GTPase-activating protein 1

Protein Interactions

Both full-length Sprouty and a truncated Sprouty containing residues 1-369 (i.e., without the cys-rich domain and C-terminal residues) were assayed for their ability to bind in vitro translated members of the Ras pathway. Strong interactions are detected between Sprouty and Drk (an SH2-SH3 containing adaptor protein homologous to mammalian Grb2), and between Sprouty and Gap1 (a Ras GTPase-activating protein). No interactions were seen between Sprouty and several other proteins involved in the Ras pathway: Sos, Dos, Csw, Ras1, Raf, and Leo (14-3-3). The interactions with Drk and Gap1 do not require the presence of the C-terminal cysteine-rich domain, the region of Sprouty most conserved between flies and humans. Since the well-conserved cysteine-rich domain of Sprouty is not required for binding to Drk or Gap1, it might instead target the protein to the plasma membrane. To test this, two truncated forms of Sprouty were expressed in cultured cells. One form lacks the conserved cysteine-rich domain, whereas a second exclusively comprises the cysteine-rich domain. The form with the cysteine-rich domain is membrane associated and is indistinguishable from the wild-type protein. In sharp contrast, the form lacking the cysteine-rich domain is distributed uniformly throughout the cell, with no specific localization to membranes. Cell fractionation confirms these results. It is concluded that the 147-residue cysteine-rich domain in Sprouty, which corresponds to the most conserved region in the published human ESTs, is responsible for the specific localization of Sprouty to the plasma membrane (Casci, 1999).

DEVELOPMENTAL BIOLOGY

Effects of Mutation or Deletion

A Drosophila gene with similarity to the mammalian Ras GTPase activating protein has been isolated in screens for mutations that affect eye development. Inactivation of the locus, Gap1, mimics constitutive activation of the Sevenless receptor tyrosine kinase and eliminates the need for a functional Sevenless protein in the R7 cell. These results suggest that Gap1 acts as a negative regulator of signaling by Sevenless by down-regulating the activity of the Ras1 protein, which has been shown to be a key element in Sevenless signaling (Gaul, 1992). Germline clonal analysis shows that Gap1 is required in the somatic follicle cells and not the germ line for embryonic dorsoventral polarity determination (Chou, 1993).

The two central photoreceptor neurons of the Drosophila eye, R7 and R8, form a retinotopic map in the optic lobe of the fly brain. A technique has been developed that allows the visualization of the projections of these neurons with high resolution. Using this technique, a new mutant, mip (more inner photoreceptors) has been identified in which this map shows a striking hyperinnervation. The extra terminals in the brain derive from an excessive recruitment of sevenless-independent R7 photoreceptor cells during eye development. The original R7, however, remains Sevenless responsive. The behavior of this gene suggests that recruitment to the R7 pathway, and possibly to multiple programs in ommatidial assembly, is partially regulated by inhibition (Buckles, 1992).

M Loss of one copy of the recently isolated gene sextra (sxt) promotes R7 photoreceptor cell development in a genetically sensitized background, while loss of both copies results in precursors of non-neuronal cone cells transforming into R7 cells. The requirement for sxt function is cell-autonomous. The transformation of cone-cell precursors into R7 cells occurs independent of the Sevenless signal. However, the R7 precursor becomes neuronal in an sxt/sxt mutant only in a wild-type sevenless background. The genetic analysis of sxt suggests that it plays an inhibitory role, preventing cone cells from becoming neuronal. Additionally, sxt functions in R7 precursors, but the Sevenless signal is essential for specification of this fate, since loss of sextra alone is unable to impart a neural fate to this cell (Rogge, 1992).

Bottomley, J. R., et al. (1998). Structural and functional analysis of the putative inositol 1,3,4, 5-tetrakisphosphate receptors GAP1(IP4BP) and GAP1(m). Biochem. Biophys. Res. Commun. 250(1): 143-9.

Buckles, G. R., Smith, Z. D. and Katz, F. N. (1992). mip causes hyperinnervation of a retinotopic map in Drosophila by excessive recruitment of R7 photoreceptor cells. Neuron 8(6): 1015-29.

Casci, T., Vinos, J. and Freeman, M. (1999). Sprouty, an intracellular inhibitor of Ras signaling. Cell 96(5): 655-65.

Chou, T. B., Noll, E. and Perrimon, N. (1993). Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras. Development 119(4): 1359-1369.

Cullen, P. J., et al. (1995). Identification of a specific Ins(1,3,4,5)P4-binding protein as a member of the GAP1 family. Nature 376(6540): 527-30.

Cullen, P.J., et al. (1997). Inositol 1,3,4,5-tetrakisphosphate and Ca 2 + homoeostasis: the role of GAP1IP4BP. Biochem. Soc. Trans. 25, 991-996.

Feldmann P., et al. (1999). Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 ras-GTPase-activating protein. Mol. Cell. Biol. 19(3): 1928-37.

Fukuda, M. and Mikoshiba, K. (1996). Structure-function relationships of the mouse Gap1m. Determination of the inositol 1,3,4,5-tetrakisphosphate-binding domain. J. Biol. Chem. 271(31): 18838-42.

Fukuda, M., Kojima, T., Mikoshiba, K. (1997). Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III. Biochem. J. 323, 421-425.

Gaul, U., Mardon, G. and Rubin, G. M. (1992). A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase. Cell 68(6): 1007-19.

Lai, Z.C. and Rubin, G.M. (1992). Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell 70: 609-620.

Lockyer, P. J., et al. (1999). Tissue-specific expression and endogenous subcellular distribution of the inositol 1,3,4,5-tetrakisphosphate-binding proteins GAP1(IP4BP) and GAP1(m). Biochem. Biophys. Res. Commun. 255(2): 421-6.

Loomis-Husselbee, J. W. et al. (1998), Modulation of Ins(2,4,5)P3-stimulated Ca2+ mobilization by ins(1,3,4, 5)P4: enhancement by activated G-proteins, and evidence for the involvement of a GAP1 protein, a putative Ins(1,3,4,5)P4 receptor. Biochem J. 331 ( Pt 3): 947-52.

Maekawa, M., et al. (1994). A novel mammalian Ras GTPase-activating protein which has phospholipid-binding and Btk homology regions. Mol. Cell. Biol. 14(10): 6879-85.

Powe, A. C., et al. (1999). In vivo functional analysis of Drosophila Gap1: involvement of Ca2+ and IP4 regulation. Mech. Dev. 81(1-2): 89-101.

Rogge, R., et al. (1992). Neuronal development in the Drosophila retina: the sextra gene defines an inhibitory component in the developmental pathway of R7 photoreceptor cells. Proc. Natl. Acad. Sci. 89(12): 5271-5.

R¯rth, P. (1996). A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc. Natl. Acad. Sci. 93: 12418-12422.

Rubio, I. and Wetzker, R. (2000). A permissive function of phosphoinositide 3-kinase in Ras activation mediated by inhibition of GTPase-activating proteins Curr. Biol. 10: 1225-1228. 11050394

Thackeray, J. R., et al. (1998). small wing encodes a phospholipase C-gamma that acts as a negative regulator of R7 development in Drosophila. Development 125: 5033-5042.

The, I., et al. (1997). Rescue of a Drosophila NF1 mutant phenotype by protein kinase A. Science 276(5313): 791-4.

date revised: .

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