Gdp

Sos promotes dissociation of GDP from Ras; GTP binds and active Ras dissociates from Sos

Signaling

Signaling

Genetic Studies in Drosophila Identify Key Signal-Transducing Proteins Downstream from Receptor Tyrosine Kinases

The compound eye of the fly is composed of some 800 individual eyes called ommatidia (Figure 14-17a). Each omma-tidium consists of 22 cells, eight of which are photosensitive neurons called retinula, or R cells, designated R1-R8 (Figure 14-17b). An RTK called Sevenless (Sev) specifically regulates development of the R7 cell and is not essential for any other known function. In flies with a mutant sevenless (sev) gene, the R7 cell in each ommatidium does not form (Figure 14-17c). Since the R7 photoreceptor is necessary for flies to see in ultraviolet light, mutants that lack functional R7 cells but are otherwise normal are easily isolated.

During development of each ommatidium, a protein called Boss (Bride of Sevenless) is expressed on the surface of the R8 cell. This membrane-tethered protein is the ligand for the Sev RTK on the surface of the neighboring R7 precursor cell, signaling it to develop into a photosensitive neuron (Figure 14-18a). In mutant flies that do not express a functional Boss protein or Sev RTK, interaction between the Boss and Sev proteins cannot occur, and no R7 cells develop (Figure 14-18b).

To identify intracellular signal-transducing proteins in the Sev RTK pathway, investigators produced mutant flies expressing a temperature-sensitive Sev protein. When these flies were maintained at a permissive temperature, all their om-matidia contained R7 cells; when they were maintained at a nonpermissive temperature, no R7 cells developed. At a par

▲ FIGURE 14-17 The compound eye of Drosophila melanogaster. (a) Scanning electron micrograph showing individual ommatidia that compose the fruit fly eye. (b) Longitudinal and cutaway views of a single ommatidium. Each of these tubular structures contains eight photoreceptors, designated R1-R8, which are long, cylindrically shaped light-sensitive cells. R1-R6 (yellow) extend throughout the depth of the retina, whereas R7 (brown) is located toward the surface of the eye, and R8 (blue) toward the backside, where the axons exit. (c) Comparison of eyes from wild-type and sevenless ticular intermediate temperature, however, just enough of the Sev RTK was functional to mediate normal R7 development. The investigators reasoned that at this intermediate temperature, the signaling pathway would become defective (and thus no R7 cells would develop) if the level of another protein involved in the pathway was reduced, thus reducing the activity of the overall pathway below the level required to form an R7 cell. A recessive mutation affecting such a protein would have this effect because, in diploid organisms like Drosophila, a heterozygote containing one wild-type and one mutant allele of a gene will produce half the normal amount of the gene product; hence, even if such a recessive mutation is in an essential gene, the organism will be viable. However, a fly carrying a temperature-sensitive mutation in the sev gene and a second mutation affecting another protein in the signaling pathway would be expected to lack R7 cells at the intermediate temperature.

By use of this screen, researchers identified the genes encoding three important proteins in the Sev pathway (see Figure 14-16): an SH2-containing adapter protein exhibiting 64 percent identity to human GRB2; a guanine nucleotide-exchange factor called ,Sos (Son of Sevenless) exhibiting 45 percent identity with its mouse counterpart; and a Ras protein exhibiting 80 percent identity with its mammalian counterparts. These three proteins later were found to function in other signaling pathways initiated by ligand binding to different RTK receptors and used at different times and places in the developing fly.

In subsequent studies, researchers introduced a mutant rasD gene into fly embryos carrying the sevenless mutation.

mutant flies viewed by a special technique that can distinguish the photoreceptors in an ommatidium. The plane of sectioning is indicated by the blue arrows in (b), and the R8 cell is out of the plane of these images. The seven photoreceptors in this plane are easily seen in the wild-type ommatidia (top), whereas only six are visible in the mutant ommatidia (bottom). Flies with the sevenless mutation lack the R7 cell in their eyes. [Part (a) from E. Hafen and K. Basler, 1991, Development 1 (suppl.):123; part (b) adapted from R. Reinke and S. L. Zipursky, 1988, Cell 55:321; part (c) courtesy of U. Banerjee.]

(a) Wild type I R8 cell )

Boss Sev

R7 precursor

Induction

Boss Sev

R7 precursor

Induction e

(b) Single mutant (c) Double mutant

No induction

Active Ras ve

No induction ti)

R7 neuron

▲ EXPERIMENTAL FIGURE 14-18 Genetic studies reveal that activation of Ras induces development of R7 photoreceptors in the Drosophila eye. (a) During larval development of wild-type flies, the R8 cell in each developing ommatidium expresses a cell-surface protein, called Boss, that binds to the Sev RTK on the surface of its neighboring R7 precursor cell. This interaction induces changes in gene expression that result in differentiation of the precursor cell into a functional R7 neuron. (b) In fly embryos with a mutation in the sevenless (sev) gene, R7 precursor cells cannot bind Boss and therefore do not differentiate normally into R7 cells. Rather the precursor cell enters an alternative developmental pathway and eventually becomes a cone cell. (c) Double-mutant larvae (sev~ ; RasD) express a constitutively active Ras (RasD) in the R7 precursor cell, which induces differentiation of R7 precursor cells in the absence of the Boss-mediated signal. This finding shows that activated Ras is sufficient to mediate induction of an R7 cell. [See M. A. Simon et al., 1991, Cell 67:701, and M. E. Fortini et al., 1992, Nature 355:559.]

As noted earlier, the rasD gene encodes a constitutive Ras protein that is present in the active GTP-bound form even in the absence of a hormone signal. Although no functional Sev RTK was expressed in these double-mutants (sev- ; rasD), R7 cells formed normally, indicating that activation of Ras is sufficient for induction of R7-cell development (Figure 14-18c). This finding, which is consistent with the results with cultured fibroblasts described earlier, supports the conclusion that activation of Ras is a principal step in intracel-lular signaling by most if not all RTKs.

Binding of Sos Protein to Inactive Ras Causes a Conformational Change That Activates Ras

The adapter protein GRB2 contains two SH3 domains, which bind to Sos, a guanine nucleotide-exchange factor, in addition to an SH2 domain, which binds to phosphotyrosine residues in RTKs. Like phosphotyrosine-binding SH2 and PTB domains, SH3 domains are present in a large number of proteins involved in intracellular signaling. Although the three-dimensional structures of various SH3 domains are similar, their specific amino acid sequences differ. The SH3 domains in GRB2 selectively bind to proline-rich sequences in Sos; different SH3 domains in other proteins bind to proline-rich sequences distinct from those in Sos.

Proline residues play two roles in the interaction between an SH3 domain in an adapter protein (e.g., GRB2) and a pro-line-rich sequence in another protein (e.g., Sos). First, the pro-line-rich sequence assumes an extended conformation that permits extensive contacts with the SH3 domain, thereby facilitating interaction. Second, a subset of these prolines fit into binding "pockets" on the surface of the SH3 domain (Figure 14-19). Several nonproline residues also interact with the SH3 domain and are responsible for determining the binding specificity. Hence the binding of proteins to SH3 and to SH2 domains follows a similar strategy: certain residues provide the overall structural motif necessary for binding, and neighboring residues confer specificity to the binding.

Following activation of an RTK (e.g., Sevenless or the EGF receptor), a complex containing the activated receptor, GRB2, and Sos is formed on the cytosolic face of the plasma membrane (see Figure 14-16). Formation of this complex depends on the ability of GRB2 to bind simultaneously to the receptor and to Sos. Thus receptor activation leads to

SH3 domain

▲ FIGURE 14-19 Surface model of an SH3 domain bound to a short, praline-rich target peptide. The target peptide is shown as a space-filling model. In this target peptide, two prolines (Pro4 and Pro7, dark blue) fit into binding pockets on the surface of the SH3 domain. Interactions involving an arginine (Arg1, red), two other prolines (light blue), and other residues in the target peptide (green) determine the specificity of binding. [After H. Yu et al., 1994, Cell 76:933.]

▲ FIGURE 14-20 Structures of Ras bound to GDP, Sos protein, and GTP (a) In Ras-GDP the Switch I and Switch II segments do not directly interact with GDP (see Figure 13-8). (b) One a helix (orange) in Sos binds to both switch regions of Ras-GDP leading to a massive conformational change in Ras. In effect, Sos pries Ras open by displacing the Switch I region, thereby allowing GDP to diffuse out. (c) GTP is thought to bind to

Ras-Sos first through its base; subsequent binding of the GTP phosphates completes the interaction. The resulting conformational change in Switch I and Switch II segments of Ras, allowing both to bind to the GTP y phosphate (see Figure 13-8), displaces Sos and promotes interaction of Ras-GTP with its effectors (discussed later). [Adapted from P A. Boriack-Sjodin and J. Kuriyan, 1998, Nature 394:341.]

relocalization of Sos from the cytosol to the membrane, bringing Sos near to its substrate, namely, membrane-bound Ras-GDP. Biochemical and genetic studies indicate that the c-terminus of Sos inhibits its nucleotide-exchange activity and that GRB2 binding relieves this inhibition.

Binding of Sos to Ras-GDP leads to conformational changes in the Switch I and Switch II segments of Ras, thereby opening the binding pocket for GDP so it can diffuse out (Figure 14-20). Because GTP is present in cells at a concentration some 10 times higher than GDP, GTP binding occurs preferentially, leading to activation of Ras. The activation of Ras and Ga thus occurs by similar mechanisms: a conformational change induced by binding of a protein—Sos and an activated G protein-coupled receptor, respectively— that opens the protein structure so bound GDP is released to be replaced by GTP. Binding of GTP to Ras, in turn, induces a specific conformation of Switch I and Switch II that allows Ras-GTP to activate downstream effector molecules, as we discuss in the next section.

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