Agonist Action at D_2(short) Dopamine Receptors Determined in Ligand Binding and Functional Assays

Abstract

Mechanisms of agonist action at the G protein‐coupled D2(short) dopamine receptor expressed in Chinese hamster ovary cells have been investigated. Agonist binding was assayed in the presence and absence of GTP (100 µM). Data in the absence of GTP were fitted best by a two‐site model (apomorphine, dopamine, 10,11‐dihydroxy‐N‐n‐propylnorapomorphine hydrochloride, and quinpirole) or a one‐site model [bromocriptine, dihydroergocristine, and (−)‐3‐(3‐hydroxyphenyl)‐N‐propylpiperidine hydrochloride], whereas in the presence of GTP a one‐site model was the best fit for all compounds. Agonist binding parameters were used to provide a measure of the ability of the agonist to stabilise the ternary complex of agonist/receptor/G protein. Agonist stimulation of [35S]guanosine 5′‐O‐(3‐thiotriphosphate) ([35S]‐GTPγS) binding for a range of agonist concentrations was measured and the EC50 and maximal effects determined. The initial rates of [35S]GTPγS binding induced by maximally stimulating agonist concentrations were also recorded. Simultaneous inhibition of agonist‐stimulated [35S]GTPγS binding and receptor occupancy by spiperone was determined. Agonist inhibition of forskolin‐stimulated cyclic AMP accumulation was determined for a range of agonist concentrations and the EC50 and maximal inhibition recorded. The data on the maximal agonist responses showed that it was possible to detect a spectrum of agonist efficacy (partial and full agonism) in both functional assays. The data on the apparent potencies 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 measured in binding assays.

Agonist Regulation of D2 Dopamine Receptor/G Protein Interaction

“…There is no clear relationship between the maximal effects of the agonists in [ 35 S]GTP␥S binding assays and the K l /K h ratio. Therefore, stabilization of receptor/G protein coupling is not a clear predictor of agonist efficacy, and similar results were seen in other studies on the D 2 receptor expressed in CHO cells (24,25). Agonists may, therefore, influence the activity of the ternary complex as well as its formation (7,8,24,25).…”

“…Table IV. (23)(24)(25). The present system is, therefore, behaving similarly to a system in which the receptor couples exclusively with endogenous mammalian G proteins.…”

“…The K l /EC 50 ratio (or amplification ratio (24,25,46)) indicates the extent to which agonist activation of a response occurs at lower concentrations than agonist binding to the receptor and so is a measure of receptor/G protein activation. The K l /EC 50 ratio of the agonists is greater in the preparation containing G o than in the preparation containing G i2 , providing a further indication that there is a more productive interaction between the D 2 receptor and G o .…”

Mechanisms of Agonism and Inverse Agonism at Serotonin 5‐HT1A Receptors

“…The agonist affinity ratio, therefore, has some value in predicting that a compound will possess efficacy as an agonist, but it clearly is unable to predict in detail. One reason for this may be that this ratio estimates only a part of the stabilisation of the ternary complex, and the activity of the ternary complex may depend on the agonist used, as discussed elsewhere (Gardner et al, 1997;Gardner and Strange, 1998;Strange, 1998).…”

“…The agonist affinity ratio, therefore, has some value in predicting that a compound will possess efficacy as an agonist, but it clearly is unable to predict in detail. One reason for this may be that this ratio estimates only a part of the stabilisation of the ternary complex, and the activity of the ternary complex may depend on the agonist used, as discussed elsewhere (Gardner et al, 1997;Gardner and Strange, 1998;Strange, 1998).One concern about using [ 35 S]GTP␥S binding to assess agonist efficacy is that it may provide different estimates of efficacy as compared with other assays such as inhibition of adenylyl cyclase, given that the two assays are likely to be subject to different degrees of amplification. This does not seem to be the case in the present cell system.…”