Kang J-H, Hassan S A, Zhao P, Tsai-Morris C H, Dufau M L
Section on Molecular Endocrinology, Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institutes of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-4510, USA.
Center for Molecular Modeling, Division of Computational Bioscience, Center for Information Technology, National Institutes of Health, Bethesda, MD 20892-4510, USA.
Biochim Biophys Acta. 2014 Jul;1840(7):2272-80. doi: 10.1016/j.bbagen.2014.04.006. Epub 2014 Apr 13.
Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin's diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function.
Studies were conducted using mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET) and molecular modeling approaches.
Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals an increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers.
These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function.
Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure-function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.
人催乳素受体（PRLR）的长形式（LF）同型二聚体介导催乳素的多种作用。短形式S1b通过异源二聚化抑制LF功能。乳腺癌中S1b/LF比值降低可能有助于肿瘤发展/进展。目前的工作确定了S1b的D1结构域对其抑制催乳素诱导的LF功能的结构和功能相关性。
使用诱变、启动子/信号分析、生物发光共振能量转移（BRET）和分子建模方法进行研究。
D1 S1b中的E69或靠近结合口袋（S）的受体表面相邻残基发生突变会导致其抑制作用丧失，而远离该区域（A）或D2结构域中的突变则表现出与野生型相同的抑制作用。所有S1b突变体均保留催乳素诱导的Jak2激活。BRET显示，在稳定表达LF的转染细胞中，D1突变的S1b（S）同型二聚体的亲和力增加。相比之下，具有D1（A）或D2突变的S1b同型二聚体的亲和力保持不变。这有利于催乳素诱导的LF介导的信号传导。分子动力学模拟表明，突变（S）引起主要的构象变化，该变化向下传播到D1/D2界面并改变其二聚体中的相对取向。
这些发现证明了D1对S1b结构的重要作用及其对催乳素诱导的LF介导功能的抑制作用。
D1关键区域的单个突变引发受体构象和二聚化亲和力的重大变化。我们的结构-功能/模拟研究为小分子建模和设计提供了基础，以增强对LF激活的抑制作用，潜在用于乳腺癌治疗。
