MOLECULAR GENETICS OF SENSORY TRANSDUCTION IN DROSOPHILA 
Charles S. Zuker, Ph.D., Associate Investigator 
The aim of Dr. Zuker's research program is to elu- 
cidate mechanisms used for signal transduction in 
the visual system. The results obtained from these 
studies should increase understanding of the molec- 
ular basis of sensory reception and information pro- 
cessing and will be useful in understanding abnor- 
malities in the human visual and nervous systems. 
Activation of the Visual Cascade 
Dr. Zuker's laboratory has been characterizing a 
number of molecules involved in the activation of 
the phototransduction cascade. These include the 
visual pigment molecule rhodopsin, the G protein 
that couples to the effector, and phospholipase C. 
Rhodopsin is composed of a protein, opsin, cova- 
lently linked to 1 l-cis retinal. The spectral sensitiv- 
ity of human rhodopsin and color opsins appears to 
be regulated by interactions between the 1 1 -cis reti- 
nal chromophore and charged or polar amino acids 
within the opsin apoprotein. In an attempt to eluci- 
date the molecular basis of spectral specificity of 
rhodopsin in vivo, a large collection of chimeric 
visual pigments made up of parts of the blue- 
sensitive Rl-6 opsin and parts of the violet-sensitive 
R8 opsin were constructed and then reintroduced 
into flies, where their spectral behavior can be as- 
sayed in their normal cellular and organismal envi- 
ronment. Characterization of these transgenic flies 
showed that it is possible to introduce localized 
structural changes within the opsin protein that 
alter its spectral properties. Moreover, it is possible 
to retune metarhodopsin absorption, the activated 
form of the molecule, without an associated change 
in rhodopsin sensitivity. This finding demonstrated 
that different regions of the protein interact with the 
chromophore in the native and activated states and 
opens up the possibility of custom-tailoring light 
receptor molecules with defined properties. 
rdgB (retinal degeneration E) is an X-linked mu- 
tation that triggers an irreversible course of light- 
dependent degeneration of the Rl-6 photoreceptor 
cells of Drosophila. Activation of the visual cascade 
is required for the degeneration of the photorecep- 
tors to occur. As a means of identifying novel compo- 
nents involved in the activation of the visual cas- 
cade, the laboratory is screening for second site 
suppressors and enhancers of photoreceptor degen- 
eration in rdgB. A number of recessive mutations 
that protect the photoreceptors of rdgB from light- 
dependent degeneration have been identified. 
These mutants are being characterized with physio- 
logical, genetic, and molecular techniques. 
Regulation of the Visual Cascade 
Arrestins are a family of proteins thought to medi- 
ate the inactivation of G protein-coupled receptors. 
In photoreceptors, they are thought to uncouple 
rhodopsin-G protein interaction. Although much is 
known about arrestin function in vitro, little is 
known about its precise role in vivo. Two arrestin 
genes have been identified in Drosophila {arrl and 
arrZ); both are photoreceptor cell specific. Al- 
though it is tempting to speculate that arrestins 
function in catalyzing the termination of the active 
state of rhodopsin, the identification of a biochemi- 
cal activity in vitro does not necessarily mimic or 
underlie a corresponding role for those molecules 
in vivo. Thus the phenotype of mutants defective in 
arrestin function may not be easily predicted. To 
circumvent this potential problem, the laboratory 
successfully completed a screen for arrestin mutants 
that was based solely on the loss of the protein on 
Western blots. The physiological and biochemical 
characterization of these mutants should provide 
definitive evidence as to the in vivo role of arrestins. 
To carry out a detailed physiological characteriza- 
tion of the light response of Drosophila photore- 
ceptors. Dr. Zuker and his colleagues developed a 
novel preparation of isolated photoreceptors suit- 
able for patch-clamp analysis. With this preparation, 
influx of extracellular calcium was shown to be suf- 
ficient and necessary for mediating a rapid stimulus- 
dependent inactivation of the phototransduction 
cascade. The laboratory has shown that mutations in 
a gene that encodes a photoreceptor-specific iso- 
form of a protein kinase C (eye-PKC) are specifi- 
cally defective in this calcium-dependent inactiva- 
tion mechanism, and the expression of the mutant 
phenotype requires extracellular calcium. These 
data suggest a model in which the light-dependent 
calcium influx rapidly shuts off the light-activated 
electrical response through activation of eye-PKC. 
Genetic screens aimed at identifying the upstream 
regulators and downstream targets of this eye- 
specific PKC are in progress. 
Recently, in collaboration with Dr. Roger Tsien 
(HHMI, University of California, San Diego), the lab- 
oratory has begun to combine patch-clamp record- 
ings of the electrical response with high-speed 
confocal imaging of the kinetics of calcium mobili- 
NEUROSCIENCE 453 
