Multiple forms of heterotrimeric G proteins exist in the nervous system

Three types of heterotrimeric G protein were identified in early studies. G_t, termed transducin, was identified as the G protein that couples rhodopsin to regulation of photoreceptor cell function (see Chap. 47), and G_s and G_i were identified as the G proteins that couple plasma membrane receptors to the stimulation and inhibition, respectively, of adenylyl cyclase, the enzyme that catalyzes the synthesis of cAMP (see Chap. 22).

Since that time, over 35 heterotrimeric G protein subunits have been identified by a combination of biochemical and molecular cloning techniques [1–5]. In addition to G_t, G_s and G_i, the other types of G protein in brain are designated G_o, G_olf, G_gust, G_z, G_q and G_11–16. Moreover, for most of these G proteins, multiple subtypes show unique distributions in the brain and peripheral tissues.

Each G protein is a heterotrimer composed of single α, β and γ subunits

The different types of G protein contain distinct α subunits, which, in part at least, confer the specificity of functional activity. The types of G protein α subunit are listed in Table 20-1, and are categorized based on their structural and functional homologies. Current nomenclature identifies several subfamilies of G protein α subunit: G_αs, G_αi, G_αq and G_α12. The M_r of these proteins varies between 38,000 and 52,000. As a first approximation, these distinct types of α subunit share common β and γ subunits. However, multiple subtypes of β and γ subunits are now known: five β subunits of M_r 35,000 to 36,000 and seven γ subunits of M_r 6,000 to 9,000. These proteins show distinct cellular distributions, and differences in their functional properties are now becoming apparent [1–5].

Table 20-1

Heterotrimeric G Protein α-subunits in Brain.

The functional activity of G proteins involves their dissociation and reassociation in response to extracellular signals

This is shown schematically in Figure 20-1. In the resting state, G proteins exist as heterotrimers that bind GDP and are associated with extracellular receptors (Fig. 20-1A). When a ligand binds to and activates the receptor, it produces a conformational change in the receptor, which in turn triggers a dramatic conformational change in the α subunit of the G protein (Fig. 20-1B). This conformational change leads to (i) a decrease in the affinity of the α subunit for GDP, which results in the dissociation of GDP from the α subunit and the subsequent binding of GTP as the cellular concentration of GTP is much higher than that of GDP; (ii) dissociation of a βγ subunit complex from the α subunit; and (iii) release of the receptor from the G protein (Fig. 20-1B,C). This process generates a free α subunit bound to GTP as well as a free βγ subunit complex, both of which are biologically active and can regulate the functional activity of effector proteins within the cell [1–5]. The GTP-bound α subunit is also capable of interacting with the receptor and reducing its affinity for ligand. The system returns to its resting state when the ligand is released from the receptor and the GTPase activity that resides in the α subunit hydrolyzes GTP to GDP (Fig. 20-1D). The latter action leads to reassociation of the free α subunit with the βγ subunit complex to restore the original heterotrimers.

Figure 20-1

Functional cycle of heterotrimeric G proteins. A: Under basal conditions, G proteins exist in cell membranes as heterotrimers composed of single α, β and γ subunits and are associated only loosely with neurotransmitter receptors. (more...)

The structural basis of the interactions among the α, β, and γ subunits of G proteins and between the subunits and the associated receptor has become increasingly understood as the crystalline structure of these proteins has been determined [3,6]. Each α subunit has two identifiable domains. One contains the GTPase activity and the GTP-binding site. This domain also appears to be most important in binding βγ subunits as well as various effector proteins. The function of the other domain remains unknown, but it may be involved in the dramatic conformational shift that occurs in the protein upon exchanging GTP for GDP. The ability of the heterotrimeric G protein to bind to a receptor is thought to depend on sites located within all three G protein subunits. Thus, the different α subunits as well as subtypes of β and γ subunits seem to be responsible for targeting a particular type of G protein to a particular type of receptor.

G proteins couple some neurotransmitter receptors directly to ion channels

It is now clearly established that G protein subunits released from the G protein—receptor interaction can directly open or close specific ion channels [2,7]. One of the best examples of this mechanism in brain is the coupling of many types of receptors, including opioid, α₂-adrenergic, D2-dopaminergic, muscarinic cholinergic, 5HT_1a-serotonergic and GABA_B receptors, to the activation of an inward rectifying K⁺ channel (GIRK) via pertussis toxin-sensitive G proteins, that is, subtypes of G_o and/or G_i, in many types of neurons. In initial studies, it was controversial as to whether the free α subunit or the free βγ dimer was responsible for this action. Based on elegant studies in which cloned channel and G protein subunits were expressed in a variety of cell types, it is now the general consensus that the βγ complex is the more important mechanism [2,7]. In fact, the region of the channel responsible for binding the βγ complex has been identified [8]. Moreover, it seems that particular combinations of β and γ subtypes are more effective at opening this channel than others. It also appears, however, that subtypes of α_i can open the channel, although not to the same extent as the βγ subunits.

These same neurotransmitter receptors also are coupled via pertussis toxin-sensitive G proteins to voltage-gated Ca²⁺ channels, although the channels are inhibited by this interaction. In this case, available evidence supports a role for βγ in mediating this effect, although there is some evidence that α_i and α_o subunits also can be active [2,7]. Binding of the G protein subunits to the Ca²⁺ channels reduces their probability of opening in response to membrane depolarization. This mechanism is best established for L-type Ca²⁺ channels, which are inhibited by the dihydropyridine antihypertensive drugs, such as verapamil, but may also operate for other types of voltage-gated Ca²⁺ channel (see also Chaps. 6 and 23).

Still another example of direct regulation of ion channels by G proteins is the stimulation of L-type Ca²⁺ channels by G_s. In this case, free α subunits appear to bind to the channel and increase their probability of opening in response to membrane depolarization [2].

G proteins regulate intracellular concentrations of second messengers

G proteins control intracellular cAMP concentrations by mediating the ability of neurotransmitters to activate or inhibit adenylyl cyclase. The mechanism by which neurotransmitters stimulate adenylyl cyclase is well known. Activation of those neurotransmitter receptors that couple to G_s results in the generation of free G_αs subunits, which bind to and, thereby, directly activate adenylyl cyclase. In addition, free βγ-subunit complexes activate certain subtypes of adenylyl cyclase (see Chap. 22). A similar mechanism appears to be the case for G_αolf, a type of G protein structurally related to G_αs which is enriched in olfactory epithelium and striatum [9].

The mechanism by which neurotransmitters inhibit adenylyl cyclase and decrease neuronal levels of cAMP has eluded definitive identification. By analogy with the action of G_s, it was proposed originally that activation of neurotransmitter receptors that couple to G_i results in the generation of free G_αi subunits, which bind to and, thereby, directly inhibit adenylyl cyclase. However, the inhibition of adenylyl cyclase by G_αi has been difficult to demonstrate in some cell-free reconstitution experiments. Alternative possibilities are that free βγ-subunit complexes, generated by the release of G_αi, might directly inhibit certain forms of adenylyl cyclase or bind free G_αs subunits in the membrane [10]. Such sequestration of G_αs would then decrease basal stimulation of adenylyl cyclase. Indeed, there is now compelling evidence (discussed further in Chap. 22) that βγ subunits can inhibit certain forms of adenylyl cyclase, whereas other forms of the enzyme are activated by the subunits. In addition to G_i, there is evidence that G_αz, which can be considered a subtype of the G_i family based on sequence homologies, also can mediate neurotransmitter inhibition of adenylyl cyclase.

The transducin family of G proteins mediate signal transduction in the visual system (see Chap. 47) by regulating specific forms of phosphodiesterase, which catalyze the metabolism of cyclic nucleotides (see Chap. 22) [1–5]. G_αt activates PDE via direct binding to the enzyme. Gustducin (G_αgust) shares a high degree of homology with G_αt [4]. It is enriched in taste epithelium and has been speculated to mediate signal transduction in this tissue via the activation of a distinct form of phosphodiesterase.

The ability of neurotransmitter receptors to stimulate the phosphoinositide second-messenger pathway is mediated by the activation of PI-PLC, which, as mentioned above, catalyzes the hydrolysis of PIP₂ to form the second messengers inositol triphosphate (IP₃) and diacylglycerol (DAG) (see Chap. 21). It now appears that neurotransmitter-induced activation of PI-PLC is mediated via G proteins [1–5]. In most cases, G_q is involved, and it is thought that G_αq and related α subunits bind to and directly activate certain forms of PI-PLC, particularly the C_β form. In some cell types, it appears that subtypes of G_i and/or G_o may be involved. The mechanism by which G proteins mediate neurotransmitter regulation of arachidonic acid metabolism, via the activation or inhibition of PLA₂, is less well established but also may involve subtypes of G_i or G_o. In each of these cases, possible roles for βγ complexes in the regulation of enzyme activity have been proposed but remain to be established [1,3].

G proteins have been implicated in membrane trafficking

In addition to mediating signal transduction at the plasma membrane, certain heterotrimeric G proteins have been implicated in several processes that involve the trafficking of cell membranes, although the precise mechanisms involved remain obscure. For example, the G_αi subunit has been detected at relatively high concentrations in intracellular membranes, including the Golgi complex, transgolgi network and endoplasmic reticulum [11,12]. Experiments that involve activation or inhibition of this subunit with various guanine nucleotides suggest that G_αi may regulate the budding of membrane vesicles through these organelles. It also has been suggested that G_αi could be involved in the process by which portions of the plasma membrane are vesicularized into the cytoplasm via endocytosis. Synaptic vesicle trafficking is discussed in detail in Chapter 9.

Another example of the involvement of G proteins in membrane trafficking is the proposed role for G_αo in the extension of neural processes. G_αo is present at very high concentrations in nerve growth cones. Moreover, activation of G_αo by guanine nucleotides or overexpression of a constitutively active mutant form of G_αo increases nerve outgrowth in cultured neurons [12]. One interesting possibility is that G_αo may interact with growth-associated protein of 43 kDa (GAP-43), another growth coneenriched protein, to promote the growth of neural processes.

G protein βγ subunits subserve numerous functions in the cell

In early studies, G protein βγ subunits were thought to be inactive proteins that merely sequester active α subunits or anchor them to the plasma membrane. However, it has become clear that βγ subunits, acting as dimers, are highly active biological molecules that play important roles in several cellular functions.

As mentioned above, βγ subunits directly bind to and activate a class of K⁺ channels called GIRKs and bind to and modulate the activity of PI-PLC_β and certain classes of adenylyl cyclase. The βγ subunits also bind to several other proteins, including certain protein kinases as well as phosducin and Ras guanine nucleotide exchange factor (see below) [1,3,13]. The ability of such diverse cellular proteins to bind βγ subunits has led to a search for a common structural motif in these various target proteins that is responsible for this binding. One possibility is that these proteins contain a specific amino acid sequence within their primary structures, termed the pleckstrin homology (PH) domain, which binds βγ with high affinity.

One class of protein kinase that binds βγ subunits is called G protein receptor kinases (GRKs). These kinases phosphorylate G protein-coupled receptors that are occupied by ligand and thereby mediate one form of receptor desensitization (see Chap. 24). It now appears that βγ subunits play a key role in this process [13]. As shown in Figure 20-2, the GRK is normally a cytoplasmic protein that does not come into appreciable contact with the plasma membrane receptor under basal conditions. Ligand binding to the receptor activates the associated G protein, which results in the generation of free α and βγ subunits. The βγ subunits, which remain membrane-bound (as will be described below), are now free to bind to the C-terminal domain of the GRK. This draws the GRK into close physical proximity with the receptor and enables receptor phosphorylation. In this way, the βγ subunits target GRKs, which have constitutive catalytic activity, to those receptors that are ligand-bound.

Figure 20-2

Schematic illustration of the role of G protein βγ subunits in intracellular targeting of proteins. A: Under resting conditions, the receptor is associated loosely with a heterotrimeric G protein and G protein receptor kinases (GRK) are (more...)

Another important role for βγ subunits is regulation of the mitogen-activated protein kinase (MAP-kinase) pathway [14]. MAP-kinases are the major effector pathway for growth factor receptors (see Chaps. 10, 19 and 24). However, signals that act through G protein-coupled receptors, particularly those coupled to G_i, can modulate growth factor activation of the MAP-kinase pathway. This is mediated via βγ subunits. Activation of the receptors leads to the generation of free βγ subunits, which then activate the MAP-kinase pathway at some early step in the cascade. Some possibilities include direct action of the βγ subunits on Ras (see below) or on one of several “linker” proteins between the growth factor receptor itself and activation of Ras.

The molecular specificity of various subtypes of β and γ subunit is an area of intense research [15]. The five forms of β are highly structurally similar, whereas the seven forms of γ are more divergent. Different forms of β and γ subunits interact with each other with widely varying abilities in in vitro expression systems. Identifying which forms of βγ subunit complexes occur in vivo and the specificity of these complexes for various target proteins, such as adenylyl cyclases, K⁺ channels, GRKs and others, is just beginning.

The functioning of heterotrimeric G proteins is modulated by several other proteins

The function of one major class of modulator protein is to bind to G protein α subunits and to stimulate their intrinsic GTPase activity. These are termed GTPase-activating proteins (GAPs) (Fig. 20-3). GAPs had been known to exist for many years for small G proteins (see below), but only recently have analogous proteins been identified for heterotrimeric G proteins [16]. These GAPs, first identified in yeast but subsequently found in mammalian tissues, have been termed regulators of G protein-signaling (RGS) proteins. Activation of α subunit GTPase activity hastens the hydrolysis of GTP to GDP and more rapidly restores the inactive heterotrimer; thus, RGS proteins inhibit the biological activity of G proteins.

Figure 20-3

Schematic illustration of proteins that modulate the functioning of G proteins. The functional activity of G proteins is controlled by cycles of binding GDP versus GTP. This is associated with a major conformational change in the protein as depicted. (more...)

More than 18 forms of mammalian RGS protein are now known, and most are expressed in brain with highly region-specific patterns [16a]. It is thought that different families of G protein α subunits are likely to be modulated by different forms of RGS protein. Another possibility is that the various forms of RGS protein stimulate α subunit GTPase activity to different extents, which would allow for exquisite fine-tuning of G protein activity in a cell.

The function of RGS proteins in mammalian cells remains poorly understood. One exciting possibility, based on studies in yeast, is that alterations in the activity of specific RGS proteins, for example, via changes in their expression or phosphorylation, could modulate the activity of specific G proteins and, consequently, the sensitivity of specific G protein-coupled receptors.

Phosducin is another protein that modulates G protein function [17,18]. Phosducin is a cytosolic protein enriched in retina and pineal gland but also expressed in brain and other tissues. Phosducin binds to G protein βγ subunits with high affinity. The result is prevention of βγ subunit reassociation with the α subunit. In this way, phosducin may sequester βγ subunits, which initially may prolong the biological activity of the α subunit. However, eventually this may inhibit G protein activity by preventing the direct biological effects of the βγ subunits as well as regeneration of the functional G protein heterotrimer. How phosducin functions in intact cells remains incompletely understood, although the ability of phosducin to bind to βγ subunits is altered upon its phosphorylation by cAMP- or Ca²⁺-dependent protein kinases [17–19]. This raises the possibility that phosducin may be an important physiological modulator of G protein function.

G proteins are modified covalently by the addition of long-chain fatty acids

Addition of these lipid groups appears to modify the ability of the G proteins to interact with other proteins or with the plasma membrane. Several fatty acid modifications have been demonstrated: myristoyl groups, which consist of 14 carbon chains (C₁₄); farnesyl groups (C₁₅); palmitoyl groups (C₁₆); and geranylgeranyl, or isoprenyl, groups (C₂₀) [20]. Myristoyl groups are added via amide links to N-terminal glycine residues present in certain proteins. Farnesyl and isoprenyl groups are added to cysteine residues of specific C-terminal motifs. Palmitoyl groups also are added to cysteine groups within specific consensus amino acid sequences.

All G protein α subunits are modified in their N-terminal domains by palmitoylation or myristoylation [20–22]. These modifications may regulate the affinity of the α subunit for its βγ subunits and, thereby, the likelihood of dissociation or reassociation of the heterotrimer. The modifications also may help determine whether the α subunit, released upon ligandreceptor interaction, remains associated with the plasma membrane or diffuses into the cytoplasm. This could have important consequences on the types of effector proteins regulated. It is also possible that palmitoylation, but not myristoylation, is regulated dynamically. There is evidence that palmitoylation can be regulated by ligand binding, which makes this a potentially important control point. However, very little is known about palmitoyl transferases and depalmitoylases, the enzymes responsible for palmitoylation. In contrast, myristoylation appears to be a one-time event in the life cycle of an α subunit.

G protein γ subunits are modified on their C-terminal cysteine residues by isoprenylation [20,23]. There is now strong evidence that this modification plays a key role in anchoring the γ subunit and its associated β subunit to the plasma membrane. The importance of this anchoring is illustrated in Figure 20-2, which shows that the ability of βγ-subunits to target GRKs to ligand-bound receptors depends on this membrane localization.

The functioning of G proteins may be influenced by phosphorylation

G proteins have been reported to undergo phosphorylation by cAMP- and Ca²⁺-dependent protein kinases and by protein tyrosine kinases. However, the effect of phosphorylation on G protein function and its role in the regulation of physiological processes have been particularly difficult to establish with certainty. This remains an important area of future investigation.