Poster Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Roles of the intraloops and C-terminus in calcium/calcimimetic selection of G-protein dependant signalling pathways from the calcium-sensing receptor (#302)

Kimberly T Edwards 1 , Mahvash Goolam 1 , Maria Oh 1 , Katie Leach 2 , Arthur Christopoulos 2 , Arthur D Conigrave 1
  1. University of Sydney, Camperdown, NSW, Australia
  2. The Monash Institute of Pharmaceutical Sciences, Melbourne, Victoria, Australia

The calcium-sensing receptor (CaSR) is a Class C G-protein coupled receptor that contributes to the control of calcium metabolism and bone homeostasis via its expression  in various tissues, including the parathyroid glands, kidney and bone. The CaSR mediates diverse effects by selecting for signalling pathways in a ligand- and cell-type-specific manner. However, the mechanisms that underlie the selection of signalling pathways are not well understood.


To properly understand the mechanisms underlying the role of calcium, and calcimimetics including L-amino acids and the clinical compound, cinacalcet, in CaSR-mediated ligand dependant selection, we decided to perform alanine scanning site-directed mutagenesis of intraloops -1, -2 and -3, and to truncate the C-terminus. We assessed the residues critical for the coupling of the CaSR, in the presence/absence of the aforementioned calcimimetics, to distinct pathways, including downstream of PI-PLC (IP1 accumulation), phosphorylated ERK1/2 (pERK), intracellular Ca2+ (Ca2+i) mobilization, and suppression of forskolin-stimulated adenylyl cyclase (intracellular cAMP levels). An enzyme-linked immunosorbance assay (ELISA) was also performed to examine the cell surface expression of these CaSR mutants. 


The results demonstrate that distinct residues and sub-domains mediate coupling to distinct signalling pathways downstream of the receptor. In particular, the CaSR mutant constructs A642-644 (iL-1), A701-704, F706A (iL2), A793-795, A796-798, E799A, F801A, (iL-3), none of which impaired cell surface expression, markedly attenuated PI-PLC and pERK. E803A was observed to have reduced cell surface expression. Furthermore, the C-terminal truncation mutant, T876X, markedly attenuated PI-PLC and ERKp, while R891X retained sensitivity. Most strikingly, R866X exhibited complete loss of Ca2+i mobilization but retained intact suppression of adenylyl cyclase.


The results demonstrate that pathway selection arises from distinct domains and sub-domains of the receptor's intraloops.