SIGNAL TRANSDUCTION MEDIATED BY G PROTEINS 
James B. Hurley, Ph.D., Associate Investigator 
Signal transduction mechanisms mediated by G 
proteins include responses of cells to light, hor- 
mones, neurotransmitters, and chemotactic agents. 
G proteins transduce extracellular signals into reg- 
ulation of specific effectors that alter cellular prop- 
erties. Like the receptors and effectors that they in- 
teract with, G proteins are a diverse family encoded 
by many different closely related genes. Dr. Hurley 
and his colleagues are studying how the diversity of 
the G protein family generates a variety of cellular 
responses. 
L G Proteins and Phototransduction. 
Rod and cone photoreceptor cells hyperpolarize 
when exposed to light. Light acts directly on the 
chromophore of rhodopsin, retinal. The subse- 
quent response is mediated by transducins, a spe- 
cific class of G proteins. Activated rhodopsin cata- 
lyzes GTP binding to the largest subunit of 
transducin Ta. This form of transducin then inacti- 
vates an inhibitor subunit of a cyclic GMP (cGMP) 
phosphodiesterase. Cyclic GMP in the photorecep- 
tor cell is then rapidly hydrolyzed, a cGMP-depen- 
dent cation channel is shut down, and the cell 
hyperpolarizes. 
This pathway exists in both rod and cone photo- 
receptor cells. Rods are very sensitive to light, but 
slow to respond, whereas cones are insensitive to 
light but quick to respond. Distinct forms of opsins, 
transducins, and phosphodiesterases have been 
identified in both cell types. Dr. Hurley and his col- 
leagues have been studying cone transducin and a 
cone phosphodiesterase inhibitor to determine if 
properties of these enzymes are responsible for the 
physiological differences between rods and cones. 
Cone transducin and cone phosphodiesterase in- 
hibitor cDNA clones have been identified by screen- 
ing cDNA libraries at low stringency, using rod 
cDNAs as probes. Antipeptide antibodies were used 
to show that the proteins encoded by these cDNA 
clones are found only in cone cells. Anti-opsin 
probes were also used, together with anticone 
transducin probes, to demonstrate that cone trans- 
ducin is present in red-, green-, and blue-sensitive 
cones in human retinas. Recently, a monoclonal an- 
tibody raised against a cone transducin fusion pro- 
tein has been used to afifinity-purify cone trans- 
ducin from bovine retinas. The purified cone 
transducin is being used to compare its kinetic 
properties with those of rod transducin. The 
epitope recognized by this antibody has been 
mapped by testing its immunoreactivity against a 
series of fusion proteins successively deleted from 
the amino terminus. Dr. Hurley and his colleagues 
also characterized a cone-specific form of the phos- 
phodiesterase inhibitor subunit. Antipeptide anti- 
bodies raised against sequences unique to this pro- 
tein recognize only a subpopulation of cones in 
bovine retinas. These cones correspond to the 
blue-sensitive cones present in human retinas. Dr. 
Hurley and his colleagues plan to express the rod 
and cone phosphodiesterase inhibitors in bacteria 
to compare their properties. A cone-specific phos- 
phodiesterase has been purified in the laboratory 
of Dr. Joseph Beavo, and Dr. Hurley and his col- 
leagues plan to use all of these proteins to compare 
the kinetic characteristics of the rod and cone 
cGMP phototransduction cascades. 
II. Drosophila G Proteins. 
The roles of G proteins in Drosophila signal 
transduction pathways are also being investigated. 
Drosophila G protein a- and P-subunits were iden- 
tified by isolating Drosophila genomic and cDNA 
clones related to mammalian G proteins. Two Dro- 
sophila a-subunit genes and a (3-subunit gene have 
been characterized. One of the a-subunits is closely 
related to mammalian G. a-subunits, whereas the 
other is closely related to mammalian G^. Two 
forms of Drosophila G^ mRNA that are produced by 
alternative splicing encode proteins that differ only 
at the amino terminus. There appears to be only 
one (3-subunit gene that has no introns within the 
coding region. 
In situ localization analyses of mRNA corre- 
sponding to these genes demonstrate that the G. a- 
subunit is expressed primarily in the nurse cells of 
Drosophila ovaries, whereas the G^ a-subunit is ex- 
pressed primarily in nervous tissues. No G protein 
a-subunit has yet been identified in Drosophila 
eyes. G protein P-subunit mRNA is expressed pri- 
marily in nervous tissue, including brain and eyes. 
A variety of antibody probes were used to localize 
the Drosophila G. a-subunit in Drosophila sec- 
tions. These antibodies react most strongly with nu- 
clei in nurse cells and follicle cells in Drosophila 
ovaries. Only very weak reactivity is seen in other 
tissues. An antipeptide antibody that recognizes a 
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