G proteins function in the context of two interrelated cycles: a cycle of subunit association and dissociation and a cycle of GTP binding and hydrolysis (discussed in detail in the legend to Figure 1-2). G protein heterotrimers consist of G", G> , and G1" subunits at a 1:1:1 stoichiometry and are named according to decreasing mass, with the ^ subunits having an apparent mass of 40-52 kDa, ' subunits having an apparent mass of 35-36 kDa, and y subunits having an apparent mass of 5-20 kDa. The different types of G protein have been named on the basis of the distinct« subunits they possess (i.e., Gs represents G proteins containing GKS). This classification system arose from the erroneous assumption that it was only the,:' subunits that were responsible for the proteins' specific functional activity; it is now known that the ~ and * subunits exert a number of functional effects on their own (see Table 1-1) and are not simply "anchoring proteins" for «subunits. Although the ^ and subunits are not covalently bound, they are tightly linked by noncovalent coiled-coil interactions; thus, they are generally assumed to function as L dimers. It is very likely that different L ' subunits exert different effects on « subunits and effectors (e.g., ^2T2 behaves differently from n2T3), but the delineation of the differential effects of the different subunit compositions is still in its infancy.
Mediation of neurotransmitter-neurotransmitter and receptor-receptor interactions
The CNS is remarkably complex, both anatomically and chemically, with a remarkable convergence of different receptors in common cortical layers and considerable convergence of neurotransmitter action. A single neuron in the brain receives thousands of synaptic inputs on the cell body and dendrites, and neuronal response is also modulated by a variety of hormonal and neurohormonal substances that are not dependent on synaptic organization (Kandel et al. 2000). The neuron needs to integrate all the synaptic and nonsynaptic inputs impinging on it; this integration of a multitude of signals determines the ultimate excitability, firing pattern, and response characteristics of the neuron, which are then conveyed to succeeding targets via synaptic transmission. How does the single neuron decipher and integrate the multitude of signals it receives and, additionally, generate unique responses to each of these signals or combinations of signals?
Not only do G proteins amplify signals, but they also appear to form the basis of a complex information-processing network in the plasma membrane (Bhalla and Iyengar 1999; Manji 1992). Thus, the ability of G proteins to interact with multiple receptors provides an elegant mechanism to organize the signals from these multiple receptors and to transmit them to a relatively much smaller number of effectors. Signals from a variety of receptors can be "weighted" according to their intrinsic ability to activate a given G protein and integrated to stimulate a single second-messenger pathway (see Figure 1-11). Similarly, the dual (positive and negative) regulation of adenylyl cyclase by G proteins allows for stimulatory and inhibitory signals for these pathways to be "balanced" at the G protein level, yielding an integrated output. Thus, G proteins provide the first opportunity for signals from different receptors to be integrated. This complex web of interactions linking receptors, G proteins, and their effectors with signals converging to shared detectors appears to be crucial for the integrative functions performed by the CNS.
Abnormalities in a variety of human diseases have now been clearly shown to arise from primary abnormalities in G protein signaling cascades and in the G protein subunits themselves (for an excellent discussion, see Simonds 2003; Spiegel 1998). To date, the direct evidence for the involvement of G proteins in psychiatric disorders is more limited. Thus, although elevations in the levels of Gc;5 have been found in postmortem brain and peripheral tissue in bipolar disorder, a mutation in the Gr/S gene has not yet been identified (discussed in Manji and Lenox 1999). There is, however, convincing evidence that chronic lithium administration attenuates the functioning of both Gs and Gi, resulting in an elevation of basal cAMP levels but dampened receptor-mediated effects. The allosteric modulation of G proteins has been proposed to play a role in lithium's long-term prophylactic efficacy in protecting susceptible individuals from cyclic affective episodes induced spontaneously or by stress or drugs (e.g., antidepressant, stimulant) (G. Chen et al. 1999; Gould and Manji 2002).
G proteins control intracellular cAMP levels by mediating the ability of neurotransmitters to activate or inhibit adenylyl cyclase (Figure 1-12; see also Figure 1-2). The mechanism by which neurotransmitters stimulate adenylyl cyclase is well established. Activation of those neurotransmitter receptors that couple to Gs results in the generation of free Gets subunits that bind to and directly activate adenylyl cyclase. A similar mechanism appears to be the case for Gc?olf, a type of G protein (structurally related to Go:s) that is enriched in olfactory epithelium and dopamine-rich areas of the brain and mediates the ability of odorant receptors and D1 receptors to stimulate adenylyl cyclase. The mechanism by which neurotransmitters inhibit adenylyl cyclase and decrease neuronal levels of cAMP is somewhat less clear, and more than one mechanism may be operative. By analogy with the action of Gs, it was originally proposed that activation of neurotransmitter receptors that couple to Gi results in the generation of free Gtfj subunits, which could bind to, and thereby directly inhibit, adenylyl cyclase. While this mechanism may be operative, there are also data to suggest that 137 subunit complexes, generated by the release of Gttj, might directly inhibit certain forms of adenylyl cyclase or might bind and "tie up" free G&:s subunits in the membrane.
Receptors can be positively (e.g., ^-adrenergic, Di) or negatively (e.g., 5-HTia, D2) coupled to adenylyl cyclase (AC) to regulate cAMP levels. The effects of cAMP are mediated largely by activation of protein kinase A (PKA). One major downstream target of PKA is CREB (cAMP response element-binding protein). After activation, the phosphorylated CREB binds to the cAMP response element (CRE), a gene sequence found in the promoter of certain genes; data suggest that antidepressants may activate CREB, thereby bringing about increased expression of a major target gene, BDNF. Phosphodiesterase is an enzyme that breaks down cAMP to AMP. Some antidepressant treatments have been found to upregulate phosphodiesterase. Drugs like rolipram, which inhibit phosphodiesterase, may be useful as adjunct treatments for depression. Forskolin is an agent used in preclinical research to stimulate adenylyl cyclase.
Long-term changes in neuronal plasticity and function
Copyright © American Psychiatric Publishing, Inc., or American Psychiatric Association, unless otherwise indicated in figure lagend. All rights reserved.
It is now clear that there are several forms of adenylyl cyclase that make up a distinct enzyme family; these various forms are differentially regulated and display distinct distributions in nervous and nonnervous tissues. For example, type I is found predominantly in brain, whereas types II and IV, although abundantly expressed in the brain, have a more widespread distribution. The topographical structure of the adenylyl cyclase proteins resembles that of membrane transporters and ion channels. However, there is currently no convincing evidence of a transporter or channel function for mammalian adenylyl cyclases.
As would be predicted, the different forms of adenylyl cyclase are regulated by distinct mechanisms. Type I through IV enzymes differ in their ability to be most intriguing regulation is that by the G protein 13 and >' subunits. Thus, it is now clear that when type II adenylyl cyclase is concurrently stimulated by a stimulatory receptor (e.g., ^-adrenergic receptor), the subunits released from an "inhibitory receptor" (e.g., 5-HTj.a, H-i, GABAb) can, in fact, robustiy potentiate the cAMP response (Bourne and Nicoll 1993). Type II adenylyl cyclase thus serves as a "coincidence detector" in the CNS, capable of temporally and spatially integrating signals to bring about dramatically different effects. An additional important mechanism by which adenylyl cyclase can be regulated is by cross-talk with protein kinase C, thereby linking receptors linked to stimulation of adenylyl cyclase and those linked to the turnover of membrane phosphoinositides. The physiological effects of cAMP are mediated primarily by activation of protein kinase A, an enzyme that phosphorylates and regulates many proteins, including ion channels, cytoskeletal elements, transcription factors, and other enzymes. Indeed, one major CNS target for the actions of PKA is the transcription factor CREB (cAMP response element-binding protein), which plays a major role in long-term neuroplasticity and is an indirect target of antidepressants (Duman 2002) (see Figure 1-12). As we discuss in greater detail below, phosphorylation and dephosphorylation reactions play a major role in regulating a variety of long-term neuroplastic events in the CNS.
Phosphoinositide/Protein Kinase C Phosphoinositide
Although inositol phospholipids are relatively minor components of cell membranes, they play a major role in receptor-mediated signal transduction pathways. They are involved in a diverse range of responses, such as cell division, secretion, and neuronal excitability and responsiveness. In many cases, Gq/n is involved, and it is believed that GGCq/n directly binds to and activates phospholipase C (Figure 1-13). In other cases, however, it is the subunits released upon activation of receptors coupled to Gi/Go that bring about activation of the enzyme PLC to produce the intracellular second messengers sn-1,2-diacylglycerol (DAG; an endogenous activator of PKC) and inositol-1,4,5-triphosphate (IP3). IP3 binds to the IP3 receptor and facilitates the release of calcium from intracellular stores, in particular the endoplasmic reticulum (see Figure 1-13). The released calcium then interacts with various proteins in the cell, including the important family of calmodulins (Ca2+-receptor protein calmodulin, or CaM) (discussed later in this chapter; Figure 1-14). Calmodulins then activate calmodulin-dependent protein kinases (CaMKs), which affect the activity of diverse proteins, including ion channels, signaling molecules, proteins that regulate apoptosis, scaffolding proteins, and transcription factors (Miller 1991; Soderling 2000).
FIGURE 1-13. Phosphoinositide (PI) signaling pathway.
A number of receptors in the CNS (including Mi, M3, M5, 5-HT2c) are coupled, via Gctq/n, to activation of PI hydrolysis. Activation of these receptors induces phospholipase C (PLC) hydrolysis of phosphoinositide-4,5-bisphosphate (PIP2) to sn-1,2-diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP3). DAG activates protein kinase C (PKC), an enzyme that has many effects, including the activation of phospholipase A2 (PLA2), an activator of arachidonic acid signaling pathways. IP3 binds to the IP3 receptor, which results in the release of intracellular calcium from intracellular stores, most notably the endoplasmic reticulum. Calcium is an important signaling molecule and initiates a number of downstream effects such as activation of calmodulins and calmodulin-dependent protein kinases (see Figure 1-15). IP3 is recycled back to PIP2 by the enzymes inositol monophosphatase (IMPase) and inositol polyphosphatase (IPPase; not shown), both of which are targets of lithium. Thus, lithium may initiate many of its therapeutic effects by inhibiting these enzymes, thereby bringing about a cascade of downstream effects involving PKC and gene expression changes.
Source. Adapted from Gould TD, Chen G, Manji HK: "Mood Stabilizer Pharmacology." Clinical Neuroscience Research 2:193-212, 2003. Copyright 2003, Elsevier. Used with permission.
regulated by Ca and calmodulin. Types I and III are stimulated by Ca -calmodulin complexes, whereas types II and IV are insensitive. Perhaps the
FIGURE 1-14. Calcium-mediated signaling.
In neurons, Ca2+-dependent processes represent an intrinsic nonsynaptic feedback system that provides competence for adaptation to different functional tasks. Ca2+ is generally mobilized in one of two ways in the cells: either by mobilization from intracellular stores (e.g., from the endoplasmic reticulum) or from outside of the cell via plasma membrane ion channels and certain receptors (e.g., NMDA [W-methyl-D-aspartate]). The external concentration of Ca2+ is approximately 2 mM, yet resting intracellular Ca2+ concentrations are in the range of 100 nM (2 x 104 lower). Local high levels of calcium result in activation of enzymes, signaling cascades, and, at extremes, cell death. Release of intracellular stores of calcium is primarily regulated by inositol-1,4,5-triphosphate (IP3) receptors that are activated upon generation of IP3 by phospholipase C (PLC) activity, and the ryanodine receptor that is activated by the drug ryanodine. Many proteins bind Ca2+ and are classified as either "buffering" or "triggering." These include calcium pumps, calbindin, calsequestrin, calmodulin, PKC, phospholipase A2, and calcineurin. Once stability of intracellular calcium is accomplished, transient low-magnitude changes can serve an important signaling function. Calcium action is local. Because of the high concentration of calcium-binding proteins, it is estimated that the free Ca2+ ion diffuses only approximately 0.5 UM and is free for about 50 Usee before encountering a Ca2+-binding protein. Ca2+ is sequestered in the from the cell is either via sodium-calcium exchange or by means of a calcium pump.
IP3 can be metabolized both by dephosphorylation to form inositol-1,4-P2 and by phosphorylation to form inositol-1,3,4,5-P4 (IP4), which has been proposed to be involved in the entry of Ca2+ into cells from extracellular sources. Recycling of IP3 is important for continuation of phosphoinositide hydrolysis in response to extracellular signals. This is achieved by the enzyme inositol monophosphatase (IMPase), which is the rate-limiting enzyme that converts IP3 back to phosphoinositide-4,5-bisphosphate (PIP2). Without this enzyme, PIP2 cannot be recycled adequately, potentially leading to low levels of PIP2 and inhibition of the signaling cascades involving DAG and IP3. Lithium, at therapeutically relevant concentrations, is a noncompetitive inhibitor of IMPase (for a review, see Gould et al. 2003). This has led to the "inositol depletion hypothesis," which posits that lithium brings about a reduction in the levels of inositol by inhibiting the activity of this "recycling enzyme." Although lithium does reduce inositol levels in the areas of the brain in bipolar patients (Moore et al. 1999), this likely represents an upstream "initiating event," which brings about downstream changes in PKC and regulates gene expression, which may be ultimately responsible for some of its therapeutic effects (see Figure 1-13) (Brandish et al. 2005; Gould et al. 2003; Manji and Lenox 1999).
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