The standard model of GPCR signal transduction had long been restricted to a three-component system: receptor, G-protein, and effector 1
. The seven-transmembrane domain receptor is coupled to a membrane-associated heterotrimeric complex composed of a GTP-hydrolyzing Gα subunit and a Gβγ dimeric partner. Agonist-induced conformational changes enhance the guanine nucleotide exchange activity of the receptor, leading to the release of GDP (and subsequent binding of GTP) by the Gα subunit. On binding GTP, conformational changes within the three `switch' regions of Gα allow the release of Gβγ. Separated Gα·GTP and Gβγ subunits are then free to propagate intracellular signaling via diverse effectors 2
. The intrinsic GTP hydrolysis (GTPase) activity of Gα resets the cycle by forming Gα·GDP which has low affinity for effectors but high affinity for Gβγ. In this way, the inactive, GDP-bound heterotrimer (Gα·GDP/Gβγ) is reformed and capable once again to interact with activated receptor.
Based on this cycle of receptor-catalyzed GTP exchange and intrinsic GTP hydrolysis by Gα, the duration of heterotrimeric G-protein signaling is thought to be controlled by the lifetime of the Gα subunit in its GTP-bound state. After the establishment of this basic model 1
, RGS proteins (“regulators of G-protein signaling”) were subsequently discovered 3–5
to bind Gα subunits (via a conserved ~120 amino-acid RGS domain) and dramatically accelerate their intrinsic GTPase activity 6
, thereby attenuating heterotrimer-linked signaling. Nearly 40 human proteins contain at least one RGS domain, with many of these proteins (e.g.
, RGS4, RGS16) serving as GTPase-accelerating proteins (GAPs) for Gαi/o
subunits, yet others such as RGS2 and p115-RhoGEF being particularly attuned to Gαq/11
substrates, respectively 7
. The discovery of this superfamily of Gα-directed GAPs resolved apparent timing paradoxes between observed rapid physiological responses mediated by GPCRs and the slow hydrolysis activity of the cognate G-proteins seen in vitro
. Thus, in this capacity as negative regulators of GPCR signal transduction, the RGS proteins present themselves as excellent potential drug discovery targets 7
. For example, pharmacological inhibition of RGS domain GAP activity should lead to prolonged signaling from G-proteins activated by agonist-bound GPCRs.
The most direct way to detect RGS protein function is by measuring the increased GTPase activity exhibited by its target Gα protein. However, accurate in vitro
measurements of Gα-catalyzed GTP hydrolysis are difficult to obtain without laborious biochemical reconstitutions with purified Gβγ and an activated GPCR (e.g.
, ref. 8
). In the absence of GPCR-mediated nucleotide exchange, it is GDP release (rather than GTP hydrolysis) that is the rate-limiting step in the Gα nucleotide cycle 9
. Thus, to examine the effect of an RGS protein in accelerating GTP hydrolysis by an isolated Gα subunit in vitro
, a single round of hydrolysis of radiolabelled GTP is usually performed (a.k.a.
the “single-turnover GTPase assay”; ref. 6
). This standard assay for measuring RGS domain-mediated GAP activity is low-throughput and requires discrete steps of [γ-32
P]GTP loading onto Gα, protein reactant admixture (with addition of the critical cofactor Mg2+
to initiate hydrolysis), isolation (in discrete time intervals) of released [32
P]phosphate with activated charcoal precipitation and centrifugation, and finally scintillation counting. We have described an alternative single-turnover GTPase assay 10
using a coumarin-labeled, phosphate-binding protein to facilitate fluorescence-based detection of inorganic phosphate production; however, this method demands stringent controls on multiple experimental steps to eliminate phosphate contaminants that interfere with the detection of GTPase activity. Such convoluted protocols of inorganic phosphate detection are difficult for the non-specialist and especially not suited for high-throughput screening (HTS) of large compound libraries for RGS domain inhibitors. We and others have reported alternative, fluorescence-based strategies for detecting the binding between RGS protein and Gα substrate 11–13
, but none has specifically facilitated a discrete endpoint measurement of RGS domain-mediated GAP activity per se
In order to develop a facile steady-state GTPase assay for RGS domain GAP activity, we first set out to increase the spontaneous GDP release rate of Gα (koff(GDP)
) while also decreasing its intrinsic rate of GTP hydrolysis (kcat(GTPase)
), thereby allowing detection of at least a five-fold enhancement of steady-state GTP hydrolysis by RGS proteins to provide an adequate signal-to-noise ratio. Gαi1
and closely related Gα proteins have been the focus of extensive structure/function studies 14–17
, and point mutations that affect both koff(GDP)
without affecting functional interaction with the RGS domain have been identified previously 15–18
, ). Two of the most striking Gα mutations have been made to the highly-conserved active-site arginine (R178C; ref. 15
), which causes a ~100-fold reduction in GTPase activity, and to the alanine residue within the conserved TCAT loop that contacts the guanine ring (A326S; ref. 16
), which results in a ~25-fold increase in koff(GDP)
relative to wildtype yet an identical kcat(GTPase)
Figure 1 Increased GDP release and decreased GTP hydrolysis of the Gαi1(R178M/A326S) mutant compared to wildtype Gαi1 and single point-mutants, as measured by [35S]GTPγS binding and single-turnover [γ-32P]GTP hydrolysis, respectively (more ...)
To detect RGS protein-accelerated GTPase activity, we adapted a monoclonal antibody and fluorescent tracer, previously developed for the Transcreener ADP assay 19
, for selective immunodetection of GDP with a fluorescence polarization readout. Measurement of GTPase activity using this Transcreener GDP assay overcomes the signal-to-noise limitations of phosphate detection methods and has been validated as a robust HTS method in the case of ADP detection for kinases and ATPases 20–22
. Moreover, because it is a catalytic assay rather than a substrate binding assay, it should enable detection of all types of modulators of RGS protein GAP activity, including those that bind at allosteric sites and affect RGS protein catalytic activity without directly targeting the RGS domain Gα binding-site 23
In this present study, we tested multiple point-mutant Gαi1 proteins with increased GDP dissociation and/or decreased GTP hydrolysis rates for their ability to enable detection of RGS domain GAP activity using a steady-state GTPase assay format (i.e., multiple rounds of turnover of GTP to GDP). Coupling one of these variants, Gαi1(R178M/A326S), to the Transcreener GDP detection system has not only allowed facile detection of RGS protein GAP activity, but was useful in helping establish (along with surface plasmon resonance spectroscopy) that the mutant Gαi1 interacted with RGS proteins with the same specificity and affinity as the wildtype Gαi1 protein.