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Characterization of agonist-dependent somatostatin receptor subtype 2 trafficking in neuroendocrine cells

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Abstract

Background

Somatostatin (SOM) receptor subtype 2 (SSTR2) is the major receptor subtype mediating SOM effects throughout the neuraxis. We previously demonstrated that the non-selective agonist [D-Trp8]-SOM induces intracellular sequestration of SSTR2, whereas this receptor is maintained at the cell surface after treatment with the SSTR2-selective agonist L-779,976 in cells co-expressing SSTR2 and SSTR5.

Methods and results

In this study, we knocked-out SSTR5 in AtT20 cells endogenously expressing both SSTR2 and SSTR5 and used immuno-labeling and confocal microscopy to investigate the effect of SSTR5 on regulation of SSTR2 trafficking. Our results indicate that unlike [D-Trp8]-SOM-induced intracellular sequestration, L-779,976 stimulation results in the maintenance of SSTR2 at the cell surface regardless of whether SSTR5 is present or not. We then examined the trafficking pathways of SSTR2 upon stimulation by either agonist. We found that both [D-Trp8]-SOM and L-779,976 induce SSTR2 internalization via transferrin-positive vesicles. However, SSTR2 internalized upon L-779,976 treatment undergoes rapid recycling to the plasma membrane, whereas receptors internalized by [D-Trp8]-SOM recycle slowly after washout of the agonist. Furthermore, [D-Trp8]-SOM stimulation induces degradation of a fraction of internalized SSTR2 whereas L-779,976-dependent, rapid SSTR2 recycling appears to protect internalized SSTR2 from degradation. In addition, Octreotide which has preferential SSTR2 affinity, induced differential effects on both SSTR2 trafficking and degradation.

Conclusion

Our results indicate that the biased agonistic property of L-779,976 protects against SSTR2 surface depletion by rapidly initiating SSTR2 recycling while SSTR5 does not regulate L-779-976-dependent SSTR2 trafficking.

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Acknowledgements

The SSTR5 knockout cell lines were created by the MNI iPSC/CRISPR platform. Jasmine Leung provided blind quantification of SSTR2 cell surface labeling in the experiments with the SSTR5 knockout clones and Sean Goldfarb quantified the Octreotide experiments. We are most grateful to the MNI’s Microscopic Cellular Imaging Facility for their advice and support with advanced microscopy. We would also like to thank Dr Peter S. McPherson for intellectual support. The authors also wish to extend their gratitude to Naomi Takeda for administrative assistance. This work was funded through grant CIHR MOP-102713 to T.S. and grants GRK1459 and Kr1321/7-1 from Deutsche Forschungsgemeinschaft to H.J.K.; W.A. was supported by a Jeanne Timmins Costello Fellowship by the Montreal Neurological Institute.

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Authors and Affiliations

  1. Department of Neurology and Neurosurgery, McGill University, and the Montreal Neurological Institute, Montreal, QC, Canada

    Walaa Alshafie, Yingzhou Edward Pan & Thomas Stroh

  2. Institute for Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Yingzhou Edward Pan & Hans-Jürgen Kreienkamp

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  1. Walaa Alshafie
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Correspondence to Walaa Alshafie or Thomas Stroh.

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Alshafie, W., Pan, Y.E., Kreienkamp, HJ. et al. Characterization of agonist-dependent somatostatin receptor subtype 2 trafficking in neuroendocrine cells. Endocrine 69, 655–669 (2020). https://doi.org/10.1007/s12020-020-02329-x

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