Ligand-receptor interactions in the TGF-beta superfamily

说明(申请人提供)：转化生长因子�S和转化生长因子�超家族的其他结构相关蛋白，如骨形态发生蛋白、GDF和激活素，是分泌的信号蛋白，调节许多必要的过程，从胚胎发育期间身体计划的建立到适应性免疫系统的调节。30多个超家族成员的重要性被许多人类疾病所证明，这些疾病是由该途径的失调引起的，包括骨骼和心脏疾病、不孕不育和癌症。开发有效的治疗途径相关疾病的方法需要了解控制配体活动的相互作用--这包括与信号受体的相互作用，以及与许多结合配体和调节配体可及性的辅助蛋白的相互作用。这个项目的目标是利用我们目前的理解 因此，我们将研究转化生长因子�S如何与其信号受体结合，以研究膜锚定的辅助结合蛋白聚糖Betaglycan如何一方面促进转化生长因子�信号转导，另一方面抑制激活素信号转导。转化生长因子�信号被认为是由于其结合和定位转化生长因子�配体的能力而产生的。为了研究这一点，我们将使用结晶学方法确定转化生长因子�结合的两个独立的转化生长因子�结合域bge和bgu的结构，这对于这些目的是理想的，因为bge和bgu与转化生长因子�结合并形成高度稳定的络合物。这些化合物的结构所暗示的增强受体结合的机制将在功能分析中进行测试。β-多糖对激活素信号的抑制被认为是通过将激活素II型受体ActRII或接近的同系物ActRIIb隔离在与β-多糖和抑制素A形成的三元复合体中而实现的。抑制素A是一种异二聚体配体，其激活素�亚基与激活素�亚基结合在一起 它结合了BGU，即Betaglycan的C-末端结合域。为了研究这一点，我们将首先确定激活素如何与其I型受体ActRIb结合。这些研究将使用核磁共振进行，因为ActRIb与激活素A结合很弱。这将通过使用结晶学确定抑制素A与BGU结合的结构来进一步研究，这对于这些目的是理想的，因为BGU与抑制素A结合并形成高度稳定的络合物。根据络合物的结构所揭示的抑制素A拮抗激活素的机制将在功能分析中进行测试。通过对BGe：T�RII：转化生长因子�络合物的晶体衍射、分离BGUC：转化生长因子�络合物的能力以及ActRIb的核磁共振信号归属，证明了所提出的研究的可行性。这些结构的确定将为我们理解超家族的辅助蛋白之一--β-聚糖如何影响转化生长因子�-S和激活素诱导的下游信号传导提供一个重要的缺失环节。这些结构还将有助于开发新的靶向疗法，用于治疗由转化生长因子�和激活素信号异常引起的疾病和障碍，如癌症和不孕不育。

DESCRIPTION (provided by applicant): TGF�s and other structurally related proteins of the TGF� superfamily, such as BMPs, GDFs, and activins, are secreted signaling proteins that regulate many essential processes, ranging from establishment of the body plan during embryogenesis to regulation of the adaptive immune system. The importance of the more than 30 superfamily members is demonstrated by the many human diseases that result from dysregulation of the pathway, including skeletal and cardiac disorders, infertility, and cancer. The development of effective therapies for treating pathway-associated diseases requires knowledge of the interactions that govern ligand activities - this includes interactions with the signaling receptors, but as well with the many accessory proteins that bind ligands and regulate ligand accessibility. The objective of this project is to capitalize upon our current understanding of how TGF�s bind their signaling receptors to investigate how betaglycan, a membrane-anchored accessory binding proteolgycan, functions on the one hand to promote TGF� signaling, and on the other to inhibit activin signaling. The potentiation of TGF� signaling by betaglycan is thought to arise from its ability to bind and localize TGF� ligands on the cell surface. To investigate this, we will determine the structures of betaglycan's two independent TGF� binding domains, BGE and BGU, bound to TGF� using crystallography, which is ideal for these purposes, as BGE and BGU bind and form highly stable complexes with TGF�. The mechanisms for potentiation of receptor binding suggested by the structure of the complexes will be tested in functional assays. The inhibition of activin signaling by betaglycan is thought t be mediated by sequestration of the activin type II receptor, ActRII or the close homolog ActRIIb, in a ternary complex with betaglycan and inhibin A, a heterodimeric ligand with an activin � subunit that binds the activin type II receptor, ActRII/ActRIIb, and an activin � subunit that binds BGU, betaglycan's C-terminal binding domain. To investigate this, we will first determine how activins bind their type I receptor, ActRIb. These studies will be carried out using NMR as ActRIb binds activin A weakly. This will be further investigated by determining the structure of inhibin A bound to BGU using crystallography, which is ideal for these purposes as BGU binds and forms a highly stable complex with inhibin A. The mechanisms for antagonism of activin by inhibin A suggested by the structure of the complexes will be tested in functional assays. The feasibility of the proposed studies is demonstrated by diffracting crystals obtained for the BGE:T�RII:TGF� complex, the ability to isolate the BGUC:TGF� complex, and NMR signal assignments for ActRIb. The determination of these structures will provide an important missing link in our understanding of how one of the accessory proteins of the superfamily, betaglycan, influences downstream signaling induced by TGF�s and activin. The structures will also aid in the development of novel targeted-therapies for treatment of diseases and disorders, such as cancer and infertility, caused by aberrant TGF� and activin signaling.