Three general mechanisms, with varying degrees of experimental support, appear to combine to allow GAPs to potentiate receptor activity and thus balance the monocycle of : scaffolding mechanisms that maintain receptor–G protein binding and/or block GAP action; ‘kinetic scaffolding’, i.e. enhanced association based on the lifetimes of specific GTPase cycle intermediates; and allosteric potentiation of receptor function that is mediated by the Gα subunit itself.
Receptor–GAP association, either direct or mediated by scaffolding proteins, has now been described at varying levels of detail for diverse receptors and GAPs in many cells (see [4
] for reviews). These interactions no doubt contribute significantly to the selectivity of GAPs for specific receptor pathways, but most studies focused on how receptor–GAP binding promotes inhibition of signaling. To some extent, this focus reflects the relative experimental ease of measuring inhibition of downsteam signaling in cells compared with the difficulty of monitoring the kinetics of isolated G-protein functions, but selective recruitment of GAPs as inhibitors is clearly widespread.
Two studies of receptor–GAP interaction do suggest that GAP may stimulate the receptor; however. Wang et al.
] found that neurabin, a scaffolding protein that binds both RGS proteins and GPCRs, potentiated Ca2+
signaling by the α1B
-adrenergic receptor both in cell culture and parotid gland ducts. Supporting data were consistent with the idea that neurabin acts by blocking the inhibitory effects of an RGS protein while both are bound to the receptor, although a detailed mechanism was not available. The result is interesting also because spinophilin, a close paralog of neurabin, inhibits G-protein signaling by recruiting RGS proteins to form an inhibited complex [18
]. A second but more ambiguous case is RGS4-induced inhibition of Gq
-mediated signaling in pancreatic acinar cells [9
]. Here, RGS4 inhibits m3 muscarinic cholinergic receptor signaling at only about 1% of the concentration needed to inhibit signaling by cholecys-tokinin, with the bombesin receptor displaying intermediate sensitivity. RGS2 did not display such selectivity among the three receptors. All three receptors and RGS4 acted through a single pool of Gq
, suggesting that some specific interaction led to the resistance of cholecystokinin signaling to inhibition by this GAP, although the mechanism is unknown. Signal termination kinetics were not monitored in these studies.
An alternative mechanism for how GAPs potentiate stimulation of G protein by the receptor was proposed specifically to explain the problem of combining robust signaling with a fast turn-off rate [12
] (). The mechanism, called kinetic scaffolding, describes a pathway of reactions through the GTPase cycle in which a GAP promotes the continuous association of receptor and G protein (, inner cycle). Put simply, GAP-stimulated GTP hydrolysis is fast enough that receptor does not have time to dissociate from the G protein-GTP complex, such that it is still bound and available to drive a new round of GDP–GTP exchange. Kinetic scaffolding thus obviates the slow, diffusion-limited association between the receptor and the GDP-bound G protein (, outer cycle) and shifts the rate-limiting (i.e. slowest) step in G-protein activation from receptor–G protein binding to receptor-driven GDP dissociation, which is far faster (). Note that kinetic scaffolding does not imply physical scaffolding or any thermodynamic enhancement of affinity, but merely a change in reaction path that is allowed because GTP hydrolysis is accelerated by the GAP. Conversely, however, if receptor and G protein remain bound as an active complex, their relative interactions might be expected to be even more efficient than if both were merely tethered close to each other by a separate protein.
Figure 4 Kinetic scaffolding limits receptor dissociation 
Several enzymologic studies support the importance of the kinetic scaffolding pathway [12
], but the association lifetime for receptor–G protein binding during GTPase cycle turnover has not yet been measured directly. Recently, Turcotte et al.
] used an experimentally determined set of rate constants for the complete GTPase cycle to simulate the reaction pathways for the m1 muscarinic cholinergic receptor and Gq
with and without PLC, which is both a GAP for Gq
and a Gq
effector. They found that PLC increases the fraction of G protein that stably associates with receptor during steady-state GTPase turnover—the basic concept of kinetic scaffolding. These simulations also describe the dependence of kinetic scaffolding on the concentration of GAP and on its maximal activity, and thus map the period over which direct measurements of binding should be made.
In addition to scaffolding mechanisms, analysis of steady-state GTPase activity has suggested that a GAP can also directly increase the rate of receptor-promoted GDP–GTP exchange. This provides yet a third mechanism for a GAP to maintain signal output by accelerating exchange to match fast hydrolysis. Turcotte et al.
] found that the rate of dissociation of GDP from a complex of the m1 muscarinic cholinergic receptor, Gq
and PLCβ1 is about 17-fold faster than its dissociation from the receptor–Gq
complex alone. This enhancement of the intrinsic GDP–GTP exchange rate combined with kinetic scaffolding to allow the receptor to maintain about 20% of the Gq
in the active state, despite a more than 1,000-fold increase in the rate of hydrolysis of Gq
-bound GTP. The physical mechanism whereby a GAP contributes to receptor-driven GDP dissociation is not clear. For example, GAP may either make the receptor a better exchange catalyst or make the G protein more responsive to receptor. Differentiating between these two possibilities will be difficult because a complex of all three proteins is required, and there is no information on its structure. There was no effect of GAP on the nucleotide exchange rate for Gq
in the absence of receptor, as is true for many GAP–G protein combinations [4
]. Significantly, the GAP in this case is also the effector whose signaling activity is stimulated by Gq
-GTP, and it will be interesting to see whether non-effector RGS proteins also display a similar effect.