| pA2 online © Copyright 2004 The British Pharmacological Society |
117P
University of Newcastle Winter Meeting December 2004 |
Impaired cell surface expression of conserved Asp148 within the “dry” motif of the V1a vasopressin receptor Stuart R. Hawtin. Institute of Cell Signalling, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK . |
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Print abstractThe vasopressin V1areceptor (V1aR) is a member of a sub-family of related G-protein-coupled receptors (GPCRs) that are activated by the hormone vasopressin (AVP) ( Hawtin et al., 2003) . The highly conserved “DRY” triplet in the distal region of TM-III for most class I GPCRs is implicated in their activation process, G-protein signalling and internalisation ( Barak et al., 2003; Capra et al., 2004; Karnik et al., 2003 ). The aim of this study was to determine if specific features required at this locus are important for the rat V1aR. Each residue within this region was mutated to Ala, expressed in HEK293T cells and pharmacologically compared to wild-type (Wt). Radioligand binding experiments were performed utilising a 96-well MultiScreen™ assay with AVP and three structurally different antagonists ( Hawtin et al., 2003) . G-protein signalling was assessed by AVP-mediated accumulation of [3H]-inositol phosphates ( Hawtin et al., 2003 ). Cell surface expression and agonist-mediated internalisation of Wt and mutant V1aRs were quantified (in parallel experiments) by an ELISA-based assay utilising an N-terminal HA-epitope tag incorporated into each receptor.
Table 1
Binding affinities (Ki) (nM) |
Stimulation of |
Cell surface expression (% Wt) |
||||
Receptor |
AVP |
CA |
LA |
SR49059 |
||
Wt |
1.0 ± 0.1 |
0.5 ± 0.1 |
0.2 ± 0.0 |
0.7 ± 0.1 |
0.6 ± 0.2 |
100 (8) |
D148A |
ND |
ND |
ND |
ND |
ND |
2 ± 2 (7) |
R149A |
2.6 ± 0.4 |
1.4 ± 0.3 |
1.2 ± 0.1 |
1.9 ± 0.2 |
1.8 ± 0.2 |
101 ± 5 (7) |
Y150A |
1.3 ± 0.3 |
1.0 ± 0.2 |
0.4 ± 0.1 |
1.3 ± 0.2 |
1.2 ± 0.2 |
91 ± 4 (6) |
Values represent mean ± S.E.M of three separate experiments performed in triplicate unless stated (n). Ki values were corrected for [3H]AVP occupancy. CA=cyclic peptide antagonist and LA=peptide linear antagonist ( Hawtin et al., 2003) . ND= none detected
D148A was not expressed at the cell surface (Table 1). R149A displayed a 3-6 fold decrease in binding affinity with all ligands. Potency for AVP-mediated inositol phosphate signalling was also reduced 3-fold for R149A (Table 1) with no detectable increase in basal activity. R149A and Y150A mutants internalised in response to AVP (1 µM) to similar levels (58 ± 3 % n=7 and 61 ± 2 % n=6) compared to Wt(59 ± 3 % n=8) over a 60 min period and at rates (t1/2= 6.8 ± 1.0 min; 7.9 ± 1.4 min and 9.9 ± 1.0 min; (n=3) respectively). Collectively, these results show that Asp148 is critical for surface expression, whereas mutation of either Arg149 or Tyr150 does not significantly perturb receptor function.
Barak, L et al., (2003) Assay Drug Dev. Technol. 1, 409-424.
Capra, V et al., (2004) Mol. Pharmacol. 66, 880-809.
Hawtin, S et al., (2003) Mol. Endocrinol. 16, 600-609.
Karnik, S et al., (2003) Trends Endocrinol.Metab. 14, 431-437.
This work is funded by a fellowship awarded by the University of Nottingham.