Abstract
The calcitonin receptor-like receptor (CLR) is a class B G protein-coupled receptor (GPCR) which forms heterodimeric complexes with receptor activity-modifying proteins (RAMPs) to give rise to distinct receptors. These have important roles in the cardiovascular, renal and central nervous systems. A naturally occurring variant of the CLR gene where the isoleucine is substituted with a threonine at position 446 of the intracellular domain (I446T) has been recently identified as highly enriched in individuals with Māori and Pacific ancestry. So far, clinical phenotyping and studies in rodent models indicate the variant could be linked with blood pressure and kidney function, however, there is currently insufficient in vitro evidence to ascertain the molecular basis of this phenotype.
The intracellular domain of GPCRs associates with a plethora of proteins involved in cellular signalling and receptor regulation. CLR is Gαs coupled, so activation of the receptor stimulates cAMP-mediated cellular responses via G protein association with the intracellular domain. CLR receptor internalisation is also initiated by phosphorylation and β-arrestin recruitment to the C-terminal tail. It was hypothesised that the I446T variant may cause a change in these receptor-protein interactions, leading to alterations in cell signalling and receptor cell surface expression. Prior in vitro studies have attempted to characterise this by comparing the receptor function of wildtype and I446T variant human CLR constructs designed with an artificial signal sequence. It has been hypothesised however, that this artificial signal sequence may drive overexpression in cell models and mask an effect the variant is exerting, meaning the current evidence on the variant remains inconclusive. Therefore, the aims of this project were to characterise I446T CLR-comprising receptor function in HEK293S cells using new constructs designed with a native signal sequence. Specifically, this was done by comparing cAMP production and receptor expression of the human and rat variant receptors with their wildtype counterparts.
cAMP responses at the human I446T variant receptors were significantly more potent and efficacious than the human wildtype receptors in response to stimulation with physiologically relevant peptides, (calcitonin gene-related peptides, adrenomedullin and adrenomedullin 2). The human I446T variant receptors appeared to have higher expression than the wildtype, which could explain the increase in the cAMP responses, but these experiments were difficult to interpret due to very low detection of the wildtype receptor. These human receptor observations were unable to be replicated in the rat receptor data as cAMP production at rat receptors were too variable to interpret and no difference was observed between the wildtype and variant receptor expression. My in vitro results suggest that in humans, the I446T variant could create a hyperactive receptor, but further studies are needed to investigate this.