Both Gs and Gi are targets for medically important bacterial toxins. Cholera toxin, which is produced by the bacterium that causes cholera, is an enzyme that catalyzes the transfer of ADP ribose from intracellular NAD+ to the α subunit of Gs. This ADP ribosylation alters the α subunit so that it can no longer hydrolyze its bound GTP, causing it to remain in an active state that stimulates adenylyl cyclase indefinitely. The resulting prolonged elevation in cAMP concentration within intestinal epithelial cells causes a large efflux of Cl– and water into the gut, thereby causing the severe diarrhea that characterizes cholera. Pertussis toxin, which is made by the bacterium that causes pertussis (whooping cough), catalyzes the ADP ribosylation of the α subunit of Gi, preventing the protein from interacting with receptors; as a result, the G protein remains in the inactive GDP-bound state and is unable to regulate its target proteins. These two toxins are widely used in experiments to determine whether a cell’s GPCR-dependent response to a signal is mediated by Gs or by Gi.
Some of the responses mediated by a Gs-stimulated increase in cAMP concentration are listed in Table 15–1. As the table shows, different cell types respond differently to an increase in cAMP concentration. Some cell types, such as fat cells, activate adenylyl cyclase in response to multiple hormones, all of which thereby stimulate the breakdown of triglyceride (the storage form of fat) to fatty acids. Individuals with genetic defects in the Gs α subunit show decreased responses to certain hormones, resulting in metabolic abnormalities, abnormal bone development, and mental retardation.
Cyclic-AMP-Dependent Protein Kinase (PKA) Mediates Most of the Effects of Cyclic AMP
In most animal cells, cAMP exerts its effects mainly by activating cyclic-AMPdependent protein kinase (PKA). This kinase phosphorylates specific serines or
Figure 15–25 The synthesis and degradation of cyclic AMP. In a reaction catalyzed by the enzyme adenylyl cyclase, cyclic AMP (cAMP) is synthesized from ATP through a cyclization reaction that removes two phosphate groups as pyrophosphate (PPi); a pyrophosphatase drives this synthesis by hydrolyzing the released pyrophosphate to phosphate (not shown). Cyclic AMP is short-lived (unstable) in the cell because it is hydrolyzed by specific phosphodiesterases to form 5ʹ-AMP, as indicated.
Figure 15–24 An increase in cyclic AMP in response to an extracellular signal.
This nerve cell in culture is responding to the neurotransmitter serotonin, which acts through a GPCR to cause a rapid rise in the intracellular concentration of cyclic AMP. To monitor the cyclic AMP level, the cell has been loaded with a fluorescent protein that changes its fluorescence when it binds cyclic AMP. Blue indicates a low level of cyclic AMP, yellow an intermediate level, and red a high level. (A) In the resting cell, the cyclic AMP level is about 5 × 10–8
M. (B) Twenty seconds after the addition of serotonin to the culture medium, the intracellular level of cyclic AMP has increased to more than 10–6 M in the relevant parts of the cell, an increase of more than twentyfold. (From B.J. Bacskai et al., Science 260:222–226, 1993. With permission from AAAS.) threonines on selected target proteins, including intracellular signaling proteins and effector proteins, thereby regulating their activity. The target proteins differ from one cell type to another, which explains why the effects of cAMP vary so markedly depending on the cell type (see Table 15–1).