Molecular and Cellular Signaling - Martin Beckerman

266 11. Signaling in the Endocrine System

gamma phosphate group to the GDP molecule. The result is an increase in the rate of hydrolysis from one GTP molecule every 35 minutes to 102 to 103 molecules per minute.

11.15Role of Rho, Rac, and Cdc42, and Their Isoforms

Rho, Rac, and Cdc42, and their isoforms, coordinate the reorganization of the actin cytoskeleton in response to extracellular signals. Members of the Rho family of GTPases—Rho, Rac, and Cdc42—regulate cell polarity, cell morphogenesis and shape, and cell motility. Each of these GTPases operates in a different pathway. In fibroblasts, Rho promotes the formation stress fiber bundles, Rac proteins regulate actin polymerization resulting in the formation of lamellipodia, and Cdc42 proteins control the formation of filopodia. All three regulate the formation of focal adhesion complexes. These observations are not limited to fibroblasts but rather are believed to take place in all eukaryotic cells. In addition to their role in remodeling the actin cytoskeleton, Rho family members regulate gene transcription. Signaling through receptor tyrosine kinases, G protein-coupled receptors and cytokine receptors activate Rho GTPases. In many instances integrins and Rho family members work together to regulate the actin cytoskeleton.

An examination of the steps leading to bud formation in yeasts provides some insight into how cell polarity develops and is regulated by the Rho family GTPases.The Cdc42 GTPase, its GEF called Cdc24, its GAP, of which there are several, and a scaffold protein named Bem1 contribute to the start of budding.As is the case for all GTPases, Cdc42 goes through a cycle of GDP release catalyzed by its GEF immediately followed by GTP binding. This state is followed some time later by hydrolysis catalyzed by its GAP in which

GTP is cleaved leaving GDP bound to the CDc42 protein. Unlike Ras, the

Cdc42 protein does not act on multiple substrate proteins in its GTP-bound state. Rather it goes through repeated cycles of GDP-GTP-GDP binding and hydrolysis to regulate the polymerization of actin fibers. The difference between the Ras switchlike behavior and Cdc42 assembly controller-like behavior is depicted in Figure 11.8. In the Ras mode of cycling, the GTPase is on and can influence many substrate proteins before being switched off by hydrolysis. In the Cdc42 mode of operation (Figure 11.8b), hydrolysis is required. Substrate interactions are again initiated by GTP binding but the action is not completed until the hydrolysis part of the cycle is executed. The EF-Tu GTPase uses this method of repeatedly cycling on and off during the translation elongation process. This second mode allows for some checking and quality control over the process; the assembly step can be aborted prior to hydrolysis if errors are detected.

Cell polarization (and bud formation) is typically driven by environmental cues such as gradients of chemoattractants or by signals from neighboring cells. These cues tell the cell which way is “up” and which way is

“down.” In bud formation, the adapter protein Bem1 is recruited to the site of bud growth where it interacts with Ccd42 and its Cdc24 GEF. The GEF is recruited and stabilized at the plasma membrane by the Gbg subunit activated by GPCR signaling. Both Cdc42 and Cdc24 bind to the scaffold and a positive feedback loop is set up in which additional Bem1 proteins are recruited. A crucial element in the choice of bud site selection is the presence of position landmarks in the cell that identify the location of the poles. If these landmarks are absent the yeast cell is still able to form buds by a process in which a bud site is randomly selected.

11.16Ran Family Coordinates Traffic In and Out of the Nucleus

While small molecules can rapidly and freely diffuse in and out of the nucleus, macromolecules of size 40 to 50 kDa or greater cannot. Instead they are transported through selective and facilitated diffusion through nuclear pore complexes. Nuclear pore complexes (NPCs) are large units built up from more than 100 different proteins. The NPCs function as selective gates. Only the larger macromolecules containing the correct targeting or localization sequence are allowed through. Nuclear pore complexes have no motors to actively transport cargo. Instead, import and export occur by means of facilitated diffusion. This process does not consume energy and is nondirectional. Proteins, mRNAs, tRNA, ribosomal subunts, and other macromolecules are transported bound to transport molecules called importins and exportins that recognize the nuclear localization signals (NLSs) and nuclear export signals (NESs). These transport receptors together with adapter molecules, Ran GTPases, Ran GEFs, and Ran GAPs facilitate the diffusion of the macromolecules in and out of the nucleus.

Transport receptors belonging to the importin b family shuttle cargo in and out of the nucleus through the NPC. These receptors are encountered in two forms: as importins that shuttle cargo into the nucleus and as exportins that chaperone cargo the other way. The loading and release of cargo by the importin b receptors is regulated by the Ran GTPases. Ran, a 25-kDa protein, is an abundant gene product found in all eukaryotic cells. The key element in their ability to regulate cargo movement is the formation of a Ran concentration gradient across the nuclear envelope. This gradient in GTP-bound Ran is established and maintained by the Ran GEFs and Ran GAPs. The Ran GAPs are restricted to the cytoplasm and cannot enter the nucleus, while the Ran GEFs are localized exclusively in the nucleus. As a result the concentration of GTP-bound Ran is low in the cytoplasm and high in the nucleus.

The combined actions of importin b receptors and Ran GTPases are illustrated in Figure 11.9. As is the case for all GTPases its main actions occur during the GTP-bound part of the cycle, when it shuttles exportin + cargo and unbound importin molecules through the NPC from the nucleus to the

268 11. Signaling in the Endocrine System

FIGURE 11.9. Ran GTPase-mediated import and export through nuclear pore complexes (NPCs): Importin (dark square) shuttles its cargo (dark circles) through the NPC into the nucleus. In the nucleus, GTP-bound Ran GTPase triggers the release of the cargo from the importin and shuttles the importin back out to the cytoplasm. The GTP-bound Ran GTPase shuttles exportin (light squares) plus its cargo (light circles) through the NPC from the nucleus to the cytoplasm where other proteins (not shown) help dissociate the complex. The unbound exportin then diffuses back to the nucleus.

cytoplasm. It differs from the Ras switching and Cdc42 cytoskeleton assembly in so far as its GEF and GAP actions take place in different compartments. Hydrolysis is not required for movement through the pore and release of the cargo, but this aspect changes when Ran mediates assembly of the nuclear envelope. In those assembly operations hydrolysis is necessary.

11.17Rab and ARF Families Mediate the Transport of Cargo

Recall that in eukaryotes secreted soluble molecules and plasma membrane lipids and proteins are transported from compartment to compartment by transport vesicles. Newly synthesized proteins and lipids move from the ER to the Golgi apparatus and from there to the plasma membrane. The vesicles that transport these molecules are produced by budding from the membrane of the donor compartment, and upon arrival to the acceptor compartment fuse with the membrane of the acceptor compartment thereby transferring the cargo. The process of delivering cargo from the internal compartments to the plasma membrane is known as exocytosis. Cargo moves in two directions, outward and inward. Many membrane components are rapidly turned over. Vesicles transport macromolecules from

FIGURE 11.10. Rab GTPase cycle: The Rab GTPase cycle synchronizes vesicle transport and fusion between the membranes labeled A and B. In the cycle, a GDP-bound Ran protein is shuttled by its GDI to membrane A where GDP is released and GTP is bound in its place. While in its active form, a cargo vesicle pinches off from the membrane and is conveyed to membrane B where it docks and then fuses. Hydrolysis occurs and, assisted by the GDI, the GDPbound Ran protein is released from the membrane and returned to the cytosol.

the plasma membrane either to the lysosomes, where they are degraded, or to endosomes, where they are recycled back to the plasma membrane. The process of transporting plasma membrane molecules to lysosomes for degradation is known as endocytosis, and the outward and inward movement of plasma membrane molecules is said to take place in the secretory and endocytic pathways.

The Rab family with more than 30 known members is the largest group of Ras superfamily GTPases. These proteins coordinate the docking and fusion of cargo vesicles operating in the secretory and endocytic pathways. Reciprocal pairs of proteins called v-SNAREs and t-SNAREs mediate the fusion of cargo vesicles. The binding of the SNAREs is preceded by tethering/protection protein-binding steps that ensure that only the appropriate fusion events takes place. The Rab proteins are localized to the cytoplasmic face of organelles and vesicles and participate in the preliminary binding operations.

The Rab protein cycle of GDP and GTP binding and release is synchronized with the movement and fusion of cargo vesicles. The combined set of steps is illustrated in Figure 11.10. In this figure the GEFs and GAPs are joined by a third set of accessory regulators—GDP dissociation inhibitors, or GDIs. These molecules bind and maintain pools of inactive Rab proteins in the cytosol and serve as recycling chaperones. They first help to release the GDPases from the membranes and then shuttle them back to their site of origin in the cytosol in preparation for their next cycle of use.

ADP-robosylation factors (Arfs) make up the fifth family of Ras

GTPases. These 20-kDa proteins help regulate the formation of cargo vesicles through budding from donor membranes. In order for budding to take place, coat proteins must be assembled over the membrane surface. These binding agents, coat protein I (COPI), coat protein II (COPII), and clathrin, mediate the mechanical forces that pull a membrane into a bud, and they

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Rho Family of GTPases

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Ran Family of GTPases

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Rab and Arf Families of GTPases

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Recycling, and Signaling in the Endocytic Pathway

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Problems

11.1(a) Binding interactions. One of the standard questions addressed in investigations of signal transduction asks what interactions are possible in a particular molecular setting. A number of proteins

Problems 273

FIGURE FOR PROBLEM 11.1. Phosphorylated threonine and tyrosine residues are identified by a “P” enclosed in a circle. The symbol “PP” refers to proline-rich regions. Threonine and tyrosine phosphorylation sites with the correct flanking sequences for recognition by their kinases are indicated by the symbols p-Thr and p-Tyr. Note that phosphorylation at the sites located in the center of the catalytic domains of the NRTKs is required for activation.

and membranes are shown in the accompanying figure. Identify the possible interactions in a mixture containing multiple copies of each element (a) through (j).

(b)Build your own. Starting at the plasma membrane construct one or more signaling pathways using the elements (a) through (j).

11.2Nerve cells are highly polarized structures, and their terminals are located far from the cell body where the nucleus is located. Signals sent from axon terminals to the cell body must travel distances of up to a meter. These signals are referred to as retrograde signals since they move in a direction opposite to action potentials, which travel down the axon to the nerve terminal. How might a neuron generate a rapid (i.e., faster than one conveyed by passive diffusion) retrograde signal, observing that receptor tyrosone kinases such as the Trks (and also G protein-coupled receptors) are internalized following ligand binding?