Earlier in this chapter we discussed two mechanisms by which signaling from cytokine receptors is down-modulated—those involving phosphotyrosine phosphatases and SOCS proteins (see Figure 16-13). These proteins are also used to depress signaling by some RTKs, but two other mechanisms—receptor-mediated endocytosis and degradation in lysosomes—are more common.

738

Receptor-Mediated Endocytosis Prolonged treatment of cells with ligand often reduces the number of available cell-surface receptors, so that the cells have a less robust response to exposure to a given concentration of ligand than they did before ligand addition. This desensitization response helps prevent inappropriately prolonged receptor activity. In the absence of EGF, for instance, cell-surface HER1 receptors are relatively long-lived, with an average half-life of 10–15 hours. Unbound receptors are internalized via clathrin-coated pits into endosomes at a relatively slow rate, on average once every 30 minutes, and are often returned rapidly to the plasma membrane so that there is little reduction in total surface receptor numbers. Following binding of an EGF ligand, the rate of endocytosis of HER1 is increased about tenfold, and only a fraction of the internalized receptors return to the plasma membrane; the rest are degraded in lysosomes. Each time a HER1–EGF complex is internalized via the process of receptor-mediated endocytosis (see Figure 14-29), the receptor has about a 20 to 80 percent chance of being degraded, depending on the cell type. Exposure of a fibroblast cell to high levels of EGF for several hours induces several rounds of endocytosis, resulting in degradation of most cell-surface EGF receptor molecules and thus a reduction in the cell’s sensitivity to EGF. In this way, prolonged treatment with a given concentration of EGF desensitizes the cell to that level of hormone, though the cell may respond if the level of EGF is increased.

HER1 mutants that lack kinase activity do not undergo accelerated endocytosis in the presence of ligand. It is likely that ligand-induced activation of the kinase activity in normal HER1 induces a conformational change in the cytosolic tail, exposing a sorting motif that facilitates receptor recruitment into clathrin-coated pits and subsequent internalization of the receptor-ligand complex. Despite extensive study of mutant HER1 cytosolic domains, the identity of these “sorting motifs” is controversial, and most likely multiple motifs function to enhance endocytosis. Interestingly, internalized receptors can continue to signal from endosomes or other intracellular compartments before their degradation, as evidenced by their binding to signaling proteins such as GRB2 and Sos, which are discussed in the next section.

Lysosomal Degradation Several processes influence recycling of surface receptors to the plasma membrane versus lysosomal degradation; one is covalent modification by the small protein ubiquitin (see Chapter 3). There is a strong correlation between monoubiquitinylation (addition of a single ubiquitin to a given lysine of a protein) of the HER1 cytosolic domain and HER1 degradation. The monoubiquitinylation, which also occurs in other ligand-activated RTKs, is mediated by the enzyme c-Cbl. This enzyme, which is an E3 ubiquitin ligase (see Figure 3-31), contains an EGF receptor–binding domain, which binds directly to phosphorylated EGF receptors, and a RING finger domain, which recruits ubiquitin-conjugating enzymes and mediates transfer of ubiquitin to the receptor. The ubiquitin functions as a “tag” on the receptor that stimulates its incorporation from endosomes into multivesicular bodies (see Figure 14-33) that are ultimately degraded in lysosomes. A role for c-Cbl in EGF receptor trafficking emerged from genetic studies in C. elegans, which established that c-Cbl negatively regulates the function of the nematode EGF receptor (Let-23), probably by inducing its degradation. Similarly, knockout mice lacking c-Cbl show hyperproliferation of mammary gland epithelia, consistent with a role of c-Cbl as a negative regulator of EGF signaling.

Experiments with mutant cell lines have demonstrated that internalization of RTKs plays an important role in regulating cellular responses to EGF and other growth factors. For instance, a mutation in the HER1 EGF receptor that prevents it from being incorporated into coated pits makes it resistant to ligand-induced endocytosis. As a result, this mutation leads to substantially elevated numbers of EGF receptors on cells and thus increased sensitivity of cells to EGF as a mitogenic signal. Such mutant cells are prone to EGF-induced transformation into tumor cells (see Chapter 24). Interestingly, the other EGF family receptors—HER2, HER3, and HER4—do not undergo ligand-induced internalization, an observation that emphasizes how each receptor evolved to be regulated in its own appropriate manner.