BIOLOGICAL OVERVIEW

In higher vertebrates, hormones and growth factors play an important role in the control of overall growth because they orchestrate cell growth, cell cycle, and cell survival. Reducing or increasing levels of growth hormone or of the growth hormone mediators, IGF1 and its receptor (IGFR), dramatically influences body and organ size (for review see Stewart, 1996). Overall growth is affected by the availability of nutrients, as is cell size, in some cases. Many organisms have developed special survival strategies for growth during periods of low nutrition. Under inadequate nutritional conditions, yeast cells, for example, reduce growth and divide at a smaller size, whereas nematodes like C. elegans enter a diapause called the dauer stage. Raising Drosophila under adverse food conditions also results in the production of small flies with fewer and smaller cells. Still, little is known in higher organisms about how growth is controlled at the cellular level: what are the genes involved in the regulation of cell growth, and what determines the critical size at which cells undergo mitosis? In Drosophila, a class of mutations known as Minutes (M) dominantly delay development and in some cases reduce body size. Some of the M genes encode ribosomal proteins and are thought to slow down growth by reducing protein synthesis. Partial loss-of-function mutations in the Drosophila myc gene diminutive cause a reduction in overall body size (Gallant, 1996). However, it is not yet known how Drosophila myc controls growth (Böhni, 1999 and references).

The Drosophila insulin receptor (INR) pathway, and in particular the adaptor protein Chico, which is homologous to vertebrate insulin receptor substrates (IRS), plays a critical role in the control of cell proliferation, cell size, and overall body growth. In Spanish, chico means 'small boy'. chico mutant flies are smaller in size, owing to a reduction in cell size and cell number. The effect of chico mutations on cell size and cell growth is strictly cell autonomous. In addition to its overall effect on growth, Chico also controls cellular metabolism; even though chico flies are only half the size of normal flies, they show an almost 2-fold increase in lipid levels, when compared with their heterozygous siblings. These results provide evidence for a cell-autonomous requirement of the INR signaling pathway in the control of cell size and overall growth (Böhni, 1999).

Many aspects of the insulin system appear to be conserved in both flies and mammals. In mammalian cells, activation of either the insulin receptor or the IGF1 (insulin-like growth factor 1) receptor by insulin and IGF1, respectively, results in the recruitment of IRS1 or IRS2 to the receptor via interaction of the IRS phosphotyrosine binding domains, with a phosphotyrosine motif (NPXY) in the juxtamembrane region of the receptors. Phosphorylation of the multiple tyrosine residues of IRS1 triggers the activation of various signaling pathways, including the RAS/MAP kinase pathway via the SH2/SH3 adaptor GRB2 and the PI3K/PKB pathway via the p85 SH2 adaptor subunit of p110 PI3K (Yenush, 1997). The Drosophila INR shares many structural features with its human homologs, including its heterotetrameric structure and a conserved PTB consensus binding site in the juxtamembrane region. However, the Drosophila INR contains a 400-amino acid C-terminal extension not found in any of the vertebrate receptors. This C-terminal tail contains three YXXM consensus binding sites for the SH2 domain of the p60 subunit of PI3K (see Phosphotidylinositol 3 kinase 92E) and four additional NPXY consensus PTB-binding sites. The C-terminal domain is functional, since expression of a chimeric receptor consisting of the extracellular domain of the human INR and the intracellular domain of the Drosophila INR in murine 32D cells lacking endogenous IRS1 can partially activate mammalian PI3K and S6K. In contrast, the ability of the human INR to activate PI3K in this system is strictly dependent on the coexpression of IRS1 (Yenush, 1996a). These findings and the identification of Chico suggest that in Drosophila, INR couples to the downstream effector PI3K in two different ways, one using docking sites in the INR C-terminal tail and the other connecting through docking sites in Chico (Böhni, 1999).

The homology of Chico with mammalian IRS1, IRS2, IRS3 and IRS4 (collectively referred to as IRS1-4) prompted a test for genetic interactions with other components involved in signaling via IRS proteins, such as the insulin receptor and the p110 catalytic subunit of PI3 kinase. Loss-of-function mutations in Inr are lethal, but certain heteroallelic combinations survive to adulthood. Such Inr mutant flies are reduced in size (Chen, 1996). As in chico mutants, cell size is reduced by 28% in Inr³¹³/Inr³²⁷ flies. Furthermore, targeted expression of a dominant-negative variant of Drosophila p110 PI3K in the developing eye or wing causes a reduction in cell size in the eye, and in both cell size and cell number in the wing. Conversely, overexpression of a constitutively active, membrane-targeted version of PI3K increases cell size and cell number (Leevers, 1996). In flies that are homozygous for chico, heterozygosity for a hypomorphic Inr allele leads to a further reduction in cell number in the wing and the eye. Thus, in the absence of chico function, a reduction of the receptor level potentiates the growth reduction. This Chico independent signaling of INR is likely to be mediated by PI3K-binding sites in the C-terminal tail of the INR (Yenush, 1996). Similarly, expressing a catalytically inactive version of PI3K in chico homozygous wing discs leads to a further reduction in wing size by 48%. Thus, the chico mutant phenotype is modified by mutations in Inr and PI3K. This is consistent with the notion that INR, Chico, and PI3K form a conserved signaling pathway involved in the cell-autonomous control of growth and cell size in Drosophila (Böhni, 1999).

Given the role of the insulin signaling pathway in the control of cellular metabolism in vertebrates and in C. elegans, a test was performed to see whether energy stores are altered in chico mutant flies. The amount of lipid, protein, and glycogen per unit of fresh weight was determined. While there was no significant difference in levels of proteins and glycogen, lipid levels are increased significantly in chico males. In fact, despite their smaller size, chico males have almost twice as much lipids as wild-type males per milligram of fresh weight. The dramatic increase in lipids in chico mutant males is reminiscent of hypertriglyceridemia in IRS1-deficient mice (Abe, 1998) and of fat accumulation observed in C. elegans containing a mutation in the daf-2 gene, which encodes the insulin receptor homolog (Kimura, 1997). Thus, it appears that the INR signaling pathway controls cellular metabolism in vertebrates, nematodes, and insects (Böhni, 1999).

Given the fact that in vertebrates, the insulin receptor pathway plays a critical role in regulating cellular metabolism and growth, it is interesting to speculate that the insulin signaling pathway in flies is part of a nutritional sensing system for each individual cell. Growth is dependent on the availability of nutrients. The reduced body size of chico flies is similar to that of flies reared under poor nutritional conditions. Poorly fed flies also eclose later and possess fewer and smaller cells. In multicellular organisms a system of overall growth coordination is required, since nutrient conditions are unlikely to be the same for all cells in an organism. Under abundant food conditions, an insulin-like peptide and/or other growth factors may be produced and secreted into the open circulatory system to activate the INR pathway in each cell. As a result, cells grow and divide when a critical cell size has been reached. However, when the level of these growth factors drops because of adverse food conditions, the INR pathway activity is reduced. As a consequence, cells slow down cell cycle progression and cell growth, resulting in fewer and smaller cells. The increased accumulation of lipids in chico adults suggests that in addition to its growth-regulating function, Chico may also alter cellular metabolism, resulting in the accumulation of lipids. Interestingly, in homozygous chico mutant larvae no significant difference in lipid levels is observed. This may suggest that during larval stages chico function is required for cell growth and only during pupal development and in the adult Chico controls metabolism. Thus, at the end of development the INR pathway may control the physiological response required to endure periods of low nutrient availability (Böhni, 1999).

Evidence from other studies points to global hormonal regulation of cell growth in Drosophila. Although in Drosophila amino acid withdrawal prevents imaginal disc cells and larval neuroblasts from entering the cell cycle, culturing brains of starved larvae in amino acid-rich medium is not sufficient to induce cell cycle entry of quiescent neuroblasts (Britton and Edgar, 1998). When such brains were cocultured with fat body from fed larvae, however, the neuroblasts started to divide. This suggests that growth control is mediated by growth factors secreted from the larval fat body (Britton, 1998). Recently, Kawamura (1999) characterized imaginal disc growth factors (IDGFs), which are expressed primarily in yolk cells and fat body. Moreover, IDGFs have been shown to cooperate with insulin to stimulate proliferation, and it was speculated that the IDGFs, as chitin-specific lectins, could interact with the INR (Kawamura, 1999). Under abundant food conditions, an insulin-like peptide and/or other growth factors, like IDGFs, may be produced and secreted by the larval fat body into the open circulatory system to activate the INR pathway in each cell. As a result, cells grow and divide when a critical cell size has been reached. However, when the level of these growth factors drops because of adverse food conditions, the INR pathway activity is reduced. As a consequence, cells slow down cell cycle progression and cell growth, resulting in fewer and smaller cells (Böhni, 1999).

The effects on growth and cell size of chico mutants are remarkably similar to the phenotypes of mutations in genes encoding other components of the INR pathway in Drosophila. Although loss-of-function mutations in the Drosophila INR gene are lethal, certain heteroallelic combinations are viable and show delayed development, reduced body size, and decreased cell number (Chen, 1996) and cell size. Expression of dominant-negative or constitutively active variants of p110 PI3K in the developing wing and eye reduces or increases cell number and cell size, respectively (Leevers, 1996). Furthermore, viable mutations in the gene encoding Drosophila protein kinase B (Staveley, 1998), a downstream effector of PI3K, cause a reduction in cell number and cell size. The striking similarities between the phenotypes of chico and mutations in the genes encoding INR and DPKB, as well as the genetic interactions between mutations in Inr, chico, and PI3K, emphasize the specific role of the INR pathway in control of cell growth and cell number as a process independent of pattern formation (Böhni, 1999).

Chico controls growth at three different levels: it regulates the size of individual cells, of organs, and of the entire organism. The similarities observed in the phenotypes of loss-of-function mutations in the INR pathway in Drosophila extend to the phenotypes caused by defects in insulin/IGF signaling pathways in humans and mice. For example, severe insulin resistance in humans causes intrauterine growth retardation and low birth weight. Mice lacking IGF1, IGF1 receptor, IRS1, or IRS2 function are also delayed in development and have a reduced body size. Therefore, it appears that the INR/IGFR signaling pathway is conserved from vertebrates to Drosophila, not only in regard to its structure but also to its function (Böhni, 1999 and references).

GENE STRUCTURE

The putative transcriptional start site of chico lies 221 bp 3' from the end of basket encoding DJNK. ME31B, immediately terminating at the 3' end of chico, encodes a DEAD box RNA helicase (Böhni, 1999).

Exons - 9

PROTEIN STRUCTURE

Amino Acids - 967

Structural Domains

The Chico amino acid sequence exhibits the strongest similarity with members of a family of vertebrate insulin receptor substrate proteins known as IRS1-4. Vertebrate IRS family members are characterized by an N-terminal pleckstrin homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and by a number of phosphotyrosine motifs that can serve as docking sites for SH2-containing proteins (for review, see Yenush, 1997). Sequence similarity between Chico and IRS1, IRS2, IRS3 and IRS4 (collectively called IRS1-4) is confined to the N-terminal region including the PH domain and the PTB domain. The amino acid identity is 41% in the PH domain and 38% in the PTB domain. Although there is no significant overall homology within the C-terminal domain, the Chico protein contains several putative SH2-binding motifs characteristic of IRS family members. Two motifs at positions 411 and 641 fit the consensus binding site (YXXM) for the p85/p60 adaptor subunit of P110 PI(3)K, and one (at position 243) corresponds to the consensus (YXN) for GRB2/DRK binding (Böhni, 1999).

date revised: 3 August 99

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