Medicine, University of Melbourne, Heidelberg, Victoria, 3084, Australia.
BOMP, University of Melbourne, Parkville, Victoria, 3052, Australia.
F1000Res. 2021 Oct 7;10:1019. doi: 10.12688/f1000research.72845.1. eCollection 2021.
The expression of the calcitonin receptor (CT Receptor) is widespread throughout the life cycle of mammals and in many diseases, and in these contexts the functions of the common isoforms is largely unknown. The relatively recent development of anti-CT Receptor antibodies that bind separate epitopes on the CT Receptor and CT Receptor isoforms has advanced our knowledge and understanding of these events. CT Receptor at the protein level is upregulated in programmed cell death including apoptosis (as described in a previous publication) and autophagy, which is discussed in our upcoming, unpublished review. Incomplete data sets are cited in this review on the upregulation of CACLR (encoding CT Receptor) mRNA, in particular the insert-positive isoform (CT Receptor), in response to cell stress. Cell stress is induced by growth in depleted foetal bovine serum (dFBS) or without FBS, both of which induce degrees of starvation and autophagy, or dFBS plus staurosporine, which induces apoptosis. Details of the methods deployed to generate these data are described here including measurement of the upregulation of CT Receptor mRNA with qPCR and nanopore long range sequencing. An anti-CT Receptor antibody also known as CalRexin , which binds an epitope in the N-terminal domain, was conjugated to either fluorophore 568, which is accumulated into apoptotic cells as previously reported, or pHrodo Red, a pH dependent fluorescent dye, which is accumulated into autophagic and apoptotic cells. These conjugates are under development to image programmed cell death. The methods for conjugation and high content imaging on the Operetta platform are described. The high fluorescence intensity at low pH of CalRexin:pHrodo Red in both autophagic and apoptotic cells suggests localisation in autophago-lysosomes and lysosomes respectively. Overall, these observations and the methods that underpin them have contributed to our understanding of the widespread expression of CT Receptor isoforms.
降钙素受体（CT 受体）的表达在哺乳动物的整个生命周期中都很广泛，并且在许多疾病中也是如此，在这些情况下，常见同工型的功能在很大程度上尚不清楚。最近开发的抗 CT 受体抗体能够结合 CT 受体和 CT 受体同工型上的不同表位，这促进了我们对这些事件的认识和理解。CT 受体在程序性细胞死亡中（如前一篇出版物所述，包括细胞凋亡和自噬）的蛋白水平上调，而自噬在我们即将发表的未公开评论中进行了讨论。在这项评论中，关于 CACLR（编码 CT 受体）mRNA 的上调（特别是插入阳性同工型 CT 受体）以响应细胞应激的数据不完整，特别是在细胞应激时。细胞应激是由在耗尽胎牛血清（dFBS）或没有 FBS 的情况下生长引起的，这两者都会引起饥饿和自噬的程度，或者 dFBS 加 staurosporine 诱导细胞凋亡。在此处描述了生成这些数据所采用的方法的详细信息，包括使用 qPCR 和纳米孔长距离测序测量 CT 受体 mRNA 的上调。一种称为 CalRexin 的抗 CT 受体抗体，与 N 端结构域中的表位结合，与荧光团 568 缀合，如前所述，该荧光团积累在凋亡细胞中，或与 pHrodo Red 缀合，这是一种依赖 pH 的荧光染料，积累在自噬和凋亡细胞中。这些缀合物正在开发中，用于成像程序性细胞死亡。描述了在 Operetta 平台上进行缀合和高内涵成像的方法。CalRexin:pHrodo Red 在自噬和凋亡细胞中的低 pH 下的高荧光强度表明它们分别定位于自噬溶酶体和溶酶体中。总的来说，这些观察结果及其背后的方法有助于我们理解 CT 受体同工型的广泛表达。
