064P Brighton
Winter Meeting December 2008 |
A region located on the first extracellular loop of the calcitonin receptor-like receptor required for agonist mediated cAMP production and desensitisation
James Barwell, David Poyner
Aston University, Birmingham, UK
Calcitonin-gene related peptide (CGRP) is a 37 amino acid neuropeptide. The CGRP receptor has an unusual oligomeric organisation containing two calcitonin receptor-like receptors (CLR) (family-B G-protein coupled receptors), and a single-pass transmembrane protein, receptor activity modifying protein 1 (RAMP1) (Héroux et al., 2007a). The CGRP receptor can couple to Gs and to β-arrestins (Héroux et al., 2007b). Here we report that three residues, L173, V176 and A177 in the first extracellular loop of CLR are needed for CGRP-stimulated cAMP production and receptor densensitisation
Methods were as described previously (Conner et al., 2006). In brief, residues were replaced by alanine or leucine. Mutant and wild-type (WT) CLR were transiently co-transfected with RAMP1 into Cos7 cells. Cells were challenged with human α-CGRP from 10pM to 100nM prior to measurement of cAMP by a radio-receptor assay. Cell surface expression and desensitization was assessed by whole cell enzyme-linked immunosorbent assays. Receptors were desensitised by treatment with 100nM human α-CGRP for an hour at 37˚C. pEC50 values were calculated using Graphpad Prism 4.00. Independent t-tests were used to analyse pEC50 values. Cell surface expression data was normalized to make WT expression 100%: Mann Whitney U tests were used to analyse agonist mediated desensitization.
Table 1. Effects of mutants on cAMP production and desensitisation
| Mutant | n | cAMP Production | Desensitization (%) |
| WT pEC50 | Mutant pEC50 | WT | Mutant |
| L173A |
4 |
10.36±0.25 |
8.87±0.087* |
48.24±3.35 |
14.17±12.98* |
| V176A |
5 |
9.61±0.15 |
8.10±0.52* |
45.58±2.76 |
28.01±8.33* |
| A177L |
6 |
10.20±0.31 |
8.87±0.31* |
62.81±6.06 |
4.91±10.05* |
Values represent mean ± S.E.; *, significantly different from WTp<0.05
The results in table 1 demonstrate that the mutations impair both CGRP-mediated cAMP production and desensitisation. The residues are close to each other and may stabilise the active conformation of the receptor or interact directly with CGRP. Binding studies are currently being conducted to explore these residues further.
This work was funded by the British Heart Foundation (FS05/054).
Conner A.C. et al., (2006), J.Biol.Chem 281 (3) 1644-51.
Héroux M. et al., (2007a), J.Biol.Chem, 282 (43) 31610-20
Héroux M. et al., (2007b), Biochemistry, 46 (23) 7022-33
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