Abstract

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 a Receptor and CT b 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death including apoptosis (as described in a previous publication) and autophagy, which is discussed in our upcoming, unpublished review.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 b 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 b Receptor mRNA with qPCR and nanopore long range sequencing. An anti-CT Receptor antibody also known as CalRexin TM, 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.

Highlights

  • This article provides details of the methods developed to generate unpublished data, which is cited in an unpublished review article due for submission

  • The methods used to generate unpublished data cited in an upcoming review, relate to the experiments with qPCR and nanopore sequencing in which the mRNA of the CTb Receptor, which together demonstrate upregulation in stressed cells

  • CalRexinTM binds in the N-terminal domain of the CT Receptor isoforms, CTa or CTb Receptor

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Summary

METHOD ARTICLE

Methods to measure calcitonin receptor activity, upregulated in cell stress, apoptosis and autophagy [version 1; peer review: 2 approved]. Peter Wookey 1, Pragya Gupta 1, Lucas Bittencourt[1], Shane Cheung[2], David Hare[1], Sebastian Furness[3,4].

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