FIG. 1. Phototransduction in Drosophila photoreceptors. Absorption of a photon of light causes a conformational change in the rhodopsin molecule (R) and activates its catalytic properties. Active metarhodopsin (M*) catalyzes G protein activation. The G protein exchanges GDP for GTP and releases the inhibitory βγ subunits. Active G protein catalyzes the activation of the norpA-encoded PLC. PLC hydrolyzes PIP2 into the intracellular messengers IP3 and DAG. cGMP has also been implicated as a possible intracellular messenger mediating excitation. Extracellular sodium and calcium enter the cell through the light-activated conductance and cause the depolarization of the photoreceptor cells. The light-activated conductance appears to be composed of at least two types of channels. The trp gene is required for a class of channels with high calcium permeability. DAG is thought to modulate a photoreceptor cell-specific PKC (encoded by inaC) that regulates deactivation and desensitization of the light response. Metarhodopsin is inactivated via phosphorylation by rhodopsin kinase (RoK) and arrestin binding (encoded by the arr1 and arr2 genes). Inactive metarhodopsin is photoconverted back to rhodopsin and then presumably dephosphorylated by the rdgC-encoded phosphatase. The box in the upper right indicates a pathway likely to be required for synthesis of PIP2. rdgA encodes DAG-kinase and rdgB encodes a protein with significant sequence homology to phosphatidylinositol-transfer protein, cds refers to CDP-DAG synthase. dgq and gbe are the genes encoding the photoreceptor cell-specific isoforms of Gα and Gβ subunits, respectively. ninaB and ninaD are genes required for retinal biogenesis, and ninaA is a cyclophilin homolog required for rhodopsin biogenesis, rh1, rh2, rh3, and rh4 are the structural genes for the four known rhodopsins. IP3R and PIPase refer to the IP3 receptor and inositol polyphosphate phosphatase (an enzyme required to break down IP3). Mutations in all gene products highlighted in red are now available (refs.11 and 22, and unpublished work from C.S.Z. laboratory).

biochemical grounds but in which a genetic approach provided fundamental insight as to their functional requirement. Examples are the cyclophilin homologue ninaA and its role in rhodopsin biogenesis (1417), an eye-specific protein kinase C (PKC) required for deactivation and calcium feedback regulation (18,19), the role of Gβ in the termination of the light response (20), and a number of enzymes involved in inositol phospholipid metabolism and shown to be required for photoreceptor cell excitation (10,21).

Inositol Phospholipids and Phototransduction

Drosophila phototransduction is one of the best model systems for the study of G protein-coupled PLC signaling (10,22,23). Not only is the system amenable to molecular genetic analysis but also it can report activity with exquisite sensitivity and specificity: photoreceptor cells are sensitive to single photons, and the signaling pathway can be turned on and off with millisecond kinetics (phototransduction in Drosophila is the fastest known G protein cascade, taking just a few tens of milliseconds to go from light activation of rhodopsin to the generation of a receptor potential). As described above, active PLC catalyzes the hydrolysis of the minor membrane phospholipid PIP2 into the second messengers inositol IP3 and DAG. IP3 mobilizes calcium from internal stores, which affects and modulates many cellular processes, and DAG activates members of the PKC family of proteins. Given the central role of PIP2 in signaling, its levels may be expected to be tightly regulated in the cell.

Fig. 2 shows an expanded view of the PIP2 cycle. CDP-DAG synthase (CDS) is an enzyme required to convert phosphatidic acid into CDP-DAG, the acceptor for the inositol head group. Using enhancer trap technology, we identified an eye-specific form of CDP-DAG and isolated mutations in this gene (eyecds) (21). To determine whether eye-cds mutants have a defect in their signaling properties, wild-type and mutant animals were assayed for their ability to maintain a continuously activated state of the photoreceptor cells because such a state would require the continuous availability of the second messenger PIP2. Our results demonstrated that light activation depletes a pool of PIP2 necessary for excitation that cannot be replenished in eye-cds mutants (Fig. 3a–d). This phenotype is due exclusively to a defect in eye-cds, because introduction of the wild-type eye-CDS cDNA into mutant hosts fully restores wild-type physiology (Fig. 3e–f). Furthermore, inclusion of PIP2 in the patch pipette is sufficient to restore signaling in the depleted eye-cds mutants (Fig. 3g–h). These results suggest, contrary to expectations, that the pool of PIP2 required for signaling is quite small and likely synthesized “on demand”. On the basis of these findings, we reasoned that it should be possible to modulate the output of this cascade by experimentally manipulating the levels of eye-CDS. Indeed, we made



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