The two aims of this project are 1) to see if changes in Amax, EC50, and PAA with cofactors such as STAMP can be dissociated, and 2) to search for other activities of STAMP.
Aim 1 has been addressed using the ternary system of GR, the coactivator TIF2, and STAMP for two reasons. First, we previously established that TIF2 and STAMP augment the Amax and PAA, and decrease the EC50, of GR-mediated gene expression in a more than additive manner. Second, the number of permutations of potential biologically active protein surfaces is much higher with three than two factors, thereby increasing the possible combinations that could be involved in selective gene transcription. Using three differently sized fragments of each of three factors (GR, TIF2, and STAMP) in whole cell bioassays, we found that different regions of each protein can selectively alter none, some, or all three parameters (Amax, EC50, and PAA). These results reveal new potential targets for pharmacological manipulation of endocrine therapies. Equally important is the demonstration that conventional studies of just the Amax will observe only a subset of those factors involved in GR-regulated gene expression and will miss those agents that modify the EC50 and/or PAA without affecting the Amax.
Aim 2 has been pursued by preparing the STAMP KO mice in a homogeneous genetic background. STAMP is a transcriptional cofactor that augments the activity of the p160 coactivators SRC-1 and TIF2 in glucocorticoid receptor-regulated gene induction and repression. However, STAMP has also been described by others as a tyrosine tubulin ligase-like family member (TTLL5) with polyglutamylation activity. Furthermore, we previously reported steroid-independent activity of STAMP regarding the growth of several cell types (He et al., 2010, BMC Cancer, 10, 128). To determine STAMP functions in whole animals, mice containing a disrupted Stamp gene, resulting in premature termination of translation in the middle of the protein, were prepared. Homozygous targeted mutant (Stamptm/tm) females appear normal. Stamptm/tm males also seem normal except that they are almost always sterile due to a unique combination of sperm maturation and motility defects involving disruption of sperm tail axonemes that is associated with reduced tubulin polyglutamylation. The axonemes in other structures appear unaffected. There is no obvious change in the organs for sperm development of wt vs. Stamptm/tm males despite the levels of wt STAMP mRNA in the testes being 20-fold higher than in any other organ examined. This defect in male fertility is unrelated to any of the 24 genes previously identified as important for sperm function. Thus, STAMP appears to participate in a unique pathway of sperm maturation and motility that may be relevant for male fertility. In summary, we are using our recently prepared STAMP KO mice to look for phenotypic defects of KO animals. Detailed studies of the action of the ternary complex of GR/TIF2/STAMP have established that the three parameters of GR-regulated induction (EC50, PAA, and Amax) are not coordinately regulated. Instead, different protein surfaces of the three proteins can selectively modulate individual parameters, which indicates that pharmaceuticals may exist that can alter the activities of one, two, or all three parameters. 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.
|Lee, Geun-Shik; He, Yuanzheng; Dougherty, Edward J et al. (2013) Disruption of Ttll5/stamp gene (tubulin tyrosine ligase-like protein 5/SRC-1 and TIF2-associated modulatory protein gene) in male mice causes sperm malformation and infertility. J Biol Chem 288:15167-80|
|Awasthi, Smita; Simons Jr, S Stoney (2012) Separate regions of glucocorticoid receptor, coactivator TIF2, and comodulator STAMP modify different parameters of glucocorticoid-mediated gene induction. Mol Cell Endocrinol 355:121-34|
|Simons Jr, S Stoney; Chow, Carson C (2012) The road less traveled: new views of steroid receptor action from the path of dose-response curves. Mol Cell Endocrinol 348:373-82|
|He, Yuanzheng; Blackford Jr, John A; Kohn, Elise C et al. (2010) STAMP alters the growth of transformed and ovarian cancer cells. BMC Cancer 10:128|