AGONIST ACTION AT D-2(SHORT) DOPAMINE-RECEPTORS DETERMINED IN LIGAND-BINDING AND FUNCTIONAL ASSAYS

Mechanisms of agonist action at the G protein-coupled D-2(short) dopam ine receptor expressed in Chinese hamster ovary cells have been invest igated. Agonist binding was assayed in the presence and absence of GTP (100 mu M). Data in the absence of GTP were fitted best by a two-site model (apomorphine, dopamine, 10,11-dihydroxy-N-n-propylnorapomorphin e hydrochloride, and quinpirole) or a one-site model [bromocriptine, d ihydroergocristine, and (-)-3-(3-hydroxyphenyl)-N-propylpiper idine hy drochloride], whereas in the presence of GTP a one-site model was the best fit for all compounds. Agonist binding parameters were used to pr ovide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [S-35]g uanosine 5'-O-(3-thiotriphosphate) ([S-35]GTP gamma S) binding for a r ange of agonist concentrations was measured and the EC50 and maximal e ffects determined. The initial rates of [S-35]GTP gamma S binding indu ced by maximally stimulating agonist concentrations were also recorded . Simultaneous inhibition of agonist-stimulated [S-35]GTP gamma S bind ing and receptor occupancy by spiperone was determined. Agonist inhibi tion of forskolin-stimulated cyclic AMP accumulation was determined fo r a range of agonist concentrations and the EC50 and maximal inhibitio n recorded. The data on the maximal agonist responses showed that it w as possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencie s of agonists to elicit the functional responses showed that different extents of amplification of response were seen for different agonists in both assays, The maximal activity data have been compared with the stabilisation of the agonist/receptor/G protein ternary complex as me asured in binding assays.