Coordinating speed and amplitude in G-protein signaling

Abstract

G-protein-mediated signaling is intrinsically kinetic. Signal output at steady state is a balance of the rates of GTP binding, which causes activation, and of GTP hydrolysis, which terminates activation. This GTPase catalytic cycle is regulated by receptors, which accelerate GTP binding, and GTPase-activating proteins (GAPs), which accelerate hydrolysis. Receptors and GAPs similarly control the rates of signal initiation and termination. To allow independent control of signal amplitude and of the rates of turning the signal on and off, the activities of receptors and GAPs must be coordinated. Here, the principles of such coordination and the mechanisms by which it is achieved are discussed.

Figure 1. G protein switches follow a monocycle composed of GTP binding, GTP hydrolysis and GDP release

The GTP-bound state (G*-GTP) is defined as ‘active’, but G-GDP may also have its distinct regulatory and protein-binding activities. The fractional activation of G protein is described simply by the k_on and k_off rate constants, as described in the text, even though they may summarize several intermediary reactions. Each step in the GTPase cycle is unusually slow for a typical enzyme and is subject to multiple kinetic controls. Guanine nucleotide exchange factors (GEFs) accelerate GDP dissociation and, in the case of heterotrimeric G proteins, GTP binding. GEFs for heterotrimeric G proteins are cell-surface transmembrane receptors; GEFs for monomeric G proteins are heterogeneous. GTPase-activating proteins (GAPs) accelerate hydrolysis of bound GTP, which is rapidly followed by dissociation of orthophosphate. Some GAPs are also G-protein-regulated effectors. GDP dissociation inhibitors (GDIs) stabilize GDP binding and thus inhibit activation. The Gβγ subunits are GDIs, among their many other functions. ‘Effector’ refers to any protein that is regulated by a G protein, whether by the GTP-binding Gα subunit or by Gβγ. The nucleotide-free G protein, an obvious intermediate in the exchange reaction, is not shown because it is calculated to have a very short half-life at cytosolic GTP concentrations (~2 ms for Gα_q).

Figure 2. GAPs can accelerate response kinetics without substantially inhibiting steady-state signaling

(A) Currents generated by mouse photoreceptor cells decay slowly after a light flash if the GAP is absent. Rod outer segments, which contain the rhodopsin–transducin G-protein module, were taken from wild-type mice (black trace) or from mice either lacking RGS9-1 (red trace), the principle photoreceptor GAP, or heterozygous for RGS9-1 (green trace): traces show responses to a single photon. While the amplitude of the downstream current spike is essentially unchanged in the GAP^−/− cells, the current decay after the flash is extremely long-lived. (Reproduced with permission from Chen et al. [54].) (B) Co-expression of a GAP is required to reconstitute the native kinetics of a Gβγ-gated potassium (K⁺) channel that is normally expressed in cardiac myocytes. K⁺ channel subunits Kir3.1/3.2 and m2 muscarinic cholinergic receptors were co-expressed in CHO fibroblasts with or without the relatively non-selective GAP RGS4. K⁺ current was monitored during exposure to acetylcholine (ACh) for the period highlighted in grey. The lower trace shows a cell that expresses RGS4. Its response to ACh resembles that of an atrial myocyte, shown in the top trace, both in amplitude and kinetics. CHO cells that do not express RGS4 (middle trace) display a markedly slow recovery after agonist is removed. Average time constants for channel deactivation (τ_deact) are shown at the right: τ_deact is the inverse of the apparent rate constant k_app for deactivation. Deactivation rates upon removal of agonist are at least 10-fold faster in the presence of RGS4. Reference bars show 0.2 nA (vertical) and 5 s (horizontal). For a simple monocycle of the sort shown in Figure 1, k_app = k_on + k_off. Because k_on is small when agonist is absent, the difference in τ_deact reflects the difference in k_off. (Reproduced with permission from Doupnik et al. [23].) These phenomena have been observed for numerous G-protein-gated K⁺ and Ca²⁺ channels [4], but are crucially dependent on the stoichiometric relationships among receptor, G protein and GAP in the membrane [8].

Figure 3. Balancing on the monocycle

To allow regulation of turn-on and turn-off rates while independently regulating steady-state signal output, it is necessary to coordinate activities of receptors and GAPs. In principle, there are two solutions. Either the receptor must inhibit the activity of the GAP (red) or the GAP must potentiate the activity of the receptor (green). In the first mechanism, the receptor must inhibit the GAP in a way that does not depend on its activation of the G protein and that itself turns off rapidly when agonist is removed. In contrast, potentiation of the receptor by GAP may be unregulated and may be propagated by the G-protein heterotrimer or Gα.

Figure 4. Kinetic scaffolding limits receptor dissociation [4]

The two concentric loops describe stereotyped paths through the GTPase cycle: the fast inner cycle that predominates in the presence of a GAP and the slower outer cycle that is traversed in the absence of GAP. The GTPase reactions that describe kinetic scaffolding are shown in the inner cycle, with rates of key reactions shown as average lifetimes (τ = 1/k). The key branch-point species is the activated complex of receptor–G protein-GTP–GAP (R–G*-GTP–GAP). Because GTP hydrolysis is more than 100-fold faster than dissociation of receptor, the receptor remains bound after hydrolysis and can rapidly catalyze GDP/GTP exchange. This cycle can maintain about 25% of G protein in the active state as long as receptor is activated. The rate constants shown are for m1 muscarinic cholinergic receptor, G_q and PLCβ1 at 30°C [22], and the GTP association rate assumes 200 µM GTP (cyto-solic concentration). Detailed analysis shows that the GAP does not remain tightly bound throughout the GTPase cycle as shown in the inner cycle, but is in rapid binding equilibrium with R–G-GDP [22]. The slower collisional coupling path, shown in the gray outer cycle, proceeds in the absence of a GAP. Because the GTP-bound species has a long lifetime, about 10 s, receptor dissociates during every catalytic cycle and the rate-limiting step becomes the diffusion-limited rebinding of receptor and G protein [55].