Urotensin II (U-II) is the most potent vasoconstrictor identified to date (Pearson et al., 1980, Camarda et al., 2002). Recently the human gene encoding for U-II has been cloned and U-II was identified as the endogenous ligand of the G_q-protein coupled receptor GPR14 (Ames et al., 1999) now referred to as the UT receptor (Douglas et al., 2000). Here we describe a viable GTP[³⁵S] binding assay for the UT receptor and compare potency measures with those downstream at the level of intracellular Ca²⁺.

GTP[³⁵S] binding to homogenates from CHO cells stably expressing GPR14 (CHO_GPR14) was measured at 30oC in 0.5ml of buffer consisting 50mM Tris-HCl, 0.2mM EGTA, 1mM MgCl₂, 100mM NaCl, 1mg/ml BSA, 0.15mM bacitracin, varying GDP concentration, 10µM peptidase inhibitors (amastatin, bestatin, captropril and phosphoramidon) containing ~150pM GTP[³⁵S]. Whole CHO_GPR14 cells were used for calcium studies. Cells were suspended in Krebs-HEPES buffer and incubated with 5µM Fura-2AM for 30 min followed by de-esterification in fresh Krebs-HEPES buffer for 20 min. Fluorescence was measured in 2ml volumes at 37^oC as described previously (Sprague et al., 2001).

1µM U-II stimulated the binding of GTP[³⁵S] to CHO_GPR14 membranes in a GDP dependent manner (pIC₅₀; 5.50±0.05) with maximal agonist stimulated binding seen at low micromolar concentrations.

Assay/conditions	pEC50	Emax
GTP[35S] 0.1mM GDP	8.38±0.13	3554±190
GTP[35S] 5mM GDP	8.23±0.15	1153±41
Ca+2 (+extracellular Ca+2)	8.82±0.15	809±36
Ca+2 (-extracellular Ca+2)	8.25±0.07	546±85

Table 1. pEC₅₀ and E_max values for U-II stimulated GTP[³⁵S] binding and intracellular Ca²⁺ in CHOGPR14 cells. Data mean±s.e.mean for n3 experiments. Emax values are specific DPM for GTP[³⁵S] binding and nM for calcium studies.

Concentration response curves were constructed for U-II at 0.1 and 5µM GDP, table 1. In whole CHO_GPR14 cells U-II produced a biphasic increase in intracellular Ca²⁺. This was monophasic in the absence of extracellular Ca²⁺ indicating that U-II produces release from intracellular stores and Ca²⁺ entry. pEC₅₀ and Emax values for these responses are shown in table 1. Potency estimates for GTP[³⁵S] binding and intracellular Ca²⁺ responses were similar (<4-fold difference). At the level of intracellular Ca²⁺ the U-II analogue [Orn⁸]U-II behaved as a lower potency (8.14±0.14, +extracellular Ca²⁺) full agonist.In this study we have shown that activation of the G_q coupled GPR14 receptor with U-II can be measured both at the level of GTP[³⁵S] and intracellular Ca²⁺.

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