The first step in this complex signalling system involves the binding of specific ligands (hormones, neurotransmitters, growth factors, glycoproteins, cytokines, odorants and photons) at the cell surface to a GPCR, thereby activating the receptor. The signal is transmitted into the cell via a conformational change in the receptor, which results in the activation of the bound G protein. GPCRs act as guanine nucleotide exchange factors for the a subunit of the G protein, whereby activated receptor promotes the exchange of bound GDP (guanine diphosphate) for GTP on the a subunit, which is the rate-limiting step in G protein activation. The binding of GTP changes the conformation of ‘switch’ regions within the a subunit, which allows the bound trimeric G protein (inactive) to be released from the receptor, and to dissociate into active a subunit (GTP-bound) and bg dimer. The a subunit and the bg dimer go on to activate distinct downstream effectors, such as adenylyl cyclase, phosphodiesterases, phospholipase C, Src, and ion channels. These effectors in turn regulate the intracellular concentrations of secondary messengers, such as cAMP, cGMP, diacylglycerol, IP3, DAG, arachidonic acid, sodium, potassium or calcium cations, which ultimately lead to a physiological response, usually via the downstream regulation of gene transcription. The cycle is completed by the hydrolysis of a subunit-bound GTP to GDP, resulting in the re-association of the a and bg subunits and their binding to the receptor, which terminates the signal.
Heterotrimeric G protein activity is regulated by the binding and hydrolysis of GTP by the a subunit. Only the GTP-bound a subunit is active. A GDP-bound a subunit is inactive, because the ‘switch’ regions in the a subunit are free to make contacts with the bg dimer to form a stable complex that does not readily allow GDP dissociation. The association of the subunits covers the effector contact sites on both the a subunit and the bg dimer, thereby preventing all effector interactions and terminating the signal. Therefore, the length of the G protein signal is controlled by the duration of GTP-bound a subunit.
However, by itself, the intrinsic level of GTP hydrolysis by the a subunit is too slow for the efficient cycling of G proteins. The lifespan of the GTP-bound a subunit can be markedly reduced by RGS (regulator of G protein signalling) proteins (InterPro entry IPR000342). RGS proteins are multi-functional, GTPase-accelerating proteins that promote a subunit GTP hydrolysis, thereby directly terminating a subunit signalling and indirectly terminating bg dimer signalling (through a subunit binding). RGS proteins promote GTP hydrolysis by stabilising the G protein transition state, increasing the reaction rate by over two orders of magnitude. There are over thirty known RGS proteins characterised in the human proteome alone. All RGS proteins contain an ‘RGS-box’ required for activity, while some also contain additional domains that confer further functionality, such as coordinating cross-talk between heterotrimeric and Ras-like G proteins. In addition, some C-family RGS proteins contain GGL (G protein g-like) domains that can interact with b subunits to form novel dimers that prevent g subunit binding. For example, in brain tissue b5 subunit/RGS dimers have been detected, which may accelerate the deactivation of GPCR signalling by enhancing GTP hydrolysis; they may also prevent heterotrimer formation, as b subunit/RGS dimers do not appear to associate with a subunits.
The covalent modification of G proteins is another way to regulate their activity. Heterotrimeric G proteins can undergo a variety of covalent modifications, including lipid attachment and phosphorylation. Both the a subunit and bg dimer carry lipid modifications that target them to the membrane and affect subunit interactions with each other and with other proteins. For example, some a subunits can contain covalently attached myristate or palmitate moieties, which target the a subunit to the plasma membrane and contribute to the strength of interaction with the bg dimer, effectors and RGS proteins. Because the absence of lipid modifications renders a subunits inactive, the regulation of reversible palmitoylation is a means of regulating a subunit activity. Protein modifications of the b and g subunits are equally important, with the isoprenylation of the g subunit with either farnesyl or geranylgeranyl groups being required for effector regulation.
Phosphorylation of G proteins appears to play a role in signal amplitude and duration. Some a subunits can be phosphorylated by protein kinase C or by p21-activated protein kinase, which inhibits their interaction with either bg dimers or with RGS proteins, thereby prolonging the signal. Conversely, the phosphorylation of a g subunit by protein kinase C increases the strength of its interaction with an a subunit and decreases its interaction with effectors, thereby quenching a signal. The phosphorylation of bg dimers may also act as an activation step by providing receptor specificity. Receptor activation can change the pattern of G protein modifications.