The molecular mechanisms that underlie agonism have been investigated for the D₂ dopamine receptor (DAR). These studies have concluded that agonists can influence receptor mediated G-protein activation at two points in this process; i) stabilisation of ternary complex formation and ii) breakdown/activation of the ternary complex (Gardner & Strange, 1998). We have extended this analysis to the D₃ DAR to determine if agonists display a similar mechanism of action at other members of the dopamine receptor family. To enable this analysis we have quantified agonist binding and function for G-protein activation and inhibition of cAMP formation at the human D₃ DAR expressed stably in CHO cells.

Agonist affinities were measured using a scintillation proximity assay (SPA). Compounds were incubated with 1.25nM [³H]-7OH-DPAT for 120min at room temperature in buffer (50mM HEPES, 100mM NaCl, pH 7.4) with 200µg of WGA-Ysi SPA beads that had been pre-coupled to membranes from CHO cells stably expressing the human D₃ DAR (10µg membrane protein/mg bead). G-protein activation was measured in a [³⁵S]GTPS binding assay as described by Gardner & Strange (1998) with the following exceptions; 3µM GDP and 0.2nM [³⁵S]GTPS. Inhibition of cAMP was quantified in a whole cell assay. Briefly 5x10⁵ cells/well were incubated with 10µM forskolin and compound for 15 min at 37°C. Cells were lysed and cAMP quantified using a cAMP Flashplate® assay kit (Perkin Elmer).

The affinities (pKi) and functional parameters (pEC₅₀) and intrinsic activity (IA: expressed as % of 1µM dopamine) of three agonists was determined (Table 1). It was observed that preclamol and ropinirole were partial agonists in the GTPS assay but had higher efficacy in the cAMP assay. However, pramipexole was a full agonist, relative to dopamine, in both assays (Table 1).

From the values in Table 1 operational efficacies () were calculated (=(K_i/EC₅₀)-1). It is clear from these results that the expected correlation between and %IA is not observed (%IA=/(+1)) (Black & Leff, 1983). This is similar to the earlier observations with the D₂ DAR (Gardner & Strange, 1998) and consistent with the conclusion that agonism results from at least two independent steps; i) promotion/stabilisation of the ternary complex and ii) breakdown/activation of the ternary complex.

Table 1: Characterisation of the human D₃ DAR using binding, GTPS and cAMP assays. Results are mean ± sem (n).

Assay		Agonist
		Pramipexole	Ropinirole	Preclamol
Binding	pKi	8.01 ± 0.02 (66)	6.92 ± 0.10 (14)	6.59 ±0.03 (5)
GTPS	pEC50	7.85 ± 0.06 (12)	6.79 ± 0.17 (6)	6.66 ±0.35 (3)
	%IA	97 ± 2	60 ± 6	28 ± 4
cAMP	pEC50	8.91 ± 0.03 (143)	8.35 ± 0.07 (9)	7.96 ±0.21 (5)
	%IA	108 ± 1	89 ± 7	94 ± 12

Gardner, B. & Strange, P.G. (1998) Brit. J. Pharmacol. 124, 978-984.