▲ FIGURE 13-17 Regulation of glycogen metabolism by cAMP in liver and muscle cells. Active enzymes are highlighted in darker shades; inactive forms, in lighter shades. (a) An increase in cytosolic cAMP activates PKA, which inhibits glycogen synthesis directly and promotes glycogen degradation via a protein kinase cascade. At high cAMP PKA also phosphorylates an inhibitor of phosphoprotein phosphatase from the inactive form of glycogen synthase, thereby activating it, and from the active forms of glycogen phosphory-lase kinase and glycogen phosphorylase, thereby inactivating them (Figure 13-17b).

Phosphoprotein phosphatase itself is regulated by PKA. Activated PKA phosphorylates an inhibitor of phosphopro-tein phosphatase; the phosphorylated inhibitor then binds to phosphoprotein phosphatase, inhibiting its activity (see Figure 13-17a). At low cAMP levels, when PKA is inactive, the inhibitor is not phosphorylated and phosphoprotein phosphatase is active. As a result, the synthesis of glycogen by glycogen synthase is enhanced and the degradation of glycogen by glycogen phosphorylase is inhibited.

Epinephrine-induced glycogenolysis thus exhibits dual regulation: activation of the enzymes catalyzing glycogen degradation and inhibition of enzymes promoting glycogen synthesis. Such coordinate regulation of stimulatory and inhibitory pathways provides an efficient mechanism for achieving a particular cellular response and is a common phenomenon in regulatory biology.

(PP). Binding of the phosphorylated inhibitor to PP prevents this phosphatase from dephosphorylating the activated enzymes in the kinase cascade or the inactive glycogen synthase. (b) A decrease in cAMP inactivates PKA, leading to release of the active form of phosphoprotein phosphatase. The action of this enzyme promotes glycogen synthesis and inhibits glycogen degradation.

Signal Amplification Commonly Occurs Downstream from Cell-Surface Receptors

The cellular responses induced by G protein-coupled receptors that activate adenylyl cyclase may require tens of thousands or even millions of cAMP molecules per cell. Thus the hormone signal must be amplified in order to generate sufficient second messenger from the few thousand receptors for a particular hormone present on a cell. .Signal amplification is possible because both receptors and G proteins can diffuse rapidly in the plasma membrane. A single receptor-hormone complex causes conversion of up to 100 inactive Gsa molecules to the active form. Each active Gsa-GTP, in turn, probably activates a single adenylyl cyclase molecule, which then catalyzes synthesis of many cAMP molecules during the time Gsa-GTP is bound to it. Although the exact extent of this amplification is difficult to measure, binding of a single hormone molecule to one receptor molecule can result in the synthesis of at least several hundred cAMP molecules before the receptor-hormone complex dissociates and activation of adenylyl cyclase ceases. Similar amplification probably occurs in signaling from receptors coupled to other G proteins and some other types of receptors whose activation induces synthesis of second messengers.

A second level of amplification is illustrated by the cAMP-mediated stimulation of glycogenolysis. As we just discussed, cAMP promotes glycogen degradation via a three-stage cascade, that is, a series of reactions in which the enzyme catalyzing one step is activated (or inhibited) by the product of a previous step (see Figure 13-17a). The amplification that occurs in such a cascade depends on the number of steps in it.

Both levels of amplification are depicted in Figure 13-18. For example, blood levels of epinephrine as low as 10~10 M can stimulate liver glycogenolysis and release of glucose. An epinephrine stimulus of this magnitude generates an intra-cellular cAMP concentration of 10~6 M, an amplification of 104. Because three more catalytic steps precede the release of glucose, another 104 amplification can occur. In striated muscle, the concentrations of the three successive enzymes in the glycogenolytic cascade—protein kinase A, glycogen phosphorylase kinase, and glycogen phosphorylase—are in a 1:10:240 ratio, which dramatically illustrates the amplification of the effects of epinephrine and cAMP.



.^Epinephrine (10"10 M)

A A Adenylyi cyclase






Protein kinase A

Activated enzyme cAMP (10"6 M)

Protein kinase A

Activated enzyme


▲ FIGURE 13-18 Amplification of an external signal downstream from a cell-surface receptor. In this example, binding of a single epinephrine molecule to one Gs proteincoupled receptor molecule induces synthesis of a large number of cAMP molecules, the first level of amplification. Four molecules of cAMP activate two molecules of protein kinase A (PKA), but each activated PKA phosphorylates and activates multiple product molecules. This second level of amplification may involve several sequential reactions in which the product of one reaction activates the enzyme catalyzing the next reaction. The more steps in such a cascade, the greater the signal amplification possible.

Although such a cascade may seem overcomplicated, it not only greatly amplifies an external signal but also allows an entire group of enzyme-catalyzed reactions to be coordi-nately regulated by a single type of signaling molecule. In addition, the multiple steps between stimulus and final response offer possibilities for regulation by other signaling pathways, thereby fine-tuning the cellular response. We will encounter other examples of cascades in signaling pathways discussed in the next chapter.

Several Mechanisms Regulate Signaling from G Protein-Coupled Receptors

Several factors contribute to termination of the response to hormones recognized by p-adrenergic receptors and other receptors coupled to Gs. First, the affinity of the receptor for hormone decreases when the GDP bound to Gsa is replaced with a GTP following hormone binding. This increase in the Kd of the receptor-hormone complex enhances dissociation of the hormone from the receptor. Second, the GTP bound to Gsa is quickly hydrolyzed, reversing the activation of adenylyl cyclase and production of cAMP (see Figure 13-11). Third, cAMP phosphodiesterase acts to hydrolyze cAMP to 5'-AMP, terminating the cellular response. Thus the continuous presence of hormone at a high enough concentration is required for continuous activation of adenylyl cyclase and maintenance of an elevated cAMP level. Once the hormone concentration falls sufficiently, the cellular response quickly terminates.

When a Gs protein-coupled receptor is exposed to hormonal stimulation for several hours, several serine and thre-onine residues in the cytosolic domain of the receptor become phosphorylated by protein kinase A (PKA). The phosphorylated receptor can bind its ligand, but ligand binding leads to reduced activation of adenylyl cyclase; thus the receptor is desensitized. This is an example of feedback suppression, in which the end product of a pathway (here activated PKA) blocks an early step in the pathway (here, receptor activation). Because the activity of PKA is enhanced by the high cAMP level induced by any hormone that activates Gs, prolonged exposure to one such hormone, say, ep-inephrine, causes desensitization not only of p-adrenergic receptors but also of Gs protein-coupled receptors that bind different ligands (e.g., glucagon). This cross-regulation is called heterologous desensitization.

Additional residues in the cytosolic domain of the p-adrenergic receptor are phosphorylated by a receptor-specific enzyme called ft-adrenergic receptor kinase (BARK), but only when epinephrine or an agonist is bound to the receptor. Because BARK phosphorylates only activated p-adrenergic receptors, this process is called homologous desensitization. Prolonged treatment of cells with epinephrine results in extensive phosphorylation and hence desensitization of the p-adrenergic receptor by both PKA and BARK.

Phosphorylated (desensitized) receptors are constantly being resensitized owing to dephosphorylation by constitutive phosphatases. Thus the number of phosphates per receptor molecule reflects how much ligand has been bound in the recent past (e.g., 1-10 minutes). This means that if a cell is constantly being exposed to a certain concentration of a hormone, that hormone concentration will eventually cease to stimulate the receptor. If the hormone concentration is now increased to a new value, the receptor will activate downstream signaling pathways but to a lesser extent than would occur if the cell were switched from a medium without hormone to one with this hormone level. If the hormone is then completely removed, the receptor becomes completely dephosphorylated and "reset" to its maximum sensitivity, in which case it can respond to very low levels of hormone. Thus a feedback loop involving receptor phos-phorylation and dephosphorylation modulates the activity of ^-adrenergic and related Gs protein-coupled receptors, permitting a cell to adjust receptor sensitivity to the hormone level at which it is being stimulated.

Another key participant in regulation of ^-adrenergic receptors is p-arrestin. This cytosolic protein binds to receptors extensively phosphorylated by BARK and completely inhibits their interaction with and ability to activate Gs. An

▲ FIGURE 13-19 Role of p-arrestin in GPCR desensitization and signal transduction. p-Arrestin binds to phosphorylated serine and tyrosine residues in the C-terminal segment of G protein-coupled receptors (GPCRs). Clathrin and AP2, two other proteins bound by p-arrestin, promote endocytosis of the receptor. p-Arrestin also functions in transducing signals from activated receptors by binding to and activating several cytosolic protein kinases. c-Src activates the MAP kinase pathway, leading to phosphorylation of key transcription factors (Chapter 14). Interaction of p-arrestin with three other proteins, including JNK-1 (a Jun N-terminal kinase), results in phosphorylation and activation of the c-Jun transcription factor. [Adapted from W. Miller and R. J. Lefkowitz, 2001, Curr Opin. Cell Biol. 13:139, and K. Pierce et al., 2002, Nature Rev. Mol. Cell Biol. 3:639.]

additional function of p-arrestin in regulating cell-surface receptors initially was suggested by the observation that loss of cell surface p-adrenergic receptors in response to ligand binding is stimulated by overexpression of BARK and p-arrestin. Subsequent studies revealed that p-arrestin binds not only to phosphorylated receptors but also to clathrin and an associated protein termed AP2, two essential components of the coated vesicles that are involved in one type of endo-cytosis. These interactions promote the formation of coated pits and endocytosis of the associated receptors, thereby decreasing the number of receptors exposed on the cell surface (Figure 13-19). Eventually the internalized receptors become dephosphorylated in endosomes, p-arrestin dissociates, and the resensitized receptors recycle to the cell surface, similar to recycling of the LDL receptor (Chapter 17). Regulation of other G protein-coupled receptors also is thought to involve endocytosis of ligand-occupied receptors and their sequestration inside the cell.

As we discuss later, p-arrestin also functions as an adapter protein in transducing signals from Gs proteincoupled receptors to the nucleus. The multiple functions of p-arrestin illustrate the importance of adapter proteins in both regulating signaling and transducing signals from cell-surface receptors.

Anchoring Proteins Localize Effects of cAMP to Specific Subcellular Regions

In many cell types, a rise in the cAMP level may produce a response that is required in one part of the cell but is unwanted, perhaps deleterious, in another part. A family of anchoring proteins localizes PKA isoforms to specific subcellular locations, thereby restricting cAMP-dependent responses to these locations. These proteins, referred to as A kinase-associated proteins (AKAPs), have a two-domain structure with one domain conferring a specific subcellular location and another that binds to the regulatory subunit of protein kinase A.

One anchoring protein (AKAP15) is tethered to the cy-tosolic face of the plasma membrane near a particular type of gated Ca2+ channel in certain heart muscle cells. In the heart, activation of p-adrenergic receptors by epinephrine (as part of the fight-or-flight response) leads to PKA-catalyzed phos-phorylation of these Ca2+ channels, causing them to open; the resulting influx of Ca2+ increases the rate of heart muscle contraction. The interaction of AKAP15 with PKA localizes PKA next to these channels, thereby reducing the time that otherwise would be required for diffusion of PKA catalytic subunits from their sites of generation to their Ca2+-channel substrate.

Another A kinase-associated protein (mAKAP) in heart muscle anchors both PKA and cAMP phosphodiesterase (PDE) to the outer nuclear membrane. Because of the close proximity of PDE to PKA, a negative feedback loop provides


G protein-coupled receptor

G protein-coupled receptor

Your Heart and Nutrition

Your Heart and Nutrition

Prevention is better than a cure. Learn how to cherish your heart by taking the necessary means to keep it pumping healthily and steadily through your life.

Get My Free Ebook

Post a comment