BIOLOGICAL OVERVIEW

Heterotrimeric G protein-coupled signaling is a form of intracellular communication that is mediated by the family of G protein-coupled receptors (also known as heptahelical/7TM receptors). In the continued presence of ligand, G protein-coupled receptors become less efficient in transducing signals, a process called desensitization. Desensitization of G protein-coupled receptors is thought to occur in several steps: binding of G protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of beta-arrestins to phosphorylated receptors.

The Drosophila G protein receptor-coupled kinase 2 (Gprk2) gene encodes a member of the G protein-coupled receptor kinase family (Cassill, 1991; Schneider, 1997). The Gprk2 protein has a high level of sequence identity to members of the mammalian GRK4 subfamily (GRK4, GRK5, and GRK6). Expression of the Gprk2 protein in the mushroom bodies of the brain is similar to the expression of genes involved in cAMP-mediated signaling pathways, such as dunce (dnc) and rutabaga (rut). The rutabaga locus encodes an adenylate cyclase and mutation of this gene results in a decrease in cAMP synthesis, whereas mutation in dnc, coding for cyclic AMP phosphodiesterase (an enzyme that degrades cAMP), causes an increase in cAMP synthesis.

Because of the potential role of the Gprk2 gene in G protein signaling it was hypothesized that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger. To examine this hypothesis, tests were performed for genetic and biochemical interactions between dunce and Gprk2 mutants; dunce is able to suppress the sterility defect of a gprk2 mutant. Similarly, gprk2 mutation is able to rescue the viability and sterility defects of dunce mutants. Results of the biochemical analysis of cAMP levels in the dunce;gprk2 mutant combinations further strengthen the genetic findings. These results suggest that Gprk2 is involved in a receptor-mediated signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

The genetic interaction between dunce and Gprk2 was examined through the effects of these two genes on oogenesis. A mutant allele of Gprk2, called gprk2⁶⁹³⁶, has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2⁶⁹³⁶. Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2⁶⁹³⁶ background suppresses all of these defects to a significant degree. Suppression is also observed when a single copy of gprk2⁶⁹³⁶ is introduced into a dunce background. Like rutabaga mutants (rutabaga encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2⁶⁹³⁶ has reduced levels of cAMP. Ovaries from gprk2⁶⁹³⁶ females contain about one third the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels (Lannutti, 2001).

Homozygous gprk2⁶⁹³⁶ females produce eggs with dorsal appendages that are malformed or truncated, and nurse dumping is often incomplete. The most severely affected egg chambers are not laid. Of the eggs that are laid, many have a flaccid appearance and there is a significant reduction in the rate of hatching. The embryos that fail to hatch show a range of defects including twisted gastrulation, fusion of adjacent segments, and a perforated cuticle (Schneider, 1997). Although some embryos do hatch, the number of animals that survive to adulthood is very low. The defects seen in gprk2⁶⁹³⁶ egg chambers are primarily the result of a lack of expression in the germline. Egg chambers from gprk2⁶⁹³⁶ germline clones display all of the defects seen in gprk2⁶⁹³⁶ homozygotes and they hatch at a similarly reduced rate. The gprk2⁶⁹³⁶ mutant is the only allele of Gprk2 that has been identified and there are no deficiencies that uncover this locus. However, expression of wild type Gprk2 under the control of either a heat shock or germline-specific promoter is sufficient to rescue the sterility defect of gprk2⁶⁹³⁶ (Lannutti, 2001).

Because of the possibility that dunce may be acting in a common pathway with Gprk2, the dunce ovarian phenotype was reexamined. Females homozygous for hypomorphic (dnc²) and null (dnc^M14 ) alleles have been shown to lay few or no eggs. This has been attributed to defects in the chorion and vitelline membrane. In addition to these defects, nurse cell dumping is incomplete in egg chambers from dnc²homozygous females. This incomplete cytoplasmic transfer may cause or contribute to the formation of misshapen dorsal appendages. Incomplete cytoplasmic dumping is also apparent in dnc^M14 homozygotes and dnc²/dnc^M14 transheterozygotes. In both of these genotypes, egg chambers have severely truncated dorsal appendages or the dorsal appendages fail to form altogether. The percentage of egg chambers that fail to complete cytoplasmic dumping does not increase significantly in the stronger allelic combinations, probably because many egg chambers fail to develop to that stage (Lannutti, 2001).

In addition to the dumping and dorsal appendage defects, ovaries from dunce mutant mothers contain degenerating egg chambers. In dnc² homozygotes, 70% of stages 10B and 11 egg chambers are degenerating, as determined by the presence of condensed, DAPI-bright nuclei. In dnc²/dnc^M14 and dnc^M14 homozygotes, egg chamber degeneration is detected in earlier stages (beginning at stage 6) and occurs more frequently. All three aspects of the dunce ovary phenotype, the incomplete cytoplasmic dumping, the malformed dorsal appendages, and the degeneration of egg chambers, resemble those of the gprk2⁶⁹³⁶ mutant (Lannutti, 2001).

Mutations in three genes, chickadee, quail, and singed, cause a block in nurse cell dumping and disrupt dorsal appendage formation. These three genes encode proteins whose mammalian homologs (profilin, villin, and fascin, respectively) are involved in actin bundling. In all three mutants, there is a failure in the formation of the cytoplasmic fibers that tether the nurse cell nuclei during cytoplasmic dumping. Oocytes from homozygous gprk2⁶⁹³⁶ mothers have dorsal appendages that are malformed, and nurse cell dumping is often incomplete. Immunofluorescence analysis of gprk2⁶⁹³⁶ egg chambers that are stained with DAPI reveal that some nuclei of nurse cells have a defect in tethering during cytoplasmic dumping. In stage 10B egg chambers from wild type mothers, cytoplasmic actin filaments extend from the nucleus to the plasma membrane. These filaments anchor the nuclei during dumping, thus preventing their movement into the ring canals. In contrast, in 31% of stage 10B egg chambers from gprk2⁶⁹³⁶ nuclei, stretching has been observed in the direction of cytoplasmic dumping and extending through the ring canals. More than two nuclei affected in a single egg chamber are never observed, and the nurse cells that dump directly into the oocyte are usually not affected. Phalloidin staining in gprk2⁶⁹³⁶ egg chambers demonstrates that cytoplasmic actin fibers do form in the mutant. However, these fibers often appear more clumped and irregular than those in wild type egg chambers. A tethering defect is never observed in the dnc² and dnc^M14 alleles (Lannutti, 2001).

There is a marked increase in fertility in allelic combinations of dunce and gprk2. This increase suggests that there should be a corresponding improvement in the ovary morphology of combinations of both mutants. To test this, a quantitative analysis of the ovaries from the different mutant combinations was performed. The feature of the dunce phenotype that is most readily quantified is egg chamber degeneration; this defect can be assayed by the presence of DAPI-bright nuclei. In dnc² homozygotes, DAPI-bright, nurse cell nuclei were observed in 39% of stages 9 and 10 egg chambers. This defect is more severe in dnc²/dnc^M14 transheterozygotes and dnc^M14/dnc^M14 homozygotes. In these genotypes 52% (dnc²/dnc^M14) and 58% (dnc^M14/dnc^M14) of stages 9 and 10 egg chambers display a degenerating phenotype. These numbers are an underestimate of the level of degeneration in dnc²/dnc^M14 and dnc^M14/dnc^M14 allelic combinations because egg chambers often degenerate earlier in oogenesis. As a result, ovaries from dnc²/dnc^M14 and dnc^M14/dnc^M14 females produce few egg chambers past stage 11. One copy of gprk2⁶⁹³⁶, which suppresses the sterility of the two dunce alleles, also suppresses degeneration in the dnc²/dnc², dnc²/dnc^M14, and dnc^M14/dnc^M14 mutants. Although degeneration is dramatically reduced in all three genotypes, rescue is not complete. These results support the suppression of sterility that is observed in dunce;gprk2⁶⁹³⁶ allelic combinations (Lannutti, 2001).

Mutations in the dunce gene cause an increase in embryonic and adult cAMP levels (two- to fivefold, depending on the allele) and most dunce alleles are sterile. In contrast, mutations in rutabaga cause a slight decrease in cAMP levels but are completely fertile. Double mutants of rutabaga and dunce have intermediate levels of cAMP and fertility is partially restored. Similarly, one copy of gprk2⁶⁹³⁶ partially restores fertility in dunce mutants. By analogy to the dunce-rutabaga interaction, the simplest explanation for the mutual suppression of gprk2⁶⁹³⁶ and dunce is that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are lower than those in wild type flies. In agreement with this suggestion, it was found that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are almost threefold lower than those in wild type females. Furthermore, the suppression of the gprk2⁶⁹³⁶ phenotype by dunce (and vice versa) is reflected in the cAMP levels of the dunce, gprk2⁶⁹³⁶ allelic combinations. Introducing a single copy of the dnc² or dnc^M14 mutant into gprk2⁶⁹³⁶ homozygotes resulted in a wild-type cAMP content, threefold higher than that in gprk2⁶⁹³⁶ homozygotes. The gprk2⁶⁹³⁶ mutant also suppresses the elevated levels of cAMP seen in mutant dunce alleles. In short, in every mutant combination in which an increase in fertility is observed, the cAMP content in the ovaries changes in the expected direction. Taken together, these results strongly support the hypothesis that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

Mutations in rutabaga result in a decrease in cAMP synthesis. By analogy, it is possible that there is a decrease in the synthesis of cAMP in the gprk2⁶⁹³⁶ mutant. This could be explained if the Gprk2 protein regulates receptors that inhibit adenylate cyclase through a Galphai protein. When the receptor becomes activated it would first decrease cAMP levels by inhibiting adenylate cyclase. Then, when the receptor becomes desensitized through the action of Gprk2, the inhibition of adenylate cyclase would be relieved and cAMP levels would increase. This model would explain the mutual suppression between gprk2⁶⁹³⁶ and the dunce alleles. In the gprk2⁶⁹³⁶ mutant, receptor activity (and, consequently, Galphai activity) would be prolonged and adenylate cyclase activity would be inhibited to a greater extent, thus leading to reduced levels of cAMP. Decreasing the dose of dunce would decrease the amount of phosphodiesterase activity. Thus the cAMP that is produced would be degraded more slowly, resulting in a greater accumulation than that in gprk2⁶⁹³⁶ homozygotes (Lannutti, 2001).

Similarly, in dunce mutants, cAMP levels are elevated as the result of a lack of cAMP metabolism. Decreasing the dose of Gprk2 would alleviate this problem, to some degree, by causing a reduction in the amount of cAMP that is synthesized. One Galphai (G-oalpha 65A) gene has been characterized in Drosophila and analysis of the Drosophila genome sequence suggests that there are no others. The Galphai protein is expressed in the follicle cells and is present in granules in the oocyte. There are no reported loss-of-function mutants of this gene. However, a dominant gain-of-function line has been generated using the UAS/GAL4 expression system. This mutant has altered signaling in the nervous system, as shown by its increase in sensitivity to cocaine (Li, 2000). If this simple model of Gprk2 function is correct, it would suggest that overexpression of activated Galphai would decrease fertility. An alternative mechanism is that the Gprk2 protein desensitizes receptors that couple to other G protein subunits that inhibit adenylate cyclases. Mammalian Galphao and certain combinations of betagamma have been shown to inhibit type I adenylate cyclases (rutabaga is a type I cyclase). Galphao, beta, and gamma subunits have all been identified in Drosophila and searches of the Drosophila genome suggest that there are six Galpha genes (including Galphas, Galphai, Galphao, Galphaq, and Galpha12/13), two beta-subunit genes, and two gamma-subunit genes). The expression of most of the alpha subunits in the ovaries has not been described. The exceptions are Galphai and Galphao, whose mRNA is expressed at high levels in the nurse cells, although there is no detectable protein expression. The expression of beta and gamma genes in the ovaries has not been reported (Lannutti, 2001).

It is interesting that gprk2⁶⁹³⁶ and dunce mutants have similar ovary phenotypes, even though they have opposite effects on cAMP levels in the ovaries. It appears that cAMP levels must be maintained at an optimum level; both an increase and a decrease cause defects in dorsal appendage formation, cytoplasmic dumping, and probably other maternal and zygotic functions. This effect has also been documented in assays of learning in the fly. Both rutabaga and dunce mutants cause defects in learning and memory, although they cause opposite changes in cAMP levels. Apparently, the level of cAMP must be tightly regulated for proper functioning of cAMP-dependent pathways (Lannutti, 2001 and references therein).

Another parallel with the dunce, rutabaga studies is the observation that the degree of suppression does not always correlate with the level of cAMP in the ovaries. In earlier studies it has been shown that a dunce, rutabaga double mutant (homozygous for both alleles) is still defective in learning, although cAMP levels are rescued. Similarly, rescue of fertility is not complete, even in combinations that produced cAMP levels very close to those of wild type. For example, the ovaries from dnc²/+;gprk2⁶⁹³⁶ /gprk2⁶⁹³⁶ females have cAMP levels that are not statistically different from wild type levels. However, the fertility of these females is still statistically less than that in wild type. There are several possible explanations for this effect. (1) It has been suggested that the kinetics of cAMP metabolism are as important as the level of cAMP per se. Similarly, the subcellular distribution of cAMP may play an equally important role in fertility. (2) The Gprk2 and dunce genes may have functions that are not dependent on one another. For example, because there are many heptahelical receptors in Drosophila and only two known GRKs, it is likely that Gprk2 protein phosphorylates many different receptor types. If some of these receptors act through second messengers other than cAMP, then they would not be rescued by a decrease in the dosage of dunce. Similarly, Dunce is the major source of phosphodiesterase activity in Drosophila. Therefore, a decrease in phosphodiesterase activity could disrupt signaling from Gprk2-independent receptors. (3) Gprk2 and Dunce could carry out functions that are not directly related to production or metabolism of second messengers. For example, mammalian GRKs have been shown to interact with cytoskeletal proteins, suggesting that they have a potential scaffolding or docking function (Lannutti, 2001 and references therein).

In germline clones of dnc^M14, eggs are laid but fail to hatch, suggesting that dunce activity is required in the somatic cells for egg laying and in the germline for hatching. This appears to contradict the data suggesting a germline requirement for dunce in egg laying. However, there are several possible explanations for this apparent discrepancy. (1) The analysis of egg laying from females with dnc^M14germline clones could not be quantitative because the ovarioles do not all contain clones. Therefore, while the data demonstrate that somatic expression of dunce is necessary for egg laying, it does not exclude a role in the germline as well. (2) By the same argument, a role for Gprk2 activity in the somatic cells has not been ruled out. Although Gprk2 expression in the follicle cells cannot be detected, it is possible that a low level of somatic expression plays a role in egg laying. (3) Although the Gprk2 gene plays a role in regulating the level of cAMP in the ovaries, it has not been directly shown that Gprk2 and dunce are acting in the same molecular pathway. Perhaps dunce and Gprk2 act in different signaling pathways that are active in somatic and germline cells, respectively, and the interaction of the two pathways is necessary for normal levels of egg laying (Lannutti, 2001).

Cytoplasmic dumping in nurse cells involves both tubulin-based microtubules and actin-based microfilaments. The submembranous cytoskeleton provides the contractile forces for dumping, and cytoplasmic actin fibers tether the nurse cell nuclei so that they do not become lodged in the ring canals. In the gprk2⁶⁹³⁶ mutant, a small number of nurse cell nuclei fail to remain tethered and, instead, begin to extend into the ring canals. This phenotype resembles the mutants quail, singed, and chickadee. The proteins encoded by these genes (homologs of villin, fascin, and profilin, respectively) are structural components of the cytoplasmic actin fibers, and in the mutants the fibers fail to form. In strong alleles of these loci the 'dumpless' phenotype is completely penetrant. In contrast, blocked ring canals are observed in the gprk2⁶⁹³⁶ mutant at a low frequency, although incomplete cytoplasmic dumping is much more common. Furthermore, the cytoplasmic actin fibers do form in gprk2⁶⁹³⁶, even in nurse cells whose nuclei are extending through ring canals. The clumped appearance of these fibers in these nurse cells suggests that they are not arranged in a normal fashion. At this time, whether the relationship between the cytoplasmic actin fibers and the lack of nuclear tethering is causative or correlative cannot be determined. The localization of the Gprk2 protein to a submembranous region suggests that Gprk2 may play a role in the interaction between the cytoplasmic filaments and the membrane, rather than a structural role in the filaments themselves (Lannutti, 2001).

Mammalian GRKs have also been shown to interact with the cytoskeleton. GRK5 (the closest mammalian homolog to Gprk2) phosphorylates ß-tubulin and microtubules, although the functional significance of this interaction is unknown (Carman, 1998). In addition, GRK5 binds to actin monomers and filaments, and stabilizes polymerized actin filaments in vitro. Actin binding, in turn, decreases the activity of GRK5 toward receptors (Freeman, 1998; Pitcher, 1998). The sequence of the actin-binding region in GRK5 is conserved in Gprk2, suggesting that Gprk2 could also bind to actin at this site. The low penetrance of the tethering defect in the gprk2⁶⁹³⁶ mutant suggests that the lack of cytoplasmic dumping cannot be completely explained by blockage of the ring canals. In addition, the dunce mutants have a similar dumping defect, although no nurse cell nuclei extending through the ring canals has been seen in these mutants. The formation of cytoplasmic fibers and nuclear breakdown that accompany normal cytoplasmic dumping fail to occur in germline clones that lack expression of Drosophila Caspase protein-1 (DCP-1), suggesting that the events that occur during cytoplasmic dumping are part of an apoptotic pathway. It has been hypothesized that DCP-1 activity leads to two parallel pathways: one resulting in the formation of cytoplasmic actin bundles and the other resulting in nuclear envelope permeabilization, elevation of cytoplasmic Ca2+, and contraction of the submembranous actin network. Because egg chambers from gprk2⁶⁹³⁶ and dunce mutants form cytoplasmic fibers and undergo contraction, it suggests that these genes are not involved in initiation of apoptosis. It is possible that Dunce and Gprk2 could affect cytoplasmic dumping through signaling processes that regulate Ca2+ levels during the apoptotic process (Lannutti, 2001 and references therein).

The rescue of the tethering defect, by reducing the dosage of dunce, suggests that this aspect of the gprk2⁶⁹³⁶ phenotype is also sensitive to the level or subcellular distribution of cAMP. It might be expected that a similar phenotype would be seen in mutant alleles of DCO, the gene encoding the catalytic subunit of PKA. DCO is an essential gene; however, combinations of certain weak alleles are semilethal and sterile. These egg chambers have an earlier defect than observed in gprk2⁶⁹³⁶. Mutant DCO egg chambers have unstable ring canals and, beginning at stage 7, nurse cells begin to fuse, forming binucleate cells. Therefore, their egg chamber morphology is partially disrupted by stage 10B, when cytoplasmic dumping begins. Nevertheless, the nurse cell nuclei appear to remain anchored in the center of the cell. Certain allelic combinations of chickadee also form bi- and multinucleate nurse cells, suggesting that cAMP signaling and the cytoskeleton both play critical roles in maintaining membrane integrity. Binucleate cells have never been observed in the gprk2⁶⁹³⁶ mutant. However, it is likely that there are receptors that contribute to cAMP signaling that are not regulated by Gprk2. These receptors could regulate PKA in the absence of Gprk2 activity (Lannutti, 2001 and references therein).

In summary, analysis of the interaction between gprk2⁶⁹³⁶ and dunce mutants demonstrates that the regulation of cAMP synthesis and metabolism is critical for development. The gprk2⁶⁹³⁶ mutant has not only reduced levels of cAMP but also decreased egg laying and hatching. These defects were all suppressed by weak and null alleles of dunce. Similarly, the increased levels of cAMP and sterility of dunce are suppressed by the gprk2⁶⁹³⁶ mutant. Further analysis of the developmental functions of gprk2⁶⁹³⁶ in vivo will continue to complement the biochemical characterization of this protein (Lannutti, 2001).

GENE STRUCTURE

Gprk2 is encoded by at least 13 exons. The Gprk2 gene utilizes at least three polyadenylation sites. Whether use of these three sites gives rise to the three transcript sizes will require further confirmation (Schneider, 1997).

Bases in 5' UTR - 1118

PROTEIN STRUCTURE

Amino Acids - 714

Structural Domains

Gprk-2 encodes a 50 kDA polypeptide displaying 60% amino acid similarity and 39% identity to BARK (ß-adrenergic receptor kinase) and 60% amino acid similarity with Drosophila GPRK-1. The most conserved region of GPRK-2 is the catalytic domain, which displays 68% similarity to the corresponding domain of BARK. Although GPRK-2 is significantly smaller that BARK (or GPRK-1), it contains all the conserved domains found in protein-serine/threonine kinases. Indeed homology region 1 of the catalytic domain begins at residue 29. The equivalent domains of GPRK-1 and BARK begin at residues 199 and 198, respectively. Since the N-terminal domains of many types of S/T kinases contain sequences involved in auto-regulation of the enzyme, it is possible that GPRK-2 represents a variant that may require an additional subunit (Cassill, 1991).

The Gprk2 gene was originally isolated based on its sequence similarity to the human ßARK1 gene (Cassill, 1991). The predicted protein from Gprk2 is 47% identical to ßARK1 protein in the kinase domain. Outside of this region there is no significant similarity. In contrast, the Gprk2 protein predicted by the longer currently accepted sequence shares a high level of sequence identity with two other mammalian members of the GRK family, GRK5 and GRK6. This similarity extends over the entire protein, except for a 121 amino acid region that is unique to Gprk2. The physiological functions of GRK5 and GRK6 have not yet been established, although both proteins are able to phosphorylate and desensitize activated G protein-coupled receptors (Schneider, 1997).

Drosophila GPRK2 was compared to members of the three mammalian GPRK subfamilies in humans and the highest amino acid identity was found in the kinase catalytic domain, with lower levels of similarity in the flanking N-terminal and C-terminal regions. GPRK2 was most similar to human GPRK4, with 87% identity in the kinase domain and 45% and 47% identity in the N- and C-terminal regions, respectively. GPRK2 displays substantially less sequence identity to mammalian GPRK3, which is necessary to desensitize odorant receptors (Peppel, 1997). The N-terminal region of GPRK2 includes a unique stretch of amino acids (Gly124-Gly261, 138 amino acids) containing asparagine-rich and glycine-rich clusters. This unique amino acid region is not present in other GPRK subfamilies and is not homologous to sequences in other proteins, on the basis of Basic Local Alignment and Search Tool (BLAST) searches. These data suggest that GPRK2 is a member of the GPRK4 family from mammals but may have additional functions compared to other GPRK4 family members (Tanoue, 2008).

date revised: 15 May 2001

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