Down-Modulation of Receptor Signaling

We have already seen several ways that signal-transduction pathways can be regulated. The levels of hormones produced and released from signaling cells are adjusted constantly to meet the needs of the organism. For example, kidney cells make and secrete more erythropoietin when the oxygen level is low and more red blood cells are needed. Intracellular proteins such as Ski and SocS are induced following stimulation by TGFp or cytokines, and then negatively regulate their respective signal-transduction pathways. Phosphorylation of receptors and downstream signaling proteins are reversed by the carefully controlled action of phosphatases. Here we discuss two other mechanisms by which signaling pathways are down-regulated: removal of receptors from the cell surface by endocytosis, and secretion of proteins that bind and sequester hormones, thus preventing their interaction with cell-surface receptors.

Endocytosis of Cell-Surface Receptors Desensitizes Cells to Many Hormones

In previous sections we discussed several signal-transduction pathways activated immediately after stimulation of cytokine receptors and receptor tyrosine kinases (RTKs). If the level of hormone in the environment remains high for several hours, cells usually undergo desensitization, such that they no longer respond to that concentration of hormone. This prevents inappropriate prolonged receptor activity, but under these conditions cells usually will respond if the hormone level is increased further. Ligand-dependent receptor-mediated endocytosis, which reduces the number of available cell-surface receptors, is a principal way that cells are desensitized to many peptides and other hormones.

In the absence of EGF ligand, for instance, the EGF receptor is internalized at a relatively slow rate by bulk mem brane flow. Besides activating the receptor's protein tyrosine kinase, binding to EGF induces a conformational change in the cytosolic tail of the receptor. This exposes a sorting motif that facilitates receptor recruitment into clathrin-coated pits and subsequent internalization. After internalization, some cell-surface receptors (e.g., the LDL receptor) are efficiently recycled to the surface (see Figure 17-28). In contrast, internalized receptors for many peptide hormones, together with their bound hormone ligands, commonly are transported to lysosomes wherein they are degraded, rather than being recycled to the cell surface.

For example, each time an EGF receptor is internalized with bound EGF, it has about a 50 percent chance of being degraded. Exposure of a fibroblast cell to high levels of EGF for 1 hour induces several rounds of endocytosis, resulting in degradation of most receptor molecules. If the concentration of extracellular EGF is then reduced, the number of EGF receptors on the cell surface recovers by synthesis of new receptor molecules, a slow process that may take more than a day. In this way a cell can become desensitized to a continual high level of hormone and, after hormone removal, reestablish its initial level of cell-surface receptors, thereby becoming sensitive again to a low level of hormone.

Experiments with mutant cell lines demonstrate that in-ternalization of RTKs plays an important role in regulating cellular responses to EGF and other growth factors. For instance, a mutation in the EGF receptor that prevents it from being incorporated into coated pits, and thus makes it resistant to ligand-induced endocytosis, substantially increases the sensitivity of cells to EGF as a mitogenic signal. Such mutant cells are prone to EGF-induced cell transformation. Interestingly, internalized receptors can continue to signal from intracellular compartments prior to their degradation.

In most cases, peptide hormones that are internalized bound to their receptors are degraded intra-ILUUJ cellularly. If the initial extracellular hormone level is relatively low, this process may reduce the hormone level sufficiently to terminate cell signaling after a few hours or so. For instance, IL-2, a cytokine that stimulates growth of immune T cells, normally is depleted from the extracellular environment by this mechanism, leading to cessation of signaling. Mutant forms of IL-2 have been obtained that bind to the IL-2 receptor normally at pH 7.5, that of the extracellular medium, but poorly at pH 6, that of the initial en-docytic vesicle, or endosome. These mutant IL-2 proteins dissociate from the receptor in the endosome and are "recycled"; that is, they are secreted back into the extracellular medium rather than accompanying the receptor to the lyso-some for degradation. Because the lifetime of these mutant IL-2 proteins is longer than normal, they are more potent than their normal counterparts and may be useful therapeu-tically for stimulating production of T cells. I

Secreted Decoy Receptors Bind Hormone and Prevent Receptor Activation

Another way of reducing the activity of cell-surface receptors is secretion of a protein that contains a hormone-binding segment but no signal-transducing activity. As might be expected, hormone binding to such proteins, called decoy receptors, reduces the amount of hormone available to bind to receptors capable of signaling. This type of regulation is important in controlling bone resorption, a complex physiological process that integrates several molecular mechanisms.

Net bone growth in mammals subsides just after puberty, but a finely balanced, highly dynamic process of disassembly (resorption) and reassembly (bone formation), called remodeling, goes on throughout adulthood. Remodeling permits the repair of damaged bones and can release calcium, phosphate, and other ions from mineralized bone into the blood for use elsewhere in the body.

Osteoclasts, the bone-dissolving cells, are a type of macrophage that contain highly dynamic integrin-containing adhesive structures, called podosomes, in the plasma membrane (see Figure 6-27). The av^3 integrin in podosomes is crucial to the initial binding of osteoclasts to the surface of bone, since antibodies that bind to and block the activity of this integrin block bone resorption. Following their initial adhesion to bone, osteoclasts form specialized, very tight seals between themselves and bone, creating an enclosed extracellular space (Figure 14-31). An adhered osteoclast then secretes into this space a corrosive mixture of HCl and proteases that dissolves the inorganic components of the bone and digests its protein components. The mechanism of HCl generation and secretion is reminiscent of that used by the stomach to generate digestive juice (see Figure 7-28). As in gastric HCl secretion, carbonic anhydrase and an anion antiport protein are used to generate H+ ions within osteoclasts. However, osteoclasts employ a V-type proton pump to export H+ ions into the bone-facing space rather than the P-class ATP-powered H+/K+ pump used by gastric epithelial cells (see Figure 7-6).

Bone resorption by osteoclasts is carefully regulated by cell-cell interactions with neighboring osteoblasts. These bone-forming cells secrete type I collagen, the major organic component of bones. Osteoblasts express a trimeric cell-surface signaling protein termed RANKL that is a member of the TNF-a superfamily of trimeric signaling proteins. RANKL is the ligand for RANK, a cell-surface receptor expressed by osteoclasts. Interaction of RANK with RANKL initiates multiple intracellular signaling pathways in osteo-clasts, including the NF-kB pathway that also is initiated by stimulation of TNF-a receptors (see Figure 14-28). Collectively, these signals induce the differentiation of osteoclasts and changes in their shape that promote tight binding to bone and thus bone resorption.

Osteoblasts also produce and secrete a soluble decoy receptor protein called osteoprotegerin (OPG), named for its ability to "protect bone." Secreted OPG binds to RANKL on

▲ FIGURE 14-31 Bone resorption and its regulation.

Osteoclasts initially bind to bone via integrin-mediated podosomes. The subsequent activation of an osteoclast by interaction with neighboring osteoblasts via the trimeric membrane proteins RANKL and RANK 1 induces cytoskeletal reorganization, leading to formation of a specialized tight seal with bone 2 . The activated osteoclast secretes into the extracellular space generated by this seal a corrosive mixture of HCl and proteases that resorbs the bone 3. Osteoblasts can suppress bone resorption by secreting osteoprotegerin (OPG). Binding of this decoy receptor to RANKL 4 blocks RANKL binding to RANK on osteoclasts and thus their activation. See the text for discussion. [Adapted from N. Takahashi et al., 1999, Biochem. Biophys. Res. Comm. 256:449.]

the surface of osteoblasts, thereby preventing the RANKL-RANK interaction and inhibiting osteoclast activation and bone resorption (see Figure 14-31). Mice deleted for the OPG gene have weak, porous bones characteristic of excessive resorption. This finding supports the essential function of OPG in reducing bone resorption.

The rare hereditary disease osteopetrosis, marked by increased bone density, is due to abnormally low resorption. Far more common is osteoporosis, which is most prevalent among postmenopausal women. This metabolic disorder results from disproportionate bone resorption, leading to porous, less dense bones that are readily broken or fractured.

Many steroid hormones (e.g., estrogen, glucocorticoids), vitamin D, polypeptide hormones, and drugs influence bone metabolism by directly interacting with osteoblasts and altering the RANKL/RANK signaling system. Estrogen, for example, normally induces secretion of OPG and thus inhibits bone resorption. When estrogen is low, as it is in many post-

biotech biotech menopausal women, resorption increases and the bones weaken. It may be possible to develop new treatments for osteoporosis based on altering the signaling system that controls bone resorption. I

Because they bind their ligands so tightly, soluble extracellular domains of cell-surface hormone receptors are finding increasing use as therapeutics. Many cell-surface receptors are oriented in the plasma membrane such that the C-terminal signal-transducing domain extends into the cytosol and the N-terminal ligand-binding domain extends into the extracellular space. With recombinant DNA techniques a stop codon can be placed in the cDNA encoding such a receptor so that translation in an appropriate expression system generates a truncated protein corresponding to the receptor's extracellular domain, which will be secreted and can function as a decoy receptor. For example, local increases in TNF-a are frequent in rheumatoid arthritis, an inflammatory joint disease. Injection of the recombinant-produced extracellular domain of the TNF-a receptor, which "soaks up" some of the excess TNF-a and reduces inflammation, is now one of the major therapies for severe cases of this disease. I

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