BIOLOGICAL OVERVIEW

Star is a member of the spitz group also known as the ventral lateral group, defined on the basis of phenotypic similarities among mutants and genetic interactions. There are defects in the cuticular structures derived from the ventro-lateral blastoderm, and defects in the ventral cord known as the CNS. Four of the six genes in the spitz group are pointed, rhomboid, Star and spitz itself (Mayer, 1988).

Star is a component in the Torpedo/EGF-R (Epidermal growth factor receptor) pathway, and interacts genetically with this Drosophila homolog of the mammalian epidermal growth factor receptor, however its exact role is currently unknown. As with Rhomboid, another membrane protein involved in the same pathway, Star may act to either amplify or restrict EGF-receptor signaling, by modulating the signaling cascade or regulating adhesive function between cells (Heberlein, 1993).

Cleavage of the ubiquitously expressed transmembrane form of Spi (mSpi) precedes EGF receptor activation. The Star and Rhomboid (Rho) proteins are necessary for Spi cleavage in Drosophila cells. Complexes between the Spi and Star proteins, as well as between the Star and Rho proteins were identified, but no Spi-Star-Rho triple complex was detected. This observation suggests a sequential activity of Star and Rho in mSpi processing. The interactions between Spi and Star regulate the intracellular trafficking of Spi. The Spi precursor is retained in the periphery of the nucleus. Coexpression of Star promotes translocation of Spi to a compartment where Rho is present both in cells and in embryos. A Star deletion construct that maintains binding to Spi and Rho, but is unable to facilitate Spi translocation, has lost biological activity. These results underscore the importance of regulated intracellular trafficking in processing of a TGFalpha family ligand (Tsruya, 2002).

To identify the domain(s) of Star required for its biological activity, deletion constructs were generated. Because Star is a novel protein with no defined domains (except for the transmembrane domain), no clues were available for the generation of such constructs. Searching the database, a Bombyx mori cDNA sharing homology with Star was identified. Complete sequence of this cDNA identified a type II transmembrane protein of 315 amino acids, which was termed BmS. Notably, the amino-terminal cytoplasmic domain of this protein is only 74 residues long, and the major boxes of homology are located immediately carboxy-terminal to the transmembrane domain. This pattern of homology raised the possibility that most of the amino-terminal domain of Star may be dispensable for its function, and prompted an examination of constructs of Star missing most of the amino- or carboxy-terminal domains. Two truncated Star constructs were generated. NTM contains the entire amino-terminal and transmembrane domains, and is truncated 16 residues after the transmembrane domain. Conversely, in TMC, the amino-terminal 259 residues were deleted, and an initiator methionine was added (Tsruya, 2002).

The biological activity of the constructs was tested in S2 cells. Only the TMC construct was capable of promoting mSpi cleavage, but at a lower efficiency than full-length Star. sSpi could be detected in the medium of cells coexpressing mSpi, TMC and Rho, but not in the medium of cells expressing only mSpi and TMC. Stable transfected lines expressing mSpi and TMC provided a more sensitive assay and produced low levels of sSpi. The lower levels of sSpi, however, indicate that this construct is less potent than the full-length Star protein. The NTM and BmS constructs are not active alone or even in the presence of Rho. In addition, after sequencing the Star⁵⁴ null allele, a termination codon was identified at position Q387 in the extracellular domain. This protein is 67 residues longer than NTM and yet is inactive, indicating that the carboxy-terminal region of Star is essential for its biological function (Tsruya, 2002).

The TMC, NTM, and BmS constructs were also tested in flies heterozygous for the Star mutation: such mutants display rough eyes due to haplo-insufficiency. Again, only ectopic expression of the TMC construct in eye discs (by GMR-Gal4) is capable of rescuing the phenotype, giving rise to wild-type eyes. High levels of expression of full-length Star (in Star heterozygous flies, as well as in wild-type flies) leads to the appearance of rough eyes and to the formation of extra wing veins, consistent with Egfr hyperactivation. The reduced biological activity of TMC is evident, since it is not capable of eliciting these hyperactivation phenotypes. It is concluded that, in cells and in flies, only the Star construct containing the carboxy-terminal domain retains the essential activities of Star, but is less potent than full-length Star (Tsruya, 2002).

Having shown that Star is essential for mSpi cleavage, it was asked whether Star interacts directly with the mSpi protein. The capacity of Star to form protein complexes with mSpi was examined. Cells were cotransfected with constructs expressing mSpi-FLAG, Star-HA, and, as a negative control, CD8-GFP. mSpi was immunoprecipitated by anti-FLAG, and coprecipitation was followed by anti-HA and anti-GFP immunoblotting. Indeed, immunoprecipitation of mSpi gives rise to significant coprecipitation of Star. The robust interaction between mSpi and Star was also demonstrated in the reciprocal experiment, where immunoprecipitation of Star-TAP results in coprecipitation of mSpi-GFP (Tsruya, 2002).

In view of the association between Spi and Star, whether the biological activity of the Star deletion constructs correlates with the capacity to bind Spi was examined. The NTM, TMC, or BmS constructs were coexpressed with mSpi in S2 cells. Although only the TMC construct, containing an intact carboxy-terminal domain, is active in cells and flies, all three proteins coprecipitate mSpi with similar efficiencies. The basis for loss of Star biological activity, in spite of the ability to bind Spi, is described below (Tsruya, 2002).

To identify the domain(s) of mSpi mediating the interaction with Star, mSpi-GFP was constructed. GFP was inserted amino-terminal to the EGF domain. This construct retains the capacity to undergo Star- and Rho-dependent cleavage in S2 cells. It is also biologically active in flies, since coexpressed mSpi-GFP and Star in wing discs give rise to abnormal, wrinkled wings. mSpi-GFP and several deletion constructs were tested for their ability to interact with Star. A secreted Spi protein lacking the cytoplasmic and transmembrane domains retains the capacity to bind Star, pointing to the extracellular domain of Spi as the mediator of binding. In accordance with this conclusion, an mSpi protein lacking the EGF domain shows only residual levels of binding to Star. Further deletions show that the EGF domain of Spi is necessary for the interaction, since secreted constructs lacking this domain show only background association with Star. Thus, the EGF domain of Spi mediates the interaction with Star (Tsruya, 2002).

The interaction between Star and mSpi prompted an examination of whether Star also associates with Rho. Star-HA was coexpressed with Rho-TAP in S2 cells. Immunoprecipitation of Rho-TAP showed coprecipitation of Star. In the reciprocal experiment, immunoprecipitation of Star-HA or Star-TAP coprecipitates Rho. To identify the regions of Star necessary for this interaction, the experiment was repeated with the three Star constructs. The NTM and BmS constructs retain the interaction with Rho, whereas the TMC construct shows no detectable interaction or only weak interaction. It appears that sequences within the amino-terminal cytoplasmic domain of Star that are not present in TMC mediate the binding to Rho (Tsruya, 2002).

The capacity of Star to bind both Spi and Rho, raises the possibility that Star may function as a scaffold protein, to form a trimeric protein complex. Examination of possible direct interactions between mSpi and Rho failed to detect any significant coprecipitation. If a trimeric complex is formed, it would be expected that Star would promote coprecipitation of mSpi by Rho. However, no elevation in mSpi coprecipitation with Rho was detected when Star was also expressed. Under these conditions, Star retains the capacity to bind mSpi, regardless of the expression of Rho. Therefore, it appears that whereas mSpi-Star or Star-Rho complexes are formed in the cells, no trimeric complex is present (Tsruya, 2002).

Because coexpression of Rho and Star significantly promotes processing of mSpi, it is possible that short-lived trimeric complexes are formed as a result of cleavage of mSpi. To circumvent this problem, the formation of complexes with a noncleavable mSpi precursor was examined. An mSpi protein in which 16 amino acids between the EGF and transmembrane domains were deleted, does not undergo cleavage in S2 cells. A similar construct fails to be cleaved in the Xenopus assays. Like mSpi, the deleted mSpi protein readily forms complexes with Star, but not with Rho. Again, even in the presence of Star, Rho is not capable of coprecipitating this uncleavable Spi construct, arguing against the presence of even a transient triple complex. This experiment suggests that Star and Rho function sequentially in mSpi processing (Tsruya, 2002).

In view of the apparent sequential roles of Star and Rho, attempts were made to identify the cellular compartments in which they are active. The subcellular localization of Star and Rho may provide a clue as to their order of activity. Star protein has been reported to be in the periphery of the nucleus, consistent with an ER localization. Immunostaining of S2 cells to follow Star overexpression confirms this localization pattern. Immunostaining of Rho in S2 cells overexpressing Rho shows a punctate distribution that does not colocalize with a 120-kD integral Golgi membrane protein, as well as plasma membrane staining. This pattern is in accordance with previous observations in embryos showing punctate endogenous Rho distribution. Following overexpression, plasma membrane staining is also detected. Expression of Star and Rho shows only a restricted overlap. The distinct cellular distribution of these proteins suggests that a key feature in the regulation of mSpi processing may be its cellular trafficking. This possibility is in accord with the sequential activities of Star and Rho implied by the coprecipitation results (Tsruya, 2002).

The distribution of mSpi-GFP was followed in cells. mSpi-GFP shows a peripheral nuclear localization in S2 cells, as well as diffuse cytosolic staining, but it is not detected in the Golgi or plasma membrane. Therefore, it appears that the mSpi protein is retained in the ER, a compartment in which it cannot undergo cleavage (Tsruya, 2002).

To identify the domain(s) of mSpi required for its ER retention, a construct containing intact extracellular and transmembrane domains, but lacking the intracellular domain was examined. This protein shows only residual levels of peripheral nuclear localization and is detected primarily in a punctate pattern, consistent with exit from the ER. Therefore, the cytoplasmic domain of mSpi is necessary for its ER retention (Tsruya, 2002).

The peripheral nuclear distribution of Star and mSpi appears similar, whereas the punctate localization of Rho suggests that it is present in a more advanced compartment along the secretory network. Because Rho is required for efficient mSpi cleavage, how does mSpi reach the Rho compartment? One option is that Star forms a complex with mSpi and alters its intracellular localization. The distribution of mSpi-GFP was examined following coexpression with Star. Indeed, the original peripheral nuclear distribution of mSpi-GFP disappears, and instead it is found in a prominent punctate pattern, which does not correspond to the Golgi and only partly overlaps with the lysosomes and endosomes. The alteration in mSpi distribution in the presence of Star was observed in all cells (Tsruya, 2002).

In contrast, expression of Rho with mSpi-GFP does not alter its distribution, in agreement with a lack of coprecipitation. However, coexpression of Rho with mSpi-GFP and Star reduces dramatically the levels of mSpi-GFP observed in the cells, in accordance with the results obtained in anti-Spi blots. The residual mSpi-GFP is found in a punctate staining colocalizing with Rho. These results show that Rho can alter the levels of mSpi in the cells, but only in the presence of Star. They are consistent with sequential activity of Star and Rho, where Star is required first to transport mSpi from the ER to the compartment containing Rho, and Rho subsequently facilitates the cleavage and secretion (Tsruya, 2002).

To test the distribution of mSpi and the effect of Star in embryos, the mSpi-GFP construct was expressed in embryos. After crossing several independent transgenes of the UAS-mSpi-GFP construct to embryonic Gal4 drivers, only very weak fluorescence was seen, in contrast with other GFP constructs such as UAS-CD8-GFP. This result may indicate a high turnover and low steady-state levels of the noncleaved, mSpi precursor. Only in a small percentage of embryos could the mSpi-GFP be visualized. Embryos expressing mSpi-GFP show a peripheral nuclear distribution colocalizing with the nuclear membrane protein Lamin. In contrast, embryos expressing mSpi-GFP and Star show a punctate distribution. Although Star is normally expressed in all embryonic cells, it is assumed that the overexpression of mSpi-GFP alone generates an excess of this protein and thus essentially represents the mSpi distribution in the absence of Star (Tsruya, 2002).

In an alternative way to circumvent the high mSpi turnover, the distribution of mSpi-GFP was followed after transient injection of the UAS-mSpi-GFP construct to embryos carrying prd-Gal4 or actin-Gal4. Monitoring fluorescence several hours after injection, a bright peripheral nuclear distribution of mSpi-GFP was seen. Coinjection of mSpi-GFP and Star constructs eliminates this distribution and leads to the accumulation of mSpi-GFP in a punctate pattern, similar to the distribution in S2 cells. Conversely, coinjection of mSpi-GFP and Rho does not alter the mSpi peripheral nuclear distribution. Finally, coinjection of mSpi-GFP, Star, and Rho also gives rise to the mSpi-GFP plaques, but their intensity is decreased. It is concluded that the effect of Star on mSpi localization observed in S2 cells also occurs in embryos and is likely to reflect the essential role of Star in mSpi intracellular trafficking (Tsruya, 2002).

It was of interest to test whether the biological activity of Star indeed corresponds to the capacity to promote mSpi trafficking. Although the two Star deletion constructs retain the ability to bind mSpi, only the TMC construct containing an intact carboxy-terminal domain is biologically active in cells and flies. Each of the two Star constructs was coexpressed with the mSpi-GFP construct. In the TMC expressing cells, mSpi-GFP displays the same punctate distribution observed when coexpressed with full-length Star. In contrast, mSpi-GFP remains in the peripheral nuclear pattern in the presence of NTM and BmS. These results imply that the carboxy-terminal domain is required for the ability of Star to target mSpi to the proper compartment. Without this property, Star is not biologically active (Tsruya, 2002).

How is the cellular distribution of Star itself regulated? In cells, Star is found in a peripheral nuclear distribution. Examination of the distribution of the Star deletion constructs shows that TMC retains the same distribution as full-length Star. In contrast, NTM is not seen in the ER and is detected instead in a weak punctate and prominent membrane staining. Thus, the cellular distribution of Star itself is regulated and residues in the carboxy-terminal domain may promote interactions with other proteins that regulate the peripheral nuclear localization of Star (Tsruya, 2002).

In conclusion, this work shows that the intracellular localization and trafficking of mSpi is crucial for its regulated cleavage. mSpi is normally retained in a peripheral nuclear compartment where it does not undergo cleavage and may be rapidly degraded. Star, which is also enriched in this compartment, associates with mSpi and translocates it to a compartment in which Rho is enriched, to allow cleavage. The association between Star and Rho may allow Star to efficiently deliver mSpi. The cleavage process thus entails orchestration of trafficking and protein-protein associations to ensure tight regulation of mSpi processing and, hence, Egfr activation (Tsruya, 2002).

GENE STRUCTURE

Exons - three

Bases in 3' UTR - approximately 1 kb

PROTEIN STRUCTURE

Amino Acids - 598

Structural Domains

The Star gene encodes a novel protein with a centrally located putative transmembrane domain. There are histidine rich and glycine rich regions. Sequence characteristics suggest a type II membrane protein in which the amino terminal region is cytoplasmic. In the amino terminal region there are two PEST sequences present in proteins with short half lives (Kolodkin, 1994).

date revised: 25 August 98

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