Yang Ming, Yang Ming, Lin Ying, Meng Fengyan, Du Xiaogang, Zeng Xianyin, Bu Guixian
Isotope Research Laboratory, College of Life Science, Sichuan Agricultural University, Xinkang Road, Ya'an 625014, PR China.
Department of Bio-engineering and Applied Biology, College of Life Science, Sichuan Agricultural University, Xinkang Road, Ya'an 625014, PR China.
Gen Comp Endocrinol. 2022 Jan 15;316:113941. doi: 10.1016/j.ygcen.2021.113941. Epub 2021 Oct 27.
Prolactin (PRL) plays crucial roles in many physiological and pathological processes through activating its specific membrane-anchored receptor (PRLR). Although this ligand-receptor pair has been extensively studied in mammals, birds and fishes, researches examining their significance is rather scarce in reptiles. Additionally, the interaction mechanism of PRL-PRLR has abortively understood across vertebrates, since two tandem repeated ligand-binding domains of PRLR have been identified in birds and few reptiles. To lay the foundation to clarify their roles and ligand-receptor interaction in reptiles, using Chinese soft-shelled turtle as model, the cDNAs containing open reading frame of PRL and PRLR were cloned. The cloned PRL consisted of 710 bp and encoded a precursor of 228 amino acid (-aa), while PRLR was 2658 bp in length and predicted to generate a 828-aa precursor. Furthermore, the recombinant PRL protein with high-purity was prepared from Escherichia coli (E. coli) strain Rosetta gamiB (DE3) by using cobalt resin. Using the 5 × STAT5-Luciferase reporter system, we found PRLR and PRLR-M2 (the PRLR-mutant lacking membrane-distal ligand-binding domain) could be dose-dependently activated by recombinant PRL, thereby triggering the intracellular JAK2-STAT5 signaling cascade, suggesting PRL-PRLR is functional in Chinese soft-shelled turtle, and the membrane-proximal ligand-binding domain of PRLR is the critical domain involving in PRL-binding. Quantitative real-time PCR revealed that PRL was predominantly and abundantly expressed in pituitary, while PRLR exhibited ubiquitous expression in all of the tissues examined. Collectively, our data indicate the PRL-PRLR pair may function in reptiles including Chinese soft-shelled turtle, in a way similar to that in birds.
催乳素（PRL）通过激活其特定的膜锚定受体（PRLR）在许多生理和病理过程中发挥关键作用。尽管这种配体-受体对在哺乳动物、鸟类和鱼类中已得到广泛研究，但在爬行动物中对其重要性的研究却相当匮乏。此外，由于在鸟类和少数爬行动物中已鉴定出PRLR的两个串联重复配体结合结构域，PRL-PRLR的相互作用机制在整个脊椎动物中尚未完全明确。为了奠定阐明它们在爬行动物中的作用和配体-受体相互作用的基础，以中华鳖为模型，克隆了包含PRL和PRLR开放阅读框的cDNA。克隆的PRL由710 bp组成，编码一个228个氨基酸（-aa）的前体，而PRLR长度为2658 bp，预计产生一个828-aa的前体。此外，通过使用钴树脂从大肠杆菌（E. coli）菌株Rosetta gamiB（DE3）中制备了高纯度的重组PRL蛋白。使用5×STAT5-荧光素酶报告系统，我们发现PRLR和PRLR-M2（缺乏膜远端配体结合结构域的PRLR突变体）可被重组PRL剂量依赖性激活，从而触发细胞内JAK2-STAT5信号级联反应，表明PRL-PRLR在中华鳖中具有功能，并且PRLR的膜近端配体结合结构域是参与PRL结合的关键结构域。定量实时PCR显示，PRL主要在垂体中大量表达，而PRLR在所有检测的组织中均呈现普遍表达。总体而言，我们的数据表明PRL-PRLR对可能在包括中华鳖在内的爬行动物中发挥作用，其方式与鸟类相似。
