Ada banyak sekali GTP-binding protein, atau biasanya disebut protein G, yang berperan penting dalam pensinyalan sel sebagai molekul penukar. G proteins store and transmit information based on their conformational state, and thus provide another canonical example of the tight coupling between protein activity and conformation.
Serving as the basis for one of the most widely and important signaling mechanisms in eukaryotes, G proteins play crucial roles in diverse signaling pathways including hormone signaling, cytoskeletal regulation, and nuclear import and export.
G proteins are named for their ability to bind guanine nucleotides-both guanosine triphosphate (GTP) and guanosine diphosphate (GDP). The primary function of G proteins is to serve as conformational switches: they adopt significantly different conformations depending on whether GTP or GDP is bound (Fig 3.25).
The GDP-bound conformation of the G protein is inactive, while the GTP-bound conformation is active and able to bind downstream effectors. Both nucleotide exchange and GTP hydrolysis are extremely slow in G proteins on their own. Activation (exchange of GDP for GTP) is speeded up by guanine nucleotide exchange factor (GEF) enzymmes, while inactivation is promoted by GTPase-activator protein (GAP) enzymes. The activity of GEFs and GAPs is regulated by signaling inputs.
In general, the GTP-bound state is the “active” conformation, which is capable of binding and modulating the activity of downstream effectors, while the GDP-bound state is the “inactive” conformation, with much lower affinity for these effectors.
G proteins are conformational switches controlled by two opposing enzymes
In the absence of regulatory inputs, cycling between the active and inactive states of G proteins is very slow. G proteins can hydrolyze bound GTP to GDP (thereby switching themselves from the active to inactive conformation), but they are very poor enzymes. In fact, the reaction half-time for GTP hydrolysis (the time at which there is a 50% chance that GTP will have been cleaved to GDP and Pi) ranges from several minutes to more than an hour for different G proteins. In addition, the release of bound GDP after hydrolysis also very slow-both GDP and GTP bind G proteins with high affinity (Kd in the nM to pM range), and thus their dissociation rates (Koff) are necessarily low.
This system is very useful for signaling, however, because regulatory enzymes can overcome these kinetic barriers. The nucleotide-binding state of G proteins is controlled by two opposing enzymes: guanine nucleotide exchange factors (GEFs), which activate G proteins, and GTPase-activator proteins (GAPs), which inactivate them.
GEFs activate G proteins by catalyzing the release of GDP and the subsequent binding of GTP. GAPs inactivate G proteins by catalyzing the hydrolysis of bound GTP to GDP. Thus, these opposing enzymes form a kinetically controlled “writer/eraser” system analogous to kinase/phosphatase systems described earlier in this chapter.
G protein regulation is also similar to phosphorylation because the cycle of regulatory reactions, while under tight kinetic control, is thermodynamically favorable.
Inactivation of a G protein is favorable because it is coupled to hydrolysis of the high-energy phosphodiester bond of GTP. Activation of a G protein, via rebinding of GTP, is favorable because the cell provides a constant excess of GTP over GDP (approximately tenfold excess), while the affinities and association rates for GTP or GDP are similar.
Thus, the cell’s production of GTP provides the energy to drive G protein signaling, while GEFs and GAPs provides the kinetic controls that harness this energy for regulatory control.
The presence of the GTP ɣ-phosphate determines the structure of G protein switch I and II regions
The nucleotides GTP and GDP differ only by the presence of a terminal phosphate on GTP (the three phosphates on GTP are designated as alpha, beta, and ɣ, starting from the nucleotide ring). Despite its small size, the highly charged ɣ-phosphate moiety plays a critical role in controlling the conformation of G proteins (Fig 3.26).
The guanine nucleotide binds in a conserved pocket. The ɣ-phosphate, when present, interacts with two loops known as switch I and switch II, forming hydrogen bonds with the main-chain atoms of two invariant Gly and Thr residues. These interactions cause significant structural rearrangements in switch I and II. Because these regions form a critical part of the binding site for downstream effectors, these conformational changes dramatically affect the ability of the G protein to bind such effectors. Thus, in a sense, the G protein functions to convert a single phosphate difference into a large conformational change.
(a) The GDP-bound conformation of a G protein is depicted schematically.
(b) In the GP-bound form, the gamma-phosphate group on the bound nucleotide hydrogen-bonds with the main-chain atoms of conserved Thr and Gly residues, leading to conformational rearrangements of the switch I and II regions of the protein. These arrangements create an effector-binding site. The amino acid numbering corresponds to the small GTPase Ras. See Figure 13.8 for a comparison of the X-ray crystal structures of the GDP- and GTP-bound forms of Ras.
In its GTP-bound conformation, a particular G protein often is capable of binding to many different downstream effectors. Thus, its activation can induce a variety of downstream effects through its interaction with different effectors. Although the switch I and II regions are critical for binding to all receptors, the other residues on the surface of the G protein that contribute to the binding site may vary from effector to effector.
For this reason, it has possible to construct specific point mutant of G proteins that bind to some effectors, but not to others. Such mutants can be helpful in teasing out which effectors are important for a particular downstream effect of G protein activation.
There are two major classes of signaling G proteins
A typical eukaryotic cell contains well over 150 different G proteins, involved in diverse signaling pathways. These G proteins can be divided into several distinct superfamilies, including two families that play key roles in cell signaling. The first family is the small G proteins; these monomeric G proteins are often referred to as small GTPases.
The second family is the heterotrimeric G proteins, which have alpha, beta, and gamma subunits. A third family of G proteins, which we will not discuss here, plays a central role in the translation (this family includes elongation factor EF-tu).
All of the G protein superfamilies have at their core a 20 kD G domain, which binds the guanine nucleotide and can adopt alternative conformations depending on whether GDP or GTP is bound. The small G proteins essentially consist of a single G domain, while the heterotrimeric G proteins contain a G domain in their Galpha subunits (Fig 3.27). Below, we describe the mechanisms of regulation of both classes of G proteins.
X-ray crystal structures of (a) Ras, a small G protein, and (b) a heterotrimeric G protein complex. Bound GDP (orange) is shown in ball-and-stick format. The alpha subunit of the heterotrimeric G protein consists of a G domain (purple) homologous to small G proteins, and an additional helical domain (blue). The beta (yellow) and gamma (pink) subunits are tightly associated via a coiled-coil interaction.
Subfamilies of small G proteins regulate diverse biological functions
The small G proteins consist of a single 20-25 kD domain. The founding member of this family is Ras, a central regulator cell proliferation and differentiation. Ras was first identified as an oncogene (a gene whose dysregulated activity can lead to the uncontrolled growth characteristic of cancer). There are approximately 150 G proteins in humans, and these can be further subdivided into at least five majors subfamilies: the Ras, Rho, Rab, Arf, and Ran families (Table 3.1).
Each of these subfamilies is, in general, involved in regulating distinct cellular functions:
- Ras proteins regulate the cytoskeleton and control cell shape and movement;
- Rab and Arf protein regulate membrane vesicle-associated processes including vesicle formation, trafficking, and secretion;
- Ran proteins control nuclear export and import, formation of the nuclear envelope, and mitotic spindle formation.
Table 3.1 Small G protein subfamilies; functions, downstream effectors, and upstream GEF and GAP domain
Not only do G protein families differ in the particular set of downstream effector functions that they control, but even within these families there are further functional subdivisions. For example, the Rho family G proteins, in their active state, interact uniquely with key cytoskeletal regulatory proteins.
The approximately 25 human Rho subfamily members can be further divided, however, into the RhoA, Rac1, and Cdc42 subclasses. Each subclass is associated with distinct functions in cytoskeletal regulation. For example, Rac 1-related G proteins are associated with formation of actin-based protrusive structures such lamellipodia, while RhoA-related G proteins are associated with formation actin-myosin contractile structures (Fig 3.28).
Rac activity (green) is concentrated at the leading edge of the cell, where it promotes actin-mediated protrusion. Rho activity (orange) is concentrated at the back of the cell, where it promotes actin-myosin-mediated contraction.
Many upstream receptors feed into a small set of common heterotrimeric G proteins
Heterotrimeric G proteins contain three subunits- alpha, beta, and gamma. The 50 kD Gaplha subunit contains the conserved 20-25 kD G domain that is homologous to the small G proteins. This domain binds GTP or GDP and regulates the conformational change of the protein. The Galpha subunit also contains an unrelated helical domain. The Gbeta and Ggamma subunits do not themselves have any enzymatic activity.
When the Galpha subunit is bound to GDP, it also associates with the Gbeta and Ggamma subunits to form heterotrimer. However, when the Galpha subunit is in the GTP-bound, or “active,” state, it dissociates from the Gbeta and Galpha subunits (which stay tightly associated to each other) (Figure 3.29).
Dissociation of the subunits occurs because the switch I and II regions in the Galpha subunit-the regions that undergo the largest nucleotide-dependent conformational shifts- from part of the heterotrimer binding interface.
In the basal state, the Galpha subunit is bound to GDP and in complex with the Gbeta gamma subunits. Activation of the GPCR by ligand binding leads to conformational changes that induce its guanine nucleotide exchange factor (GEF) activity, resulting in exchange of GDP tor GTP in the Galpha subunit. The resulting conformational changes in the Galpha subunit lead to its dissociation from the receptor and from the Gbeta gamma subunits. The Galpha and Gbeta gamma subunits then are competent to bind downstream effectors. Reversion to the GDP-bound state leads to reassociation of the hetertrimeric complex. GAP, GTPase-activator protein; RGS, regulator of G protein signaling.
When dissociated, both the Galpha and Gbeta gamma subunits can bind to various downstream effectors (channels and enzymes like adenylyl cyclase), changing their activity and thus leading to biological effects. Heterotrimeric G proteins are activated by G-protein-coupled receptors (GPCRs). In humans, there are many hundreds of distinct GPCRs, making them the most highly represented class of signaling proteins.
There are only 16 genes for Galpha subunits in humans, which can be divided into four main families: Gs alpha, Gi alpha, Gq/11 alpha, and G12/13 alpha. Although these Galpha subunits have a similar mechanism of activation, they have different effector-binding properties (Table 3.2).
For example, the Gs alpha isoform is primarily responsible for activation of adenylyl cyclase and production of the signaling mediator cAMP. Thus, a large number of diverse upstream receptors feed into set of common G proteins.
Table 3.2 Families of Galpha subunits and their effectors
The mechanism by which downstream signaling is directed to specific functional outputs using this limited set of common G proteins is still unclear. However, there is growing evidence that mechanisms such as cell-spesific expression of receptors and restricted subcellular localization mediated by scaffold proteins can help to limit and direct the downstream effectors that are targeted by specific receptor-G protein complexes.