▲ FIGURE 13-14 Structure of mammalian adenylyl cyclases and their interaction with GsaGTP (a) Schematic diagram of mammalian adenylyl cyclases. The membrane-bound enzyme contains two similar catalytic domains on the cytosolic face of the membrane and two integral membrane domains, each of which is thought to contain six transmembrane a helices. (b) Three-dimensional structure of GsaGTP complexed with two fragments encompassing the catalytic domain of adenylyl cyclase determined by x-ray crystallography. The a3-p5 loop and the helix in the switch II region (blue) of GsaGTP interact simultaneously with a specific region of adenylyl cyclase. The darker-colored portion of Gsa is the GTPase domain, which is similar in structure to Ras (see Figure 13-8); the lighter portion is a helical domain. The two adenylyl cyclase fragments are shown in orange and yellow. Forskolin (green) locks the cyclase fragments in their active conformations. [Part (a) see W.-J. Tang and A. G. Gilman, 1992, Cell 70:869; part (b) adapted from J. J. G. Tesmer et al., 1997, Science 278:1907]

Adenylyl Cyclase Is Stimulated and Inhibited by Different Receptor-Ligand Complexes

The versatile trimeric G proteins enable different receptor-hormone complexes to modulate the activity of the same effector protein. In the liver, for instance, glucagon and epinephrine bind to different receptors, but both receptors interact with and activate the same Gs, which activates adenylyl cyclase, thereby triggering the same metabolic responses. Activation of adenylyl cyclase, and thus the cAMP level, is proportional to the total concentration of Gsa-GTP resulting from binding of both hormones to their respective receptors.

▲ FIGURE 13-15 Hormone-induced activation and inhibition of adenylyl cyclase in adipose cells. Ligand binding to Gs-coupled receptors causes activation of adenylyl cyclase, whereas ligand binding to Grcoupled receptors causes inhibition of the enzyme. The Gp7 subunit in both stimulatory and inhibitory G proteins is identical; the Ga subunits and their corresponding receptors differ. Ligand-stimulated formation of active G„GTP complexes occurs by the same mechanism in both Gs and G| proteins (see Figure 13-11). However, Gs„ GTP and Gia GTP interact differently with adenylyl cyclase, so that one stimulates and the other inhibits its catalytic activity. [See A. G. Gilman, 1984, Cell 36:577]

Positive and negative regulation of adenylyl cyclase activity occurs in some cell types, providing fine-tuned control of the cAMP level. For example, stimulation of adipose cells by epinephrine, glucagon, or ACTH activates adenylyl cyclase, whereas prostaglandin PGE1 or adenosine inhibits the enzyme (Figure 13-15). The receptors for PGE1 and adenosine interact with inhibitory G(, which contains the same p and y subunits as stimulatory Gs but a different a subunit (Gla). In response to binding of an inhibitory ligand to its receptor, the associated Gi protein releases its bound GDP and binds GTP; the active Gia-GTP complex then dissociates from Gpy and inhibits (rather than stimulates) adenylyl cyclase.

cAMP-Activated Protein Kinase A Mediates Various Responses in Different Cells

In multicellular animals virtually all the diverse effects of cAMP are mediated through protein kinase A (PKA), also called cAMP-dependent protein kinase. As discussed in Chapter 3, inactive PKA is a tetramer consisting of two regulatory (R) subunits and two catalytic (C) subunits. Each R subunit has two distinct cAMP-binding sites; binding of cAMP to both sites in an R subunit leads to release of the associated C subunit, unmasking its catalytic site and activating its kinase activity (see Figure 3-27a). Binding of cAMP by an R subunit occurs in a cooperative fashion; that is, binding of the first cAMP molecule lowers the Kd for binding of the second. Thus small changes in the level of cytosolic cAMP can cause proportionately large changes in the amount of dissociated C subunits and, hence, in kinase activity. Rapid activation of an enzyme by hormone-triggered dissociation of an inhibitor is a common feature of various signaling pathways.

Most mammalian cells express receptors coupled to Gs protein. Stimulation of these receptors by various hormones leads to activation of PKA, but the resulting cellular response depends on the particular PKA isoform and on the PKA substrates expressed by the cell. For instance, the effects of epi-nephrine on glycogen metabolism, which are mediated via cAMP and PKA, are confined mainly to liver and muscle cells, which express enzymes for making and degrading glycogen. In adipose cells, epinephrine-induced activation of PKA promotes phosphorylation and activation of the phos-pholipase that catalyzes hydrolysis of stored triglycerides to yield free fatty acids and glycerol. These fatty acids are released into the blood and taken up as an energy source by cells in other tissues such as the kidney, heart, and muscles. Likewise, stimulation of G protein-coupled receptors on ovarian cells by certain pituitary hormones leads to activation of PKA, which in turn promotes synthesis of two steroid hormones, estrogen and progesterone, crucial to the development of female sex characteristics.

Although PKA acts on different substrates in different types of cells, it always phosphorylates a serine or threonine residue that occurs within the same sequence motif: X-Arg-(Arg/Lys)-X-(Ser/Thr)-$, where X denotes any amino acid and $ denotes a hydrophobic amino acid. Other serine/ threonine kinases phosphorylate target residues within different sequence motifs.

Glycogen Metabolism Is Regulated by Hormone-Induced Activation of Protein Kinase A

The first cAMP-mediated cellular response to be discov-ered—the release of glucose from glycogen—occurs in muscle and liver cells stimulated by epinephrine or other hormones whose receptors are coupled to Gs protein. This response exemplifies how activation of PKA can coordinate the activity of a group of intracellular enzymes toward a common purpose.

Glycogen, a large glucose polymer, is the major storage form of glucose in animals. Like all biopolymers, glycogen is synthesized by one set of enzymes and degraded by another (Figure 13-16). Three enzymes convert glucose into uridine diphosphoglucose (UDP-glucose), the primary intermediate in glycogen synthesis. The glucose residue of UDP-glucose is transferred by glycogen synthase to the free hydroxyl group on carbon 4 of a glucose residue at the end of a growing glycogen chain. Degradation of glycogen involves the stepwise removal of glucose residues from the same end by a phosphorolysis reaction, catalyzed by glyco-gen phosphorylase, yielding glucose 1-phosphate.

In both muscle and liver cells, glucose 1-phosphate produced from glycogen is converted to glucose 6-phosphate. In muscle cells, this metabolite enters the glycolytic pathway and is metabolized to generate ATP for use in powering muscle contraction (Chapter 8). Unlike muscle cells, liver cells contain a phosphatase that hydrolyzes glucose 6-phosphate to glucose, which is exported from these cells in part by a glucose transporter (GLUT2) in the plasma membrane (Chapter 7). Thus glycogen stores in the liver are primarily broken down to glucose, which is immediately released into the blood and transported to other tissues, particularly the muscles and brain.

The epinephrine-stimulated increase in cAMP and subsequent activation of PKA enhance the conversion of glycogen to glucose 1-phosphate in two ways: by inhibiting glycogen synthesis and by stimulating glycogen degradation (Figure 13-17a). PKA phosphorylates and thus inactivates glycogen synthase, the enzyme that synthesizes glycogen. PKA promotes glycogen degradation indirectly by phospho-rylating and thus activating an intermediate kinase, glycogen phosphorylase kinase (GPK), that in turn phosphorylates and activates glycogen phosphorylase, the enzyme that degrades glycogen. The entire process is reversed when epi-nephrine is removed and the level of cAMP drops, inactivating PKA. This reversal is mediated by phosphopro-tein phosphatase, which removes the phosphate residues

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