GTP Binding Assays

A GTPgS binding assay detects the level of G protein activation following agonist stimulation. In the resting stage, G proteins exist as Ga(GDP)pg heterotrimers with GDP bound to Ga; there is a low rate of GDP-GTP exchange. Subsequent to agonist stimulation of the receptor, GDP dissociates from Ga, which allows GTP to bind to Ga. Agonist-mediated GPCR activation increases the rate of guanine nucleotide exchange and allows the dissociated G protein subunits to interact with effector proteins to produce functional responses. In the GTPgS binding assay, occupancy of an agonist to a receptor can be measured as an increase in the binding of the nonhydrolyzable GTP analog [35S] GTPgS to cell membranes. Generally, in the GTPgS binding assay, [35S] GTPgS replaces the endogenous GTP and binds to the Ga subunit to form Ga[35S] GTPgS. [35S] GTPgS binding assays are best suited for Gi-coupled receptors because Gi proteins are expressed at higher levels and have higher GDP-GTP exchange rates than other G proteins.29

A critical aspect of the [35S] GTPgS binding assays is the suppression of basal binding of the labeled nucleotide to unstimulated G proteins using high concentrations of GDP. The amount of GDP needed for optimal agonist stimulation of [35S] GTPgS binding varies across several receptor systems. For the agonist-induced responses at muscarinic Ml and M3 receptors expressed in CHO or human embryonic kidney cells, low levels (0.1 |M) of GDP are required. In membranes from the same cell type, however, muscarinic M2 and M4 receptor-stimulated GTP binding requires 10-fold higher levels of GDP.30,31 Therefore, a user should titrate concentrations of GDP ranging from 0.1 to 10 |M for each membrane receptor.

GTP binding is always performed in presence of magnesium and sodium ions and the concentrations of these ions should be optimized for individual assays. Mg2+ increases both basal and agonist-stimulated [35S] GTPgS binding and has a preferential effect on the activated state that results in higher signals.32,33 Typically Mg2+ ions are kept at a 1 to 10 mM range.

Na+ ions decrease the basal [35S] GTPgS binding and can improve the signal-to-noise ratio.34 Na+ ions exert their effects through two possible mechanisms: allosteric regulation of receptor properties and changing the affinity of G proteins for GDP. In some systems, the concentration of sodium ions is inversely related to agonist efficacy. An agonist can behave partially or fully at high and low sodium concentrations, respectively. Therefore, care should be taken in fixing the concentration of sodium in the assay. Typically, sodium concentrations are optimized in the range of 0 to 200 mM. Some membranes may require mild detergents such as saponin in order for the GTP to reach the G protein.

Optimization of membrane protein reactions should be tested to achieve maximal stimulation with the agonist, but this varies with the receptor expression level in the membrane preparation. The increased background observed with Gs-and Gq-coupled receptors could be reduced by utilizing C-terminal anti-Gas or anti-Gaq antibodies to immunoprecipitate the [35S] GTPgS-bound a-subunit or to bring it in close proximity to the SPA beads. Another approach is to fuse the Gs or Gq receptor of interest to Gi to couple the activation of Gi indirectly to make the assay feasible in GTPgS-binding format.35 In homogeneous assay formats, the receptor preparation is immobilized onto WGA-coated SPA beads or onto the surfaces of FlashPlates.36 Results from the conventional GTPgS binding assay correlated well with FlashPlate and SPA formats; therefore, the application of these homogeneous methods would increase throughput for HTS campaigns using the GTPgS binding assay.

A nonradioactive GTP binding assay with a europium-labeled GTP derivative has been developed for a variety of GPCRs. This format requires a filtration step to remove the unbound Eu-GTP, and therefore its use for primary screening may be rather limited. The amount of Eu-GTP bound to the receptor is retained on the filter and detected by time-resolved fluorometry.37 GTPgS binding assays can be used to search for candidate compounds for known, orphan, and constitutively activated GPCRs. Because this method measures the signal earliest in receptor-mediated events, it is not susceptible to feedback or amplification mechanisms that occur in downstream signaling pathways. However, as [35S] GTPgS does not cross the cell membrane, this assay cannot be used to evaluate receptor activation in intact cells.

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