Seeking the Physical Basis of Achondroplasia

描述（由申请人提供）：受体酪氨酸激酶（RTK）通过质膜中的侧向二聚化传导生化信号。RTKs的跨膜（TM）结构域在二聚化过程中起重要作用。RTK TM结构域中的单个氨基酸突变可引起细胞信号传导的缺陷并导致病理表型。例如，软骨发育不全，人类侏儒症的最常见形式，与一个RTK，成纤维细胞生长因子受体3（FGFR 3）的TM段中的单个氨基酸突变（Gly 380-to-Arg）有关，超过97%的所有研究病例。 在发现侏儒症的遗传原因九年后，我们提出了一个关于软骨发育不全的结构决定因素的可检验的假说。我们试图验证这一假设，并阐明软骨发育不全突变的结构和热力学后果。我们建议：（1）测定野生型和突变型TM结构域的二聚化倾向（2）使用定点诱变、FRET、NMR和分子建模确定野生型和突变型TM二聚体的结构，（3）确定Arg 380是否驻留在双层的烃核心内，或在其界面区域中，和（4）评估突变的TM结构域抑制成纤维细胞中未调节的FGFR 3信号传导的能力。 拟议的工作将（1）增强我们对RTK二聚化机制和一般质膜细胞信号传导机制的认识，（2）阐明软骨发育不全的分子基础，（3）为软骨发育不全的新治疗方案铺平道路。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane. The transmembrane (TM) domains of RTKs play an important role in the dimerization process. A single amino acid mutation in RTK TM domains can cause a defect in cell signaling and result in pathological phenotype. For instance, achondroplasia, the most common form of human dwarfism, is linked to a single amino acid mutation (Gly380-to-Arg) in the TM segment of one RTK, fibroblast growth factor receptor 3 (FGFR3) in more than 97% of all studied cases. Nine years after the discovery of the genetic cause of dwarfism, we have put forward a testable hypothesis for the structural determinants of achondroplasia. We seek to test this hypothesis, and elucidate the structural and thermodynamic consequences of the achondroplasia mutation. We propose to: (1) Determine the dimerization propensities for wild-type and mutant TM domains (TMwt and TMmut) in model systems using Fluorescence Resonance Energy Transfer (FRET), (2) Determine the structures of wild-type and mutant TM dimers using site-directed mutagenesis, FRET, NMR, and molecular modeling, (3) Determine whether Arg380 resides inside the hydrocarbon core of the bilayer, or in its interfacial region, and (4) Assess the ability of mutant TM domain to inhibit unregulated FGFR3 signaling in fibroblasts. The proposed work will (1) enhance our knowledge of the mechanism of dimerization of RTKs, and of cell-signaling across the plasma membrane in general, (2) shed light on the molecular basis of achondroplasia, and (3) pave the way for new treatment options for achondroplasia.