G protein-coupled receptor kinase 2
A developmental analysis of expression has shown that the 4.0 and 5.0 kb transcripts are abundantly expressed in 0-3 hour embryos, as a result of maternal expression. The 5.0 kb transcript is no longer detectable after 0-3 hours while the 4.0 kb transcript continues to be expressed at a low level. The 5.5 kb transcript is expressed at a low level in early embryogenesis, with an increase in expression from 12 to 24 hours. Thus the three transcripts are differentially expressed during development (Schneider, 1997).
The expression of Gprk2 protein was analyzed using a polyclonal antibody. This antibody was generated against a bacterially expressed protein that contained the final 145 amino acids of the kinase domain and the entire 139 amino acid carboxyl region (Cassill, 1991). DNA probes from the corresponding genomic region hybridize to all three transcripts. In immunoblot analysis of adult tissues, a single band of 80 kDa was detected in ovaries and whole females, in close agreement to the 80.7 kDa protein predicted by the B6936 cDNA. This band is absent in ovaries from gprk26936 homozygotes whereas expression in whole females remains. Thus, as expected, Gprk2 is expressed in the ovary and its expression is disrupted in the gprk26936 mutant. In larval tissues, an 80 kDa band was observed in both the central nervous system (CNS) and in carcasses (the entire animal minus the CNS). In homozygous gprk26936 larvae, expression is no longer detectable in CNS tissue but appears to be unaffected in carcasses. These results demonstrate that gprk26936 selectively disrupts Gprk2 expression in some but not all tissues (Schneider, 1997).
The tissue distribution of the protein was studied by staining whole-mount tissues with the same antibody. The anti-Gprk2 antibody labels developing wild-type egg chambers, and most of the staining is eliminated in gprk26936 mutant ovaries. Expression is first seen in region 2B of the germarium, the stage where germline cysts are being enveloped by follicle cells. In early egg chambers, membranes between adjacent nurse cells and between nurse cells and the oocyte are labeled. In addition, staining around the germinal vesicle is sometimes observed. Membrane-associated staining persists through stage 6 but decreases in intensity at stage 7, except between the nurse cells and the oocyte. In these early stages no staining is observed in follicle cells or in the region of the nurse cells that lies adjacent to the follicle cells. During stages 8-11 membrane-associated staining decreases in the nurse cells and appears around the entire circumference of the oocyte. Weak cytoplasmic staining is observed in nurse cells and follicle cells of these stages; however, the cytoplasmic (and germinal vesicle) staining sometimes persists in gprk26936 ovaries. Thus Gprk2 protein appears to be preferentially associated with nurse cell and oocyte plasma membranes during much of oogenesis. Although in situ hybridization studies also detected GPK2 RNA only in the nurse cells, a lower level of expression in somatic cells can not be ruled out by these experiments (Schneider, 1997).
Specific staining using anti-Gprk2 is also detected in non-ovarian tissues, consistent with a role for this gene outside of the ovaries. The central nervous system (CNS) of both larvae and adults stains intensely. In larvae, staining is present in axon fascicles, especially large ones, including nerves projecting to the optic lobes, the longitudinal connectives, and portions of the mushroom bodies. Little staining is detected in cell bodies within the CNS. However, strong staining is consistently observed in the cell bodies and nerves of the corpus allatum of the ring gland. In the adult CNS, staining is restricted to two major structures within the brain. (1) The nerves within and projecting to the ellipsoid body of the central complex are consistently stained. The ellipsoid body contributes to higher brain function in flies, such as locomotion. (2) Strong staining is also observed in the mushroom bodies that included the Kenyon cells and nerve processes within the peduncles and the alpha lobes. The mushroom bodies have been implicated in memory and learning. Interestingly, the learning mutants dunce, rutabaga, and DCO, which all disrupt genes involved in G protein signaling, are predominately expressed in the mushroom bodies. In the mutant, Gprk2 staining is abolished from both the larval and adult CNS, and from the larval ring gland (Schneider, 1997).
Most of the other tissues in the larva and adult are only stained weakly and it is not possible to determine if staining is affected in the mutant. One exception to this is in the wing imaginal disc where staining is observed in the notum and in a stripe that parallels the anterior/posterior boundary of the wing blade. Within this stripe, staining is always weakest in the region corresponding to the tip of the wing. This staining is eliminated in Gprk26936 wing discs (Schneider, 1997).
The disc staining was particularly interesting because dpp is also expressed along the anterior/posterior boundary of the wing disc. To determine how Gprk2 staining corresponds to the anterior/posterior boundary, expression of Gprk2 was compared with Engrailed (En) and dpp. En protein, which is expressed in the posterior compartment of the wing disc, was visualized with a monoclonal antibody. dpp expression was visualized by ß-gal immunoreactivity in flies bearing a dpp-lacZ fusion gene. Gprk2 and dpp are co-expressed along the anterior/posterior boundary with two exceptions. (1) The stripe of Gprk2 staining is thinner, about one cell in thickness as opposed to a 2-3 cell thickness of dpp expression. The stripe of Gprk2 expression coincides with the dpp-expressing cells closest to the boundary. (2) Near the wing tip, dpp is not expressed in the cells immediately adjacent to the boundary. In this region Gprk2 staining is present in cells that do not express dpp. At the tip of the wing En staining crosses the anterior/posterior boundary. Therefore, in this region Gprk2 and En are co-expressed whereas outside of this region the patterns of the two proteins abut one another. These results confirm that Gprk2 is expressed along the anterior side of the anterior/posterior border in a pattern that overlaps dpp (Schneider, 1997).
Adult viable mutations of decapentaplegic, or its putative receptor saxophone, cause homozygous females to produce short rounded eggs with abnormal anterior structures. A new female sterile mutation that effects G protein-coupled receptor kinase 2 function, fs(3)06936, has been isolated in a P element mutagenesis screen that causes similar effects on egg shape. Mature oocytes produced by homozygous fs(3)06936 females are slightly shorter and more rounded than wild type. Although positioned normally, dorsal appendages in fs(3)06936 eggs are generally shorter and broader than wild type, and the two appendages on a single egg chamber frequently differ in length. The operculum is oriented more vertically than in wild type, giving the eggs a 'square-ended' appearance, but the chorion within the operculum retains its distinctive appearance and the micropyle forms. Nurse cells often fail to completely transfer their contents into the oocyte, leaving residual material that may interfere with anterior end formation. Thus fs(3)06936 appears to affect specific aspects of egg formation without grossly altering the major pattern axes of the egg (Schneider, 1997).
Two additional ovarian defects have suggested that fs(3)06936 also functions at earlier stages of oogenesis. (1) Homozygous fs(3)06936 egg chambers degenerate during vitellogenic stages (stages 8-10A) much more frequently than expected. 26.8% of ovarioles from 4-day-old fs(3)06936 females contained a degenerating vitellogenic chamber compared to only 0.7% of wild-type ovarioles. (2) Egg chamber formation slows or ceases entirely within a significant number of fs(3)06936 ovarioles. 5.2% of the mutant ovarioles contained only 0-2 egg chambers instead of the 6-7 that are present in wild type. Germaria in such ovarioles are often smaller and thinner than in wild type, like the germaria of agametic ovarioles. Although cyst production normally declines in old females, 4-day-old wild-type females contained no similar ovarioles (Schneider, 1997).
The fs(3)06936 mutant exhibits additional defects that indicate roles for this gene outside of the ovaries. Homozygous fs(3)06936 females lay a small number of eggs, but those that are laid display a maternal effect that is partially rescued by zygotic fs(3)06936+ expression. 23.7% of embryos produced by homozygous females hatch when crossed to wild-type males, compared to only 10.3% following crosses to homozygous males. The unhatched eggs displayed a wide variety of defects including twisted gastrulation, fused adjacent segments, and perforated dorsal and ventral cuticle. These defects are more severe when the embryos lack both maternal and zygotic fs(3)06936+ function (Schneider, 1997).
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date revised: 15 May 2001
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