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J. Biol. Chem., Vol. 276, Issue 16, 12736-12743, April 20, 2001
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From the Western Australian Institute for Medical Research,
University of Western Australia and Keogh Institute of Medical
Research, Sir Charles Gairdner Hospital, Nedlands,
Perth, Western Australia 6009, Australia
The ability of G-protein-coupled receptors
(GPCRs) to interact to form new functional structures, either forming
oligomers with themselves or forming associations with other
intracellular proteins, has important implications for the regulation
of cellular events; however, little is known about how this occurs.
Here, we have employed a newly emerging technology, bioluminescence resonance energy transfer (BRET), used to study protein-protein interactions in living cells, to demonstrate that the
thyrotropin-releasing hormone receptor (TRHR) forms constitutive
homo-oligomers. This formation of TRHR homo-oligomers in the absence of
ligand was shown by demonstration of an energy transfer between TRHR
molecules fused to either donor, Renilla luciferase (Rluc)
or acceptor, enhanced yellow fluorescent protein (EYFP) molecules. This
interaction was shown to be specific, since energy transfer was not
detected between co-expressed tagged TRHRs and either complementary
tagged gonadotropin-releasing hormone (GnRH) or
Constitutive and Agonist-dependent
Homo-oligomerization of the Thyrotropin-releasing Hormone
Receptor
DETECTION IN LIVING CELLS USING BIOLUMINESCENCE RESONANCE ENERGY
TRANSFER*
2-adrenergic receptors. Furthermore, generation of a
BRET signal between the TRHRs could only be inhibited by co-expression
of the wild-type TRHR and not by other GPCRs. Agonist stimulation led
to a time- and dose-dependent increase in the amount of
energy transfer. Inhibition of receptor internalization by
co-expression of dynamin mutant K44A did not affect the interaction
between TRHRs, suggesting that clustering of receptors within
clathrin-coated pits is not sufficient for energy transfer to occur.
BRET also provided evidence for the agonist-induced oligomerization of
another GPCR, the GnRH receptor (GnRHR), and the presence of an
agonist-induced interaction of the adaptor protein, -arrestin, with
TRHR and the absence of an interaction of -arrestin with GnRHR. This
study supports the usefulness of BRET as a powerful tool for studying
GPCR aggregations and receptor/protein interactions in general and
presents evidence that the functioning unit of TRHRs exists as
homomeric complexes.
*
This work was supported by grants from the National Health
and Medical Research Council and the Raine Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence must be addressed: Karin A. Eidne, WAIMR, B
Block, Sir Charles Gairdner Hospital, Hospital Ave., Nedlands, Perth,
WA 6009, Australia. Tel.: 61-08-9346-1980; Fax: 61-08-9346-1818; E-mail: keidne@waimr.uwa.edu.au.
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