Embryonic lethality and defective mammary gland development in activator function impaired conditional knock-in ErbB3V943R mice

ERBB3, a pseudo-kinase domain containing human epidermal growth factor receptor (ERBB/HER) family member, plays a key role in multiple cellular processes and is required for normal embryonic development. Following ligand binding, ERBB3 heterodimerizes preferentially with ERBB2, leading to allosteric kinase activation and signaling. ERBB3 has been shown to function primarily as an activator kinase that activates the receiver kinase in the asymmetric dimer activation complex. Structural studies of the activator-receiver interface have identified V943 as a key residue required for ERBB3 mediated activation of ERBB2. Here we report the generation of a knock-in mouse model where the endogenous ErbB3 allele was modified to allow tissue-specific conditional expression of ErbB3V943R (ErbB3CKI-V943R). Activation of ErbB3V943R expression during development resulted in embryonic lethality at around E12.0. Analysis of the embryos reveals thinning of the myocardium and reduced mesenchyme within the endocardial cushion of the heart, reminiscent of ErbB3 knockout mice. Tissue specific expression of ErbB3V943R in the epithelial cells of the mammary gland using an MMTV-Cre driver results in delayed elongation of the ductal network during puberty. Consistent with this, single cell RNA-seq analysis showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells. Our studies establish the importance in vivo of the allosteric interface in activation of the ERBB family and also provides an important tool that can be used to understand the role of ERBB3 in cancer development. Single-cell RNA sequencing of luminal epithelial cells from 6-week old wild-type or ErbB3 V943R conditional mutant mammary glandsERBB3是一类包含伪激酶结构域的人表皮生长因子受体（ERBB/HER）家族成员，在多种细胞生物学过程中发挥关键作用，且为正常胚胎发育所必需。配体结合后，ERBB3优先与ERBB2形成异二聚体，进而引发变构激酶激活与信号转导。已有研究表明，ERBB3主要作为激活型激酶，在不对称二聚体激活复合物中激活受体激酶。针对激活-受体界面的结构研究已证实，V943是ERBB3介导ERBB2激活所必需的关键残基。本研究构建了一种敲入小鼠模型：对内源性ErbB3等位基因进行改造，使其可实现ErbB3V943R（即ErbB3CKI-V943R）的组织特异性条件性表达。在胚胎发育过程中激活ErbB3V943R的表达，会导致胚胎在约E12.0时期致死。对胚胎的分析显示，其心肌层变薄，心脏心内膜垫内的间充质含量减少，该表型与ErbB3基因敲除小鼠一致。利用MMTV-Cre驱动元件在乳腺上皮细胞中特异性表达ErbB3V943R，会导致青春期阶段乳腺导管网络的伸长延迟。与此一致的是，单细胞RNA测序（single cell RNA-seq）分析显示，产纤维蛋白原的特定管腔上皮细胞亚群数量有所减少。本研究证实了变构界面在ERBB家族激活过程中的体内重要性，同时也提供了一种可用于探究ERBB3在癌症发生发展中作用的重要工具。本研究对6周龄野生型或ErbB3 V943R条件性突变小鼠的乳腺管腔上皮细胞开展了单细胞RNA测序。