| pA2 online © Copyright 2004 The British Pharmacological Society |
168P
GKT, University of London Winter Meeting December 2003 |
|
The role
of hydrophilic residues in transmembrane helix 2 of the CGRP1 receptor
(CL/RAMP1) in signal transduction. |
|
Print abstractThe CGRP1 receptor is a novel family-B G-protein coupled receptor (GPCR) with two components calcitonin receptor-like receptor (CL or CRLR) and an accessory protein, receptor activity modifying protein 1 (RAMP1). Both are needed for receptor function (McLatchie et al., 1998). There are a number of hydrophilic residues found within the transmembrane helices of CL and particularly in TM2. It is likely that some of these are important for function. In this study, hydrophilic residue in TM2 were mutated to alanine and the effects on CGRP-stimulated cyclic AMP production were studied.
Point mutations in
human CL were introduced by the Stratagene Quick-Change mutagenesis method.
Transfection of CL and RAMP1 into Cos-7 cells, construction of dose-response
curves to human 
CGRP
(10-12M to 10-7M),
cyclic AMP measurements and radioligand binding to cell membranes using
125I-CGRP were as described previously
(Hay et al., 2003; Howitt et al., 2003). Curve fitting to obtain pEC50
and maximum effect (Emax)
relative to wild type (WT) was by PRISM Graphpad. Mutant and WT pEC50s
were compared by paired Student's t-tests; Emax values
were compared to 100% (WT) by Mann-Whitney tests.
Only H155A and T169A altered signalling (Table 1). Both reduced the pEC50. T169A also reduced the Emax. Radioligand binding showed that H155A and T169A had similar pKd's (9.77±0.27, 8.97±0.55, n=3) and Bmax values (120±6%, 72±18%) to WT (9.34±0.58, 100% [=222±34 fmol mg-1]).
Table 1. Effects of mutants on cyclic AMP production
| Mutant |
n
|
EC50
WT
|
EC50
mutant
|
Emax
(% control)
|
| T153A |
3
|
10.57±0.28
|
10.27±0.35
|
64±12
|
| H155A |
6
|
10.60±0.27
|
9.62±0.33*
|
206±46
|
| K156A |
4
|
9.58±0.36
|
9.26±0.21
|
61±16
|
| N157A |
5
|
9.96±0.32
|
10.32±0.32
|
112±23
|
| N165A |
5
|
9.97±0.45
|
9.79±0.58
|
108±13
|
| T169A |
5
|
10.22±0.44
|
9.05±0.36***
|
69±5*
|
| H172A |
4
|
10.47±0.27
|
9.87±0.43
|
86±9
|
*, *** P>0.05, 0.001. Values are means±s.e.m. 100% (Emax) = 85±18 pmoles cAMP per 10min per well
These data suggest that H155 and T169 are important for signal transduction in the CGRP1 receptor. They are predicted to be on the same face of helix 2 and may make contacts with helix 3, implicated in many models of GPCR activation (Lu et al., 2002).
Hay, D.L. et al.,
(2003). Br. J. Pharmacol, in press.
Howitt, S.G. et al., (2003). Br. J. Pharmacol, 138,
325-332.
Lu, Z.L., et al., (2002). Trends Pharmacol Sci. 23,
140-146.
McLatchie, L.M. et al., (1998). Nature, 393, 333-339.
This work was supported by the Wellcome Trust and an MRC studentship to DLH.