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Journal of Neuroscience, Vol 15, 1261-1274, Copyright © 1995 by Society for Neuroscience
Identification and characterization of antidepressant-sensitive serotonin transporter proteins using site-specific antibodies
Y Qian, HE Melikian, DB Rye, AI Levey and RD Blakely
Graduate Program in Neuroscience, Emory University School of Medicine, Atlanta, Georgia 30322.
Serotonin (5HT) transporters (SERTs) are responsible for clearance of
synaptic and plasma 5HT and are molecular targets for multiple therapeutic
and addictive compounds. Recently brain and peripheral SERT cDNAs have been
cloned and characterized functionally in transfected cells. Antipeptide
(S365) and anti-fusion protein (CT-2) antibodies, directed at epitopes
poorly conserved among other Na+/Cl- cotransporters, have been prepared to
facilitate the identification and characterization of SERT proteins in
native and transfected cells. Immunoprecipitations and immunoblots of
rat/human SERT-transfected HeLa cells reveal specific SERT-immunoreactive
glycoproteins absent from extracts of vector-transfected cells and absent
when incubations were conducted using peptide- or fusion protein-absorbed
antibody. In SDS- PAGE of membranes prepared from rat midbrain and cortex,
SERTs migrate as single 76 kDa polypeptides with a relative abundance
consistent with the known distribution of 5HT neurons and axonal
projections. SERT- immunoreactive proteins are also detectable in platelet
and pulmonary membranes, sites of peripheral 5HT uptake, but not in liver.
Our studies also indicate that brain and platelet SERTs are formed from
identical polypeptides differing significantly in their extent of N- linked
glycosylation. Immunocytochemistry performed on rat brain sections with
CT-2 antibody revealed SERT expression associated with brainstem raphe
nuclei in a pattern virtually identical to that obtained by labeling
adjacent sections with 5HT antisera. SERT- immunoreactive fibers were found
to be widely distributed throughout the rodent brain, with highest density
in forebrain regions known to receive a dense serotonergic innervation. In
a similar manner, CT-2 antibody also detects endogenous expression of human
SERT proteins, providing an opportunity for future studies on the
modulation of transporter protein expression in neurologic and psychiatric
disorders.
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