| pA2 online © Copyright 2004 The British Pharmacological Society |
116P
University of Newcastle Winter Meeting December 2004 |
The role of the second intracellular loop in V1a vasopressin receptor activation and signalling Stuart R. Hawtin. Institute of Cell Signalling, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK. |
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Print abstract Vasopressin (AVP) activates the vasopressin V1a receptor (V1aR), a member of a sub-family of related G-protein-coupled receptors (Hawtin et al., 2003). The second intracellular loop (IC-II) of class I GPCRs has been reported to be involved in G-protein activation and internalisation ( Schumann et al., 2003 ). Chimeric V1aR/V2R studies suggested IC-II of V1aR provides epitope(s) for G
q/11 -signalling ( Liu & Wess, 1996) . The aim of this study was to determine the role of individual amino acids within the IC-II region of the V1aR. Each residue was mutated to either Ala or Gly and expressed in HEK293T cells. Mutants were pharmacologically compared to Wt by (i) radioligand binding (MultiScreen™ assay with AVP and three structurally different antagonists), (ii) measuring AVP-mediated accumulation of [3H]-inositol phosphates (Hawtin et al., 2003) and (iii), quantification of cell surface expression using an ELISA-based assay.
Table 1
Binding affinities (K i) (nM) |
Stimulation of |
Cell surface |
||||
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 |
I151A |
0.7 ± 0.3 |
0.9 ± 0.3 |
0.4 ± 0.2 |
0.7 ± 0.1 |
0.5 ± 0.1 |
37 ± 1 |
A152G |
0.5 ± 0.1 |
0.5 ± 0.1 |
0.2 ± 0.1 |
0.5 ± 0.1 |
0.9 ± 0.5 |
26 ± 4 |
V153A |
0.4 ± 0.1 |
0.6 ± 0.2 |
0.1 ± 0.1 |
0.3 ± 0.1 |
0.3 ± 0.1 |
13 ± 1 |
C154A |
1.1 ± 0.1 |
0.8 ± 0.1 |
0.2 ± 0.0 |
0.5 ± 0.1 |
0.8 ± 0.3 |
69 ± 4 |
H155A |
0.9 ± 0.1 |
0.8 ± 0.2 |
0.1 ± 0.0 |
1.4 ± 0.6 |
0.7 ± 0.1 |
87 ± 27 |
P156A |
1.0 ± 0.2 |
0.7 ± 0.1 |
0.2 ± 0.1 |
0.8 ± 0.2 |
0.7 ± 0.1 |
47 ± 2 |
L157A |
5.3 ± 0.5 |
3.9 ± 1.0 |
2.5 ± 0.3 |
6.4 ± 1.5 |
1.1 ± 0.2 |
120 ± 9 |
K158A |
1.8 ± 0.2 |
1.6 ± 0.2 |
0.5 ± 0.5 |
1.9 ± 0.4 |
0.4 ± 0.2 |
48 ± 6 |
T159A |
3.5 ± 0.4 |
2.8 ± 0.3 |
2.2 ± 0.5 |
2.5 ± 0.4 |
1.1 ± 0.1 |
118 ± 3 |
L160A |
3.5 ± 0.3 |
2.2 ± 0.2 |
1.3 ± 0.3 |
2.2 ± 0.1 |
0.7 ± 0.1 |
99 ± 1 |
Q161A |
2.3 ± 0.2 |
1.9 ± 0.3 |
0.5 ± 0.2 |
1.7 ± 0.1 |
0.6 ± 0.2 |
78 ± 16 |
Q162A |
1.3 ± 0.1 |
1.5 ± 0.1 |
0.7 ± 0.2 |
1.6 ± 0.2 |
0.4 ± 0.0 |
94 ± 2 |
P163A |
3.2 ± 0.5 |
2.3 ± 0.3 |
1.4 ± 0.2 |
2.1 ± 0.5 |
0.4 ± 0.1 |
121 ± 5 |
Values represent mean ± S.E.M of three separate experiments performed in triplicate. Ki values were corrected for [3H]AVP occupancy. CA=cyclic peptide antagonist and LA= peptide linear antagonist (Hawtin et al., 2003).
Five mutations (L157A, T159A, L160A, Q161A and P163A) showed a 3-12-fold decreased in binding affinity for specific ligands (Table 1). All mutations were able to increase inositol phosphate accumulation in response to AVP (Table 1). Mutants I151A, A152G, V153A, P156A and K158A showed a (50-90 %) decrease in cell surface expression. Overall, these results show that mutation of individual residues within this region have relatively minor roles on binding of specific ligands and signalling, but have major effects on cell surface expression.
Hawtin, S et al., (2003) Mol. Endocrinol. 16, 600-609.
Liu, J & Wess, J (1996) J. Biol. Chem. 271, 8772-8778.
Schumann, M et al., (2003) J. Pharmacol. Exp. Ther. 307, 597-607.
This work is funded by a fellowship awarded by the University of Nottingham.