the percent partial agonist activity of antisteroids by cofactors. These modulatory factors permit a continuum of responses and constitute new therapeutic targets for differential control of gene expression by steroid hormones during development, differentiation, homeostasis, and endocrine therapies. These combined findings contribute to our long-term goal of defining the action of steroid hormones at a molecular level and of understanding their role in human physiology.