Ciliary Localisation and Signal-Dependent Retrieval of Somatostatin Receptor 3 (SSTR3) from cilia in Mammalian Cells and Zebrafish

Abstract

Primary cilia are essential cellular organelles that play a role in a variety of physiological processes through the localisation of G protein-coupled receptors (GPCRs) to the cilium. The Somatostatin Receptor 3 (SSTR3) receptor is one of the GPCRs that has been identified to play a pivotal role in a variety of physiological processes. However, the mechanisms that mediate the retrieval of SSTR3 from the primary cilium after activation of its receptor have not yet been explored. In this project, site-directed mutagenesis of the SSTR3 gene was used to create mammalian cell lines that contained mutations to the lysine residues that are known to be ubiquitinated by the receptor. Through imaging and immunofluorescence assays, it was determined that lysine residues K233 and K407 are necessary for the ubiquitination of SSTR3 and its subsequent exit from the primary cilium, while mutations to lysine residues K330, K356, and K421 did not have an impact upon the exit of the receptor from the cilium. Studies are currently being performed in zebrafish to create fish with mutations in the sstr3 gene through the use of CRISPR-Cas9. Sequencing of the resulting fish has confirmed the deletion of 6 bp and 15 bp in one fish, and 2 bp deletion in the second fish. Embryos from these fish are currently being raised to perform additional experiments on these fish. These experiments will allow the researchers to determine the trafficking of the SSTR3 receptors to the primary cilia of the cells containing the gene, as well as to determine the relevance of such trafficking within the body of the patient. Furthermore, this information can help in the determination of whether disruptions in the trafficking of these receptors can lead to neurological, ciliopathic, or metabolic disorders, and in what ways those disorders may be treated.