BIOLOGICAL OVERVIEW

Learning in Drosophila involves the cyclic AMP second messenger system. Stimulation of adenylyl cyclase (Rutabaga) by neural excitation results in the production of cyclic AMP, which in turn activates Protein kinase A. PKA acts on downstream targets to bring about cellular changes that are required for learning and memory.

G proteins activate and inhibit elements in the learning pathway. Heterotrimeric G proteins transduce signals from cell surface 7 pass transmembrane serpentine receptors to downstream effectors, such as ion channels, cytoskeletal elements and other signal transduction proteins. Classification of G proteins is based historically on the functional interaction of G protein alpha subunits with specific effector proteins. For example, transducins are responsible for the activation of the cGMP phosphodiesterase in the retina. Gs alpha stimulates adenylate cyclase, while Gi alpha acts as an inhibitor.

How does the excitation of neurons stimulate the cyclic AMP second messenger system? In eukaryotic cells, serpentine receptors, excited by neurotransmitters are coupled to the activation of adenylyl cyclase by the heterotrimeric GTP binding protein complex Gs. In this pathway, the initial binding of extracellular ligands to these receptors results in the activation of the Gs complex (the heterotrimeric G protein complex) by promoting the exchange of GDP for GTP on the alpha subunit, and the dissociation of Gs alpha from beta/gamma. The GTP-bound, activated Gs alpha then mediates the activation of adenylyl cyclases, resulting in the elevation of the intracellular levels of the second messenger, cAMP. Termination of the signal occurs when GTP bound by the alpha subunit is hydrolyzed to GDP by an enzymatic activity intrinsic to the alpha subunit (Neer, 1995). For more information on the consequences of neural excitation see Cyclic AMP Second Messenger System - The Learning Pathway.

Evidence in Drosophila points to the involvement of Gs alpha in the process of associative learning. However, before examining the involvement of Gs alpha in learning, an example will be give of a developmental pathway involving Gs alpha that does not involve the cyclic AMP second messenger system.

Gs alpha can be altered by mutation to become constitutively activated. Mutation of Gs alpha in the putative S box of its guanine nucleotide-binding domain (Quan, 1991), results in Gs alpha*, which has a greatly reduced hydrolytic rate for GTP. This results in receptor independent activation of the alpha subunit and consequently in constitutive activation of downstream components coupled to G protein activation.

When Gs alpha* is expressed in flies under control of various promoters, a variety of phenotypes result, including alteration of wing morphology, smaller than normal adults and lethality. One consistent effect of Gs alpha* expressed during late pupal periods is formation of wing blisters. Phenotypes arising from activation of Gs alpha pathways would be expected to depend on the activation of PKA, given the traditional scheme developed in mammals. Consistent with this prediction, many studies have demonstrated just such a critical reliance on PKA in the response of cultured cells to activation of this pathway, through the expression of mammalian Gs alpha*. To test whether this pathway is acting in wing epithelial cells to generate blistering, a dominant-negative form of the regulatory subunit of PKA was employed. PKA functions in Drosophila wings to repress the expression of signaling molecules (for example, Decapentaplegic) that mediate subsequent growth and pattern formation in the developing wing. Thus, in the absence of PKA, inappropriate expression of dpp leads to anterior wing duplication. Expression of dominant negative PKA results, as expected, in wing duplications. By constructing flies that carry both Gs alpha* and dominant negative PKA (dnPKA), activation of Gs alpha pathways and inhibition of PKA occurs simultaneously within cells of the wing epithelium. Coexpression of both altered proteins in wing cells results in a superimposition of the Gs alpha phenotype on the dnPKA phenotype. Thus, each phenotype occurs independently when these proteins are coexpressed, indicating that PKA activity is not required to generate the blistering observed on activation of the Gs alpha pathway in wing epithelial cells (Wolfgang, 1996).

The independence of PKA expression from Gs alpha stimulation is not completely without precedence. In mammalian epithelial cells, transport of influenza virus hemagglutinin protein from the apical surface is retarded by G protein specific reagents, such as drugs or toxins. Treatment of cells with reagents known to influence the Gs class of G proteins specifically affect the apical pathway. Also, antibodies against the N-terminal domain of the alpha-subunit of Gs inhibits the transport of hemaglutinin from the trans-Golgi network to the apical surface, but not between the endoplasmic reticulum and the Golgi complex. Addition of cAMP to cells has no effect on the transport of hemaglutinin to the apical surface. Thus the effects of Gs on apical transport must be mediated by another downstream effector (Pimplikar, 1993). It seems, therefore, that Gs can act independently of the cyclic AMP second messenger pathway both in Drosophila wing blistering and in mammalian vesicular transport. For more information on Drosophila wing blistering, see Myospheroid and Serum response factor.

To test whether Gs alpha signaling is involved in learning in Drosophila, Gs alpha* was targeted either to the mushroom bodies (MB) or alternatively, to the central complex (CC) of the fly brain. To examine associative learning in these flies, a Pavlovian olfactory conditioning assay was used (see Dunce for a more complete description of this procedure). Briefly, flies are trained by exposure to electroshock paired with one odor (octanol or methylcyclohexanol) and subsequently exposured to a second odor without electroshock. Immediately after training, learning is measured by forcing flies to choose between the two odors used during training. No preference between odors results in a performance index of zero (no learning), as is the case for naive flies. Avoidance of the odor previously paired with electroshock, however, yields a performance index greater than O.

When Gs alpha* is expressed in mushroom bodies, learning is completely abolished. As a control, wild type Gs alpha was expressed in mushroom bodies, and no effect on learning was detected. In the learning-impaired lines, olfactory responses to the odors is normal, demonstrating that Gs alpha* does not affect naive sensorimotor response to electroshock or odors. When Gs salpha* is expressed in the ellipsoid body or fan-shaped body of the CC, learning is unaffected. Pan-neural expression of Gs alpha* during development produces neither lethality nor overt behavioral phenotypes, suggesting that perturbation of Gs signaling does not significantly affect basic neuronal function. Gross morphology appears normal when Gs alpha* is expressed in the MBs. Thus the abolition of learning does not appear to result from maldevelopment of underlying structures. Therefore Gs alpha* specifically impairs learning, implicating wild type Gs alpha in stimulation of the learning pathway.

Null alleles of dunce and rutabaga, two genes involved in the cyclic AMP second messenger pathways produce only a partial impairment in learning and not the complete impairment observed for Gs alpha* expression. Thus, disruption of all adenylyl cyclase regulation by Gs alpha* expression seems to have more drastic effects on signaling than removal of one form of adenylyl cyclase (as in rutabaga) or cyclic nucleotide phosphodiesterase (as in dunce). Alternatively, Gs alpha could exert signaling effects other than through the cAMP pathway, as it does in the blistering phenotype described above (Connolly, 1996).

GENE STRUCTURE

The exon-intron structure of the Drosophila Gs alpha shows substantial similarity to that of the human gene for Gs alpha. Alternative splicing of intron 7 in Drosophila, involving either the use of an unusual TG or consensus AG3' splice site, results in transcripts which code for either a long or short form of Gs alpha. These subunits differ by inclusion or deletion of three amino acids and substitution of a Ser for a Gly. The two forms of Drosophila Gs alpha differ in a region where no variation in the primary sequence of vertebrate Gs alpha subunits has been observed. Additional Gs alpha transcript heterogeneity refects the use of multiple polyadenylation sites (Quan, 1990).

cDNA clone length - 1345

Bases in 5' UTR - 303

Exons - 9

Bases in 3' UTR - 189

PROTEIN STRUCTURE

Amino Acids - 385

Structural Domains

The Drosophila G protein salpha 60A is 71% homologous to the long form of bovine Gs alpha. The level of homology to Gi alpha and Go alpha and transducin is lower but still significant (41-44%) (Quan, 1989). The similarity is highest in the four regions of homology that have been identified in the G alpha subunits, the ras oncogene proteins, and bacterial elongation factor Tu. These highly conserved regions (A, C, E and G) are thought to be responsible for guanine nucleotide binding and hydrolysis. In mammals, the alignment of the deduced amino acid sequence of the various G alpha polypeptides has shown that Gs alpha is the most divergent member of the G alpha family. Vertebrate Gs alpha is structurally heterogeneous, existing as at least two species with apparent molecular weights of 45,000 and 52,000 in SDS polyacrylamide gels. These proteins are formed by alternative splicing. The Drosophila proteins (both long and short forms) correspond most closely to the smaller vertebrate form (Quan, 1989).

The crystal structure of Gsalpha, the heterotrimeric G protein alpha subunit that stimulates adenylyl cyclase, was determined at 2.5 A in a complex with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Gsalpha is the prototypic member of a family of GTP-binding proteins that regulates the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsalpha.GTPgammaS with that of Gialpha.GTPgammaS suggests that their effector specificity is dictated primarily by the shape of the binding surface formed by the switch II helix and the alpha3-beta5 loop, despite the high sequence homology of these elements. In contrast, sequence divergence explains the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsalpha. The betagamma binding surface of Gsalpha is largely conserved in sequence and structure to that of Gialpha, whereas differences in the surface formed by the carboxyl-terminal helix and the alpha4-beta6 loop may mediate receptor specificity (Sunahara, 1997).

date revised: 9 Jan 97 

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