Molecular Aspects of Signal Transduction 
in the Visual System 
James B. Hurley, Ph.D. — Associate Investigator 
Dr. Hurley is also Associate Professor of Biochemistry at the University of Washington School of Medicine. 
He received his undergraduate degree in chemistry from the State University of New York College of 
Environmental Science and Forestry, Syracuse, and his Ph.D. degree in physiology and biophysics from 
the University of Illinois, Urbana, where he worked with Thomas Ebrey. His postdoctoral research included 
studies with Melvin Simon at both the University of California, San Diego, and the California Institute of 
Technology, and with Lubert Stryer at Stanford University. 
OUR laboratory studies molecular mecha- 
nisms responsible for visual transduction in 
vertebrate and invertebrate photoreceptors. De- 
spite the fact that these two types of cells respond 
to light via quite diverse mechanisms, they have 
many general features in common. We are inves- 
tigating mechanisms that determine such photo- 
receptor characteristics as sensitivity, rates of ac- 
tivation and deactivation, and ability to adapt to 
constant light. 
Light hyperpolarizes vertebrate photorecep- 
tors via a G protein-mediated cascade that culmi- 
nates in cyclic GMP hydrolysis. Depletion of 
cGMP reduces the activity of cGMP-gated cation 
channels in the photoreceptor plasma mem- 
brane. In darkness Ca^"^ enters the cell through 
these channels. Light blocks this entry, and the 
resulting depletion of cytosolic Ca^"^ promotes re- 
covery by stimulating guanylate cyclase to re- 
synthesize cGMP. 
Invertebrate photoreceptors respond to light 
very differently. In these cells light activates 
phospholipase C, which produces inositol tri- 
phosphate and diacylglycerol as second messen- 
gers. Few biochemical details of invertebrate 
phototransduction are well understood. 
Vertebrate Phototransduction 
Our laboratory recently identified a novel 
Ca^"^-binding protein that imparts Ca^"^ sensitivity 
to photoreceptor guanylate cyclase. This protein, 
named recoverin, promotes recovery by stimulat- 
ing guanylate cyclase when free Ca^"^ concentra- 
tions fall below 300 nM. The amino acid se- 
quence of recoverin reveals three Ca^"^-binding 
sites. Ca^"^ influences a variety of physical proper- 
ties of recoverin, including fluorescence and mo- 
bility on electrophoresis gels. We cloned re- 
coverin cDNA and expressed recombinant 
recoverin in Escherichia coli. 
The effects of Ca^"^ on recombinant recoverin 
and retinal recoverin are quite different. To ac- 
count for these differences, we compared the 
masses of recombinant and retinal recoverin di- 
rectly by ion-spray mass spectrometry. To our 
surprise, the modification turned out to be a 
novel type of heterogeneous amino-terminal acy- 
lation. Each recoverin is acylated with either a 
CI4:0, C14:1, C14:2, or C12:0 fatty acid 
residue. 
Following stimulation by light, transducin, the 
photoreceptor G protein, hydro lyzes its bound 
GTP and loses its ability to activate phosphodies- 
terase. Photoreceptor cells recover from a light 
flash within a couple of seconds, but the steady- 
state hydrolysis of GTP is slower. To clarify the 
role that GTP hydrolysis plays in recovery from a 
photoresponse, we produced transgenic mice 
that express a mutant transducin that hydrolyzes 
GTP more slowly than their normal counterparts. 
Preliminary results suggest that photoreceptors 
expressing this form of transducin are abnormally 
desensitized. This efi'ect may reflect an attempt 
by the cells to compensate for the persistent 
phosphodiesterase activity of the mutant 
transducin. 
Drosophila Vision 
Biochemical and physiological evidence sug- 
gests that a G protein mediates invertebrate pho- 
totransduction by stimulating phospholipase C. 
We characterized several Drosophila G proteins 
with the aim of understanding their role in inver- 
tebrate phototransduction. In addition to several 
G protein a-subunits, two G protein /3-subunits 
were identified. The first one was detected 
throughout the nervous system but not in the 
eyes. This prompted us to search for a photore- 
ceptor G protein /3-subunit. 
Through use of a specific type of cDNA screen- 
ing method, we were able to identify a novel type 
of G protein /S-subunit that is expressed specifi- 
cally in the Drosophila compound eye. Recently, 
in collaboration with Charles Zuker and his col- 
leagues (HHMI, University of California, San 
Diego) , two mutant Drosophila strains have been 
identified with reduced expression of this eye- 
specific |S-subunit. Biochemical analyses of eyes 
from normal Drosophila and from these mutants 
are being used to study the role of G proteins in 
phototransduction . 
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