There is increasing evidence that many G protein coupled receptors may exist as dimers or oligomers (Devi, 2001). The aim of this study was to investigate if two closely related receptors, oxytocin (OT) and vasopressin V_1A, exist as oligomers using BRET² methodology. BRET² is based on the energy transfer from a luminescent Renilla luciferase (Rluc) donor to a fluorescent GFP² acceptor protein that occurs if two proteins are in close proximity, and thus can be used as an indicator of receptor-receptor interactions (Jenson et al., 2002).

Human OT and V_1A receptor cDNAs were amplified by PCR using primers designed to remove the stop codon and then sub-cloned into pGFP²N3 and pRlucN3 expression vectors (Perkin Elmer) to produce OT and V_1A receptors C terminally tagged with GFP² and Rluc (OT-Rluc, OT-GFP², V_1A-Rluc and V_1A-GFP²). CHO cells were transiently transfected with receptor cDNA (0.12µg / cm2 cells per construct unless stated) using lipofectamine 2000. 24h after transfection, emission was read at 515 and 410nm on a Fusion microplate reader following the addition of 5µM DeepBlueC. Data are expressed as the ratio of GFP² emission/Rluc emission as mean [95% confidence interval] for n=4-6. Statistical comparisons were made on log-transformed data using t-tests or ANOVA where appropriate.

Co-expression of OT-GFP² and V_1A-Rluc in CHO cells resulted in a significant (P<0.01) increase in the GFP²/Rluc emission ratio (ER) compared to expression of V_1A-Rluc alone (ER=0.326[0.294-0.360] and 0.072[0.051-0.102], respectively) which was not affected (P>0.05) by treatment with the V_1A/OT agonist, arginine vasopressin (AVP, 1µM; ER=0.322[0.265-0.391] and 0.305[0.297-0.392] following 2 or 30 min AVP pre-treatment, respectively). Expression of OT-Rluc and V_1A-GFP² also resulted in an increase in the BRET² ratio compared to OT-Rluc alone (ER=0.184[0.146-0.231] and 0.067[0.058-0.077] respectively, P<0.01), whereas expression of Rluc and GFP² proteins lacking receptor gave a low BRET² signal (ER=0.076[0.068-0.085]). Co-transfection of cells with OT-GFP² and OT-Rluc also resulted in an increase in the BRET² signal compared to OT-Rluc alone (ER=0.292[0.213-0.389] and 0.066[0.055-0.078], respectively, P<0.01), as did expression of V_1A-GFP² with V_1A-Rluc compared to V_1A-Rluc alone (ER=0.176[0.138-0.221] and 0.068[0.061-0.075], respectively, P<0.01). As would be expected if the observed increases in BRET² signals were due to oligomerisation, co-expression with untagged OT receptor cDNA reduced the BRET² ratio of the V_1A-Rluc and OT-GFP² system in a concentration-dependent manner (ER=0.445[0.3426-0.577], 0.379[0.249-0.408] and 0.226[0.177-0.289] in the absence and presence of 0.4µg and 0.8µg OT receptor cDNA/cm² cells respectively).

These data suggest that OT and V_1A receptors constitutively form homo-and hetero-oligomers and confirm and extend recent work of Trellion et al., (2003).

Devi, L.A. (2001) Tr. Pharmacol. Sci. 22, 532-537.
Jenson, A.A. et al., (2002) Eur. J. Biochem. 269, 5076-5087.
Trellion, S. et al., (2003) Mol Endocrinol. 17, 677-691.