Cannabinoid Receptor Agonist Efficacy for Stimulating [35S]GTPγS Binding to Rat Cerebellar Membranes Correlates with Agonist-induced Decreases in GDP Affinity

Abstract

Cannabinoid receptors mediate the actions of ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC) 1 and other cannabimimetic ligands (1). To date, two types of cannabinoid receptors have been discovered, CB1 (2, 3) and CB2 (4). A splice variant of CB1, termed CB1A, has also been reported (5). Apart from a recent report of CB2 in mouse cerebellum (6), CB1 has been the only cannabinoid receptor found in brain. All cannabinoid receptors discovered to date belong to the superfamily of G-protein-coupled receptors (3, 4); their effectors include inhibition of adenylyl cyclase (7, 8), inhibition of calcium influx (9), and activation of inwardly rectifying potassium channels (10, 11). The physiological actions of cannabinoid ligands have been shown to be mediated through the activation of pertussis toxin-sensitive G-proteins (G i ␣ and G o ␣ subtypes) (7, 12), although some effects have been implicated via G s ␣ as well (13, 14).G-proteins are heterotrimeric proteins that transduce the agonist binding signal from G-protein-coupled receptors to effectors (15, 16). Upon activation by an agonist-occupied receptor, the ␣ subunit of a G-protein (G␣) releases bound GDP, binds a molecule of GTP, and dissociates from the G-protein ␤␥ subunit complex. Both G␣ and ␤␥ subunits act upon effectors until G␣ cleaves the bound GTP to GDP by its intrinsic GTPase activity, and G␣ re-associates with a ␤␥ dimer (15, 16). The cycle is then complete, and the heterotrimeric G-protein is able to be activated again. Receptors act catalytically, as one receptor can activate multiple G-proteins (17)(18)(19). The activation and dissociation of the G-protein subunits occur very rapidly and thus do not appear to be rate-limiting steps in the signal transduction cascade (20). However, since the actions of G-protein-coupled receptors are mediated strictly via the activation of G-proteins, this step plays a key role in determining overall agonist efficacy (21) and may be the most relevant step in measuring agonist efficacy at G-protein-coupled receptors (22).Agonist-stimulated binding of the hydrolysis-resistant GTP analog, [35 S]GTP␥S, to G-protein ␣ subunits measures receptor activation of G-proteins in purified and reconstituted systems (23), native cell membrane preparations (24), and brain sections (25). The present study focuses on three aspects of the role of GDP in the agonist-stimulated [35 S]GTP␥S binding assay. First, GDP has been shown to decrease basal [35 S]GTP␥S binding and allow detection of agonist stimulation. The requirement for micromolar concentrations of GDP to observe agonist effects in native membrane preparations has been reported consistently in every system for which agonist-stimulated [ 35 S]GTP␥S binding has been demonstrated (24, 26 -28

Dose-Related Differences in the Regional Pattern of Cannabinoid Receptor Adaptation and in Vivo Tolerance Development to Δ9-Tetrahydrocannabinol

“…This observation is consistent with studies in cerebellar membranes showing that CP55,940 is a high-efficacy partial agonist at CB 1 receptors, whereas WIN55,212-2 is a full agonist (Breivogel et al, 1998 (Sim et al, 1996). No other changes in basal […”

Relationship between Rate and Extent of G Protein Activation: Comparison between Full and Partial Opioid Agonists

“…35 S]GTP␥S binding is the magnitude of the decrease in the affinity of G protein for GDP (Breivogel et al, 1998). The findings could also be explained by a decreased affinity of the partial agonist-bound receptor for G protein compared with full agonist-bound receptor; such as is observed with reconstituted M2 receptor coupling to Gi (Tota and Schimerlik, 1990).…”

“…The activity of agonists acting on such systems can be determined by measuring the agonist-stimulated binding of the stable GTP analog [ 35 S]GTP␥S, to ␣-subunit proteins (Traynor and Nahorski, 1995). The potency of an agonist in this system is then defined by its EC 50 for stimulating [ 35 The differential ability of agonists to activate heterotrimeric G protein-linked receptors has been attributed to the affinity of ligand bound-receptor for G protein (Tota and Schimerlik, 1990) as well as to the ability of the agonist bound-receptor to initiate GDP dissociation from G protein (Breivogel et al, 1998). Although the relative orientation of receptors and G proteins and the surfaces through which they interact with one another have been partially defined (Bourne, 1997) our understanding of protein conformational changes that may account for differences between full and partial agonists is limited (Burgen, 1981;Kenakin, 1995a,b).…”

A Combinatorial G Protein-coupled Receptor Reconstitution System on Budded Baculovirus

“…9). Previous studies demonstrate that the agonist increases the apparent affinity of [ 35 S]GTP␥S binding sites; it also decreases the affinity of GDP binding sites (44,45). The binding of [ 35 S]GTP␥S was greatly increased by LTB 4 in BV co-expressing BLT1 and G i or G o heterotrimeric G proteins.…”