A.
SPECIFIC AIMS The TGF- signaling system is involved in many developmental and physiological processes, including earlyembryogenesis, wound healing, inflammation, immunity and reproductive function. TGF- ligands form homoorheterodimers that interact with type I and type II receptor kinases, to form signaling complexes that influencecellular activation and cell fate decisions. In addition, extracellular regulators, such as follistatin, block TGF- signaling by interfering with ligand receptor interactions. While over 50 different TGF- ligands have beenidentified, these signal through a handful of type I (7) and type II (5) receptors and our quantitativeunderstanding of how various receptorligand combinations are assembled and linked to differentialphysiological outputs remains incomplete.Several members of the TGF- family, including activins, GDF-9 and BMP-15, are critically importantfor normal reproductive function of the ovary, influencing key steps in oocyte development and release. GDF-9and BMP-15 form a unique subset of the ligand superfamily, playing relatively specialized roles in ovariandevelopment that contrast with the more widespread use of other TGF- ligands. Both GDF-9 and BMP-15mutations have been linked to aberrant reproductive function, resulting in either significantly increased fertilityor complete infertility. By virtue of their specialized usage within the reproductive axis and their central role inregulating ovarian follicle development, both GDF-9 and BMP-15 are attractive targets for potential therapeuticintervention and for use in the in vitro manipulation of follicle development. It remains to be established howGDF-9 and BMP-15 interact with their type I and type II receptors and how this may be regulated. Theextracellular antagonist follistatin, which is a key modulator of activin signaling, may also play a role in theregulation of other TGF- ligands present during ovarian development. Follistatin can interact with a broadrange of ligands, such as activins, BMPs and myostatin, and the mechanism by which it can engage suchdifferent ligands remains to be investigated. Follistatin exists in multiple isoforms that exhibit differentinteractions with heparan sulfate and TGF- ligands. We have previously focused on the interaction of activinA with its receptors and with follistatin, obtaining both structural and biochemical insights into this portion of theTGF- signaling system that is operative in the ovary. In order to gain a deeper understanding into the broadernetwork of TGF- signaling pathways that are critical to normal ovarian development and physiology, and howthese may be regulated by follistatin, we propose the following specific aims:
Specific Aim 1 : Regulation of follistatin 315 ligand interactions by its C-terminal region.
Sub aim 1. 1 Test the functional role of follistatin D3 and the D2-D3 groove in follistatin 315.
Sub aim 1. 2 Determine if the follistatin 315 C-terminal tail affects the solution conformation of follistatin,thereby reducing heparin and TGF- ligand binding affinity.
Sub aim 1. 3 Determine the structures of the free FS288 and FS315 isoforms by X-ray crystallography.
Specific Aim 2 : Quantitative studies of follistatin interactions with activins, GDF-9 and BMP-15.
Sub aim 2. 1 Measure the binding affinities of follistatin for TGF- ligands important in ovariandevelopment.
Sub aim 2. 2 Determine the role of the follistatin N-domain in binding to BMP-15, GDF-9 and other BMPmolecules.
Sub aim 2. 3 Establish whether TGF- ligand and heparin binding are coupled for BMP ligands similarlyto activin A.
Sub aim 2. 4 Provide a structural basis for understanding follistatin recognition of different TGF- ligandsthat use different sets of type I and type II receptorsSpecific Aim 3: Specificity and affinity of activins, GDF-9 and BMP-15 for type I and II signalingreceptors.
Sub aim 3. 1 Comparative analysis of activin, GDF-9 and BMP-15 interactions with ActRIIB and BMPRIIreceptors.
Sub aim 3. 2 Investigation of activin, GDF-9 and BMP-15 interactions with Alk4, Alk5, Alk6 and Alk7 typeI receptors.
Sub aim 3. 3 Structural studies of GDF-9 and BMP-15 proteins alone and in receptor-bound complexes.
Showing the most recent 10 out of 63 publications
