Principles of cell signaling - UT Southwestern

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39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 618 -strand repeats that form a cylindrical structure known as a propeller. There are five Gβ genes in mammals. Four encode strikingly similar proteins that naturally dimerize with the twelve Gγ subunits (Figure 14.24). The fifth, Gβ5, is less closely related to the others and interacts primarily with a Gγ-like domain in other proteins rather than with Gγ subunits themselves. Gγ subunits are smaller (~7 kDa) and far more diverse in sequence than are the Gβ’s. The last three amino acid residues of Gγ subunits are proteolyzed to leave a conserved C-terminal cysteine that is irreversibly S-prenylated and carboxymethylated, helping to anchor Gβγ to the membrane. Gβ and Gγ subunits can associate in most possible combinations. Because almost all cells express multiple Gβ and Gγ subunits, it has been difficult to assign specific roles to individual Gβγ combinations. The best recognized interactions of Gβγ subunits occur at sites on Gβ, although distinct functions of Gγ have also been supported. 14.21 Heterotrimeric G proteins regulate a wide variety of effectors Key concepts • G proteins convey signals by regulating the activities of multiple intracellular signaling proteins known as effectors. • Effectors are structurally and functionally diverse. • A common G-protein binding domain has not been identified among effector proteins. • Effector proteins integrate signals from multiple G protein pathways. G protein-regulated effectors include enzymes that create or destroy intracellular second messengers (adenylyl cyclase, cyclic GMP phosphodiesterase, phospholipase C-β, phosphatidylinositol-3-kinase), protein kinases, ion channels (K+, Ca2+) and possibly membrane transport proteins (see Figure 14.25). Effectors may be integral membrane proteins or intrinsically soluble proteins that bind G proteins at the membrane surface. No conserved G protein-binding domain or sequence motif has been identified among effector proteins, and most effectors are related to proteins that have similar functions but that are not regulated by G proteins. Sensitivity to G protein regulation, thus, evolved independently in multiple families of regulatory proteins. Because they can respond to a variety of Gα and Gβγ subunits, effector proteins can integrate signals from multiple G protein pathways. The different Gα or Gβγ subunits may have opposite or synergistic effects on a given effector. For example, some of the membranebound adenylyl cyclases in mammals are stimulated by Gα s and inhibited by Gα i (see Figure 14.13). Many effectors are further regulated by other allosteric ligands (e.g., lipids, calmodulin) and by phosphorylation, contributing even more to integration of information. Effectors are usually represented as multiple isoforms, and each isoform may be regulated differently, adding to the complexity of G protein networks. For example, some isoforms of adenylyl cyclase are stimulated by Gβγ, whereas others are inhibited. All phospholipase C-βs are stimulated both by Gα q family members and by Gβγ, but the potency and maximal effect of these two inputs vary dramatically among the four PLC-β isoforms. 14.22 Heterotrimeric G proteins are controlled by a regulatory GTPase cycle Key concepts • Heterotrimeric G proteins are activated when the Gα subunit binds GTP. • GTP hydrolysis to GDP inactivates the G protein. • GTP hydrolysis is slow, but is accelerated by proteins called GAPs. • Receptors promote activation by allowing GDP dissociation and GTP association; spontaneous exchange is very slow. • RGS proteins and phospholipase C-βs are GAPs for G proteins. The key event in heterotrimeric G protein signaling is the binding of GTP to the Gα subunit. GTP binding activates the Gα subunit, which allows both it and the Gβγ subunit to bind and regulate effectors. The Gα subunit remains active as long as GTP is bound, but Gα also has GTPase activity and hydrolyzes bound GTP to GDP. Gα-GDP is inactive. G proteins thus traverse a GTPase cycle of GTP binding/activation and hydrolysis/deactivation, as depicted in FIGURE 14.26. Therefore, the control of G protein signaling is intrinsically kinetic. The relative signal strength, or amplitude, is proportional to the fraction of G protein that is in the active, GTP-bound form. This fraction equals the balance of the rates of GTP binding and GTP hydrolysis, the activating 618 CHAPTER 14 Principles of cell signaling
39057_ch14_cellbio.qxd 8/28/06 5:11 PM Page 619 and deactivating arms of the GTPase cycle. Both limbs are highly regulated over a range of rates greater than 1000-fold. Receptors promote G protein activation by opening the nucleotide-binding site on the G protein, thus accelerating both GDP dissociation and GTP association. This process is referred to as GDP/GTP exchange catalysis. Exchange proceeds in the direction of activation because the affinity of G proteins for GTP is much higher than that for GDP and because the cytosolic concentration of GTP is about 20-fold higher than that of GDP. Spontaneous GDP/GTP exchange is very slow for most G proteins (many minutes), which maintains basal signal output at a low level. In contrast, receptor-catalyzed exchange can take place in a few tens of milliseconds, which allows rapid responses in cells such as visual photoreceptors, other neurons, or muscle. Because receptors are not directly required for a G protein’s signaling activity, a receptor can dissociate after GDP/GTP exchange and catalyze the activation of additional G protein molecules. In this way, a single receptor may maintain the activation of multiple G proteins, providing molecular amplification of the incoming signal. Other receptors may remain bound to their G protein targets, which means that they do not act as amplifiers. However, more tightly bound receptors can initiate signaling more quickly and promote G protein reactivation when hydrolysis of bound GTP is rapid. In the absence of stimulus, Gα subunits hydrolyze bound GTP slowly. The average activation lifetime of the Gα-GTP complex is about 10-150 seconds, depending on the G protein. This rate is far slower than rates of deactivation often observed in cells when an agonist is removed. For example, visual signaling terminates in about 10 ms after stimulation by a photon, and many other G protein systems are almost as fast. GTP hydrolysis is accelerated by GTPase-activating proteins (GAPs), which directly bind Gα subunits. In some cases acceleration exceeds 2000-fold. Such speed is necessary in systems like vision or neurotransmission, which must respond to quickly changing stimuli. Because G protein signaling is a balance of activation and deactivation, GAPs deplete the pool of GTP-activated G protein and can thereby also act to inhibit G protein signaling. GAPs can thus inhibit signaling, quench output upon signal termination, or both. What behavior predominates depends on the GAP’s intrinsic activity and its regulation. Receptor + agonist GDP G protein-GDP Pi The regulatory GTPase cycle Receptor - agonist G protein GAP GTP G protein-GTP *ACTIVE* Effector protein There are two families of GAPs for heterotrimeric G proteins. The RGS proteins (regulators of G protein signaling) are a family of about 30 proteins, most or all of which have GAP activity and regulate G protein signaling rates and amplitudes. The role of RGS proteins in terminating the G protein signal can be seen in FIGURE 14.27. Some proteins with RGS domains also act as G protein-regulated effectors. These include activators of the Rho family of monomeric GTP-binding proteins (see below) and GPCR kinases, which are feedback regulators of GPCR function. The second group of G protein GAPs are phospholipase C-βs. These enzymes are effectors that are stimulated by both Gα q and by Gβγ, but they also act as G q GAPs, probably to control output kinetics. G protein-GTP- Effector protein *ACTIVE* FIGURE 14.26 G proteins are activated when GTP binds to the G subunit, such that both G-GTP and G can bind and regulate the activities of appropriate effector proteins. G subunits also have intrinsic GTPase activities, and the primary deactivating reaction is hydrolysis of bound GTP to GDP (rather than GTP dissociation). Thus, the steady-state signal output from a receptor-G protein module is the fraction of the G protein in the GTP-bound state, which reflects the balance of the activation and deactivation rates. Both GTP binding and GTP hydrolysis are intrinsically slow and highly regulated. GDP binds tightly to G, such that GDP dissociation is rate-limiting for binding of a new molecule of GTP and consequent reactivation. Both GDP release and GTP binding are catalyzed by GPCRs. Hydrolysis of bound GTP is accelerated by GTPase-activating proteins (GAPs). Receptors and GAPs coordinately control both the steady-state level of signal output and the rates of activation and deactivation of the module. 14.22 Heterotrimeric G proteins are controlled by a regulatory GTPase cycle 619
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Principles of cell signaling - UT Southwestern

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