STRUCTURE AND FUNCTION OF THE SEROTONIN RECEPTORS 
Richard Axel, M.D., Investigator 
Serotonin is a biogenic amine that functions as a 
neurotransmitter, a hormone, and a mitogen. Sero- 
tonin-containing neurons project to most regions 
of the mammalian central nervous system and 
mediate diverse neural functions. In the brain, 
serotonergic synapses regulate sensory motor re- 
flexes and modify cortical circuits involved in affec- 
tive functions. In the periphery, serotonin activates 
chemoreceptive sensory endings and controls en- 
teric reflexes. This diverse set of physiologic pro- 
cesses results partly from the interaction of seroto- 
nin with distinct receptors. Multiple serotonin 
receptors have been defined on the basis of ligand- 
binding properties. Moreover, individual receptor 
subtypes activate different intracellular signaling 
systems. The 5HTlc and 5HT2 receptors activate 
phospholipase C, whereas 5HTla and 5HTlb re- 
ceptors regulate adenylate cyclase or couple to G 
proteins that activate ion channels directly. 
I. Structure of Serotonin Receptors. 
In collaboration with Dr. Thomas M. Jessell 
(HHMI, Columbia University College of Physicians 
and Surgeons), Dr. Axel's laboratory has cloned and 
characterized the structure and function of two se- 
rotonin receptors, in an attempt to elucidate the 
mechanism of action of serotonin in the mamma- 
lian nervous system. A cDNA expression system was 
designed that permits the identification of func- 
tional cDNA clones encoding transmitter receptors 
in the absence of protein sequence information. By 
combining cloning and RNA expression vectors 
with an electrophysiologic assay in oocytes, they 
have isolated functional clones encoding both the 
5HTlc and 5HT2 receptors. The cDNAs of both re- 
ceptors encode proteins that share numerous se- 
quence and structural properties with the family of 
receptor molecules predicted to span the lipid bi- 
layer seven times. The 5HT2 receptor shares an 
overall sequence identity of 49% with the 5HTlc re- 
ceptor, but the amino acid identity within the trans- 
membranes is 80%. The extent of sequence identity 
may reflect either a very recent gene duplication or 
evolutionary conservation required to maintain a 
common function. Comparison of the 5HTlc and 
5HT2 sequences, however, shows extensive diver- 
gence in the amino- and carboxyl-terminal regions, 
as well as in the third cytoplasmic loop. These ob- 
servations suggest that the two genes do not result 
from a recent duplication event but that the strin- 
gent conservation is required to maintain a set of 
functions shared by the two receptor subtypes. The 
striking conservation of sequence within the trans- 
membrane domains is consistent with the observa- 
tion that the two receptors exhibit common ligand- 
binding properties and couple to the same 
intracellular signaling pathway. Thus these two se- 
rotonin receptors define a new family of G protein- 
coupled receptors. 
II. Function of Serotonin Receptors in Novel Cellu- 
lar Environments. 
Previous studies have suggested that the 5HTlc 
and 5HT2 receptors activate phospholipase C in 
cells of the central nervous system. The genes 
encoding these receptors were therefore intro- 
duced into novel cellular environments to obtain 
additional evidence that the two receptors activate 
common intracellular signaling systems. In Xenopus 
oocytes injected with either 5HT2 or 5HTlc re- 
ceptor RNA, serotonin elicits a rapid inward cur- 
rent that is reversibly blocked by selective 5HT 
antagonists. This inward current is likely to be 
triggered by receptor-mediated activation of 
phospholipase C and the subsequent release of 
inositol phosphates. These second messengers pro- 
mote the mobilization of intracellular calcium and 
the opening of calcium-dependent chloride chan- 
nels. These experiments demonstrate that the 
5HT2 and 5HTlc cDNAs encode functional seroto- 
nin receptors that confer serotonin sensitivity to 
Xenopus oocytes. 
A separate series of experiments demonstrated 
that the 5HTlc and 5HT2 receptors are functional 
when introduced into NIH 3T3 fibroblasts. Fibro- 
blasts expressing either receptor on their cell sur- 
face exhibit specific high-affinity binding with sero- 
tonin. The functional response of transfected cells 
to serotonin was analyzed by loading cells with 
the calcium-sensitive dye Indo I and monitoring 
changes in the fluorescent emission spectrum of 
the dye in a flow cytometer. Cells transformed with 
either the 5HTlc or 5HT2 receptor cDNA reveal a 
marked increase in intracellular calcium when ex- 
posed to serotonin. These experiments indicate 
that this family of 5HT receptors couples to the 
same intracellular signaling systems in neurons, fi- 
broblasts, and Xenopus oocytes. 
Continued 
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