BIOLOGICAL OVERVIEW

Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin), which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE). Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration, which is believed to cause closure of the cGMP-gated ion (cyclic nucleotide gated or CNG) channel in the plasma membrane and the generation of an electrical response. In Drosophila, rhodopsin, acting through G proteins, targets the protein NorpA encoded by a phosphoinositide-specific phospholipase C (PLC). NorpA in turn catalyzes the breakdown of phospholipids and generates inositol trisphosphate (IP3) and diacylglycerol. Diacylglycerol is a potential precursor for several polyunsaturated fatty acids, such as arachidonic acid and linolenic acid. Both TRP (transient receptor potential) and TRPL (TRP-like) are cation channels that are activated in the visual transduction process. These two proteins share homology with alpha-subunits of voltage-gated calcium and sodium channels in vertebrates. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Since arachidonic acid may not be found in Drosophila, it is suggested that another polyunsaturated fatty acid, such as linolenic acid, may be a messenger of excitation in Drosophila photoreceptors (Chyb, 1999).

Of special interest is evidence for functioning of the visual cascade in Drosophila testis. DGq is the alpha subunit of the heterotrimeric GTPase (G alpha), which couples rhodopsin to phospholipase C in Drosophila vision. Three duplicated exons were identified in dgq by scanning the GenBank data base for unrecognized coding sequences. These alternative exons encode sites involved in GTPase activity and G beta-binding, NorpA-binding, and rhodopsin-binding. In vivo splicing of dgq was examined in adult flies: in all regions, other than the male gonads, only two isoforms are expressed. One isoform, dgqA, is the original visual isoform and is expressed in eyes, ocelli, brain, and male gonads. The other, dgqB, has the three novel exons and is widely expressed. Remarkably, all three nonvisual B exons are highly similar (82% identity at the amino acid level) to domains of the Gq alpha family consensus, from Caenorhabditis elegans to human, but all three visual A exons are divergent (61% identity). Intriguingly, a third isoform, dgqC, is found that is specifically and abundantly expressed in male gonads, and shares the divergent rhodopsin-binding exon of dgqA. It is suggested that DGqC is a candidate for the light-signal transducer of a testes-autonomous photosensory clock. This proposal is supported by the finding that norpA is expressed in male gonads, as well as the photoreceptor-cell-specific genes rhodopsin 2 and arrestin 1 (Alvarez, 1996).

These observations suggest that the visual DGqA is generated by the duplication of three DGqB exons. This allows the photoreceptor-specific DGqA to be optimized for phototransduction at the Gbeta-interacting/GTPase site, the NorpA-binding site and the Rh binding site. That this specialization occurs is suggested by the existence of eye-specific forms of Gbeta, NorpA, and Rh in Drosophila. NorpA, for example, has two cloned isoforms that vary at one alternatively spliced exon: one (I) is expressed at high levels and appears to be eye-specific, and the other (II) is expressed at low levels throughout development and in various tissues -- head, thorax, abdomen, and legs (Kim, 1995). The visual norpA.I is not expressed in testes, but norpA.II is. Vertebrate sensory systems also show organ specific isoform adaptation: these systems have specialized Galphas (of non-q class) for rod-vision (G_t1alpha), cone-vision (G_t2alpha), olfaction (G_olfalpha), and taste (G_gustalpha). However, squid, the only other invertebrate with a known Rh-linked (Gq_alpha), uses a Galpha more similar to DGqB than to the visual DGqA. This suggests that before the divergence of squid and fly, there was a single DGqB-like G_qalpha that mediated vision and at least one other, even more ancient, signaling pathway (Alvarez, 1996).

A central problem in sensory system biology is the identification of the signal transduction pathways used in different sensory modalities. Odorant response in the maxillary palps olfactory organ of Drosophila (see Odorant Receptors), but not the response of the antennal olfactory cells, depends on the norpA phospholipase C gene, providing evidence for use of the inositol 1,4,5-trisphosphate (IP3) signal transduction pathway. Consistent with the demonstration of a role for norpA in the maxillary palp, but not the antenna, norpA is found to be expressed in the maxillary palp, but not the antenna. Staining is localized along the lateral surface of the maxillary palp, in a region that contains a high density of olfactory hairs. Staining occurs not only on cell bodies but also in axons (the maxillary nerve). Since the norpA gene is also essential to phototransduction, this work demonstrates overlap in the genetic and molecular underpinnings of vision and olfaction. Genetic and molecular data also indicate that some olfactory information flows through a pathway that does not depend on norpA. The maxillary palp response is not abolished in norpA null flies, suggesting the existence of an additional PLC independent pathway for the odorant response (Riesgo-Escovar, 1995).

GENE STRUCTURE

A second subtype of NorpA protein has been identified that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon, exon 4, which is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. The alternatively spliced exon is outside the highly conserved box X and box Y domains shared among major types of PLC. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in the adult retina, while subtype II transcripts are most abundant in the adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, it is concluded that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways. Subtype I and II also differ in the use of a polyadenylation site by subtype I and II cDNAs (Shortridge, 1991 and Kim, 1995).

Bases in 5' UTR - 824 (subtype I)

Exons - 4

Bases in 3' UTR - 812 (subtype I)

PROTEIN STRUCTURE

Amino Acids - 1305 (subtype I) and 1312 (subtype II)

Structural Domains

Severe norpA mutations in Drosophila eliminate the photoreceptor potential and render the fly completely blind. Recent biochemical analyses have shown that norpA mutants lack phospholipase C (PLC) activity in the eye. A combination of chromosomal walking and transposon-mediated mutagenesis was used to clone the norpA gene. This gene encodes a 7.5 kb RNA that is expressed in the adult head. In situ hybridizations of norpA cDNA to adult tissue sections show that this gene is expressed abundantly in the retina. The putative norpA protein is composed of 1095 amino acid residues and has extensive sequence similarity to a PLC amino acid sequence from bovine brain. It is suggested that the norpA gene encodes a PLC expressed in the eye of Drosophila and that PLC is an essential component of the Drosophila phototransduction pathway (Bloomquist, 1991).

A Drosophila phospholipase C (PLC) gene, designated as plc-21, was isolated by screening a genomic DNA library using a cDNA for a previously isolated Drosophila PLC gene, norpA, as a probe under reduced stringency hybridization conditions. The gene maps to 21C on the left arm of the second chromosome. Two proteins of 1305 and 1312 amino acids, respectively, were deduced from two classes of cDNA that were isolated. The two putative plc-21 proteins are similar in sequence and overall structure to the beta-class of PLCs found in mammals and differ from each other only by 7 amino acid residues that are present near the C terminus of one of the proteins but not the other. Hybridization of plc-21 cDNA probes to blots of poly(A)+ RNA reveals that the gene encodes a 7.0-kilobase transcript that can be detected in the head but not in the body of adult flies and a 5.6-kilobase transcript that can be detected throughout development and in both heads and bodies of adults (Shortridge, 1991).

date revised: 1 April 99

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