gands interact with them is important for the devel- 
opment of therapeutically useful chemicals. To 
address this problem. Dr. Lerner and his colleagues 
have developed a multifaceted, G protein-coupled 
receptor bioassay. 
The G protein-coupled receptor bioassay is based 
on the ability that many animals have to change their 
color rapidly. In nature, color changes are used for 
purposes such as camouflage and to communicate 
states of emotion. A major way in which these 
changes are effected is by the controlled movement 
of pigment granules within chromatophores. When 
pigment granules in melanophores (a particular 
type of chromatophore) are aggregated, the animal 
appears light. When pigment is dispersed, the ani- 
mal appears dark. The pigment translocation appara- 
tus is controlled via second messenger systems that 
are regulated by G proteins. As a result, the state of 
pigment disposition within melanophores reflects 
the state of activity of G protein-coupled receptors. 
This laboratory has created an immortalized frog 
melanophore cell line that forms the center of an 
assay for monitoring the activity of G protein- 
coupled receptors. To test the system, (82'^drener- 
gic, substance P, and dopamine 2 receptors were 
initially expressed in the melanophores. Normally, 
receptors elevate intracellular cAMP, substance P 
receptors raise inositol 1,4,5-trisphosphate (IP3) 
and calcium, and dopamine 2 receptors lower 
cAMP. Stimulation of either the ^2 or substance P 
receptors induces pigment dispersion, while stimu- 
lation of the dopamine receptor leads to pigment 
aggregation. Eight exogenous receptors have been 
expressed in the pigment cells, and all actuate ap- 
propriate pigment translocation following stimula- 
tion. Using 96 well plates containing cells express- 
ing a receptor of interest, it is possible to obtain 
detailed dose-response curves and EC50 and IC50 val- 
ues for many drugs. 
Future studies that will involve investigating the 
effects of site-specific mutations on receptor func- 
tion will be aided by the ability to analyze the func- 
tional activation of G protein-coupled receptors in 
large numbers of cells while retaining single-cell 
resolution. For this purpose, digital image process- 
ing has been integrated into the melanophore-based 
bioassay. Translocation of melanosomes within 
thousands of individual pigment cells can be simul- 
taneously tracked by capturing gray scale video 
images before and after receptor activation. Digital 
subtraction of poststimulation from prestimulation 
images generates bitplane images containing pixels 
with nonzero gray scale values wherever melano- 
some movement occurs. Up to 200,000 plasmids 
can be evaluated in < 1 h for the presence of ones 
coding for functional receptors. 
To facilitate investigations of chemicals for their 
properties as ligands for G protein-coupled recep- 
tors, a means of creating a mobile peptide library 
(MPL) containing large numbers of soluble peptides 
that are individually localized has been developed. 
The value of such libraries stems from the observa- 
tion that the natural ligands for many G protein- 
coupled receptors are peptides. If large numbers of 
distinct peptides can be compared for their abilities 
to activate or block specific receptors, much could 
be learned. The starting point for constructing an 
MPL is a synthetic peptide combinatorial library 
(SPCL) where peptides are tethered to resin beads. 
However, for a functional assay, it is necessary for 
ligands to be capable of freely interacting with re- 
ceptors residing on living cells. To create an MPL, 
the bonds anchoring synthetic peptides to the beads 
on which they were constructed, as well as those 
between the amino acid side chain protecting 
groups and the peptides, are severed in a dry state. 
Because the peptides remain attached, albeit nonco- 
valently, to their source beads, they can be manipu- 
lated as discrete units for a variety of functional 
tests. 
The combination of the melanophore-based G 
protein-coupled receptor bioassay and the availabil- 
ity of MPLs will be useful for unraveling how ligands 
stimulate or block seven-transmembrane domain 
receptors. 
Dr. Lerner is also Associate Professor in the De- 
partments of Internal Medicine (Section of Rheu- 
matology) and of Pharmacology and the Child 
Study Center at Yale University School of Medicine. 
Articles 
Potenza, M.N., and Lerner, M.R. 1991- A recombi- 
nant vaccinia virus infects Xenopus melano- 
phores. Pigment Cell Res 4:186-192. 
Potenza, M.N., and Lerner, M.R. 1992. A rapid 
quantitative bioassay for evaluating the effects of 
ligands upon receptors that modulate cAMP levels 
in a melanophore cell line. Pigment Cell Res 
5:372-378. 
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