Characterisation of a C-terminal deletion mutant of the calcitonin receptor-like receptor.

S.Staddon 1, M.Wheatley 2, D.R.Poyner 1 & A.C.Conner 1,2. 1School of Life and Health Sciences, Aston University, Birmingham, UK; 2School of Biosciences, University of Birmingham, Birmingham, UK.

Calcitonin receptor-like receptor (CLR) is a G-protein coupled receptor (GPCR) that forms a complex with receptor activity modifying protein 1 (RAMP1) to recognize calcitonin gene-related peptide (CGRP) (McLatchie et al., 1998). The C-termini of GPCRs are generally implicated in a range of functions including cell signalling and receptor trafficking; however the C-terminus of CLR has not been examined. Here we report on the properties of human CLR where the final 61 amino acids have been deleted (D378).

Δ378 was constructed from HA-tagged wild type (WT) CLR using the Stratagene Quick-change mutagenesis method and inserted into pcDNA3.1-. Δ378 or WT receptor were co-transfected into Cos-7 cells with human RAMP1 using polyethylenimine (Conner et al; 2005). Measurement of cAMP-signalling was by a [3H]-cAMP radio-receptor assay (Conner et al; 2005) following a 15 min challenge with human α-CGRP from 10pM to 100nM. pEC 50 values were calculated using Graphpad Prism version 4.00. CGRP-stimulated ERK phosphorylation was examined by western blotting using phospho-specific antibodies ( Santa Cruz, sc-7383), as described previously (Plevin et al., 1996). Protein content of samples was measured by RCDC Protein Assay (BioRad) prior to western blotting, to ensure equal loading. Blots were quantified by densitometry. Receptor expression at the cell surface was measured by ELISA (Conner et al., 2005) after exposure to 100nM CGRP, taking time points from 3 to 60 min following agonist addition. The data were fitted to a mono-exponential curve to estimate the maximum receptor loss.

CGRP-mediated cAMP stimulation was not altered by the deletion (mean pEC 50 values ±s.e.m: Δ378, 9.82±0.28; WT, 9.94±0.365, n=5). CGRP-mediated phosphorylation of ERK was comparable for both receptors at 5 minutes ( Δ378 was 104±12 % of maximum WT stimulation, n= 5) but was sustained at 60 minutes following stimulation of Δ378 compared to WT (125±16% versus 72±8%, n=5, P<0.01, Mann Whitney). After 1hour exposure to 100nM CGRP, WT receptor surface expression was significantly reduced by 50±9% (n=4, P<0.05, Student’s t-test); by contrast, there was no significant reduction in the expression of Δ378 (6±8%). Thus the C-terminus of CLR is needed for agonist-stimulated internalization and its removal allows sustained activation of the ERK signalling cascade; however it is not involved in acute stimulation of cAMP.

Conner, A.C. et al., (2005), Mol. Pharmacol., 67, 20-31.
McLatchie, L. et al., (1998), Nature, 393, 333-339.
Plevin, R. et al., (1996), Biochem. J., 318, 657-663.

This work was funded by the Wellcome Trust.