Tài liệu Báo cáo khoa học: Constitutive oligomerization of human D2 dopamine receptors expressed in

Constitutive oligomerization of human D2 dopamine receptors

expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells

Analysis using co-immunoprecipitation and time-resolved fluorescence resonance

energy transfer

Lucien Gazi1,†, Juan F. Lo´ pez-Gime´ nez1,*†, Martin P. Ru¨ diger2 and Philip G. Strange1

1

School of Animal and Microbial Sciences, University of Reading, Reading, UK; 2

GlaxoSmithKline, New Frontiers Science Park,

Harlow, UK

Human D2Long (D2L) and D2Short (D2S) dopamine receptor

isoforms were modified at their N-terminus by the addition

of a human immunodeficiency virus (HIV) or a FLAG

epitope tag. The receptors were then expressed in Spodop￾tera frugiperda 9 (Sf9) cells using the baculovirus system,

and their oligomerization was investigated by means of

co-immunoprecipitation and time-resolved fluorescence

resonance energy transfer (FRET). [3

H]Spiperone labelled

D2 receptors in membranes prepared from Sf9 cells expres￾sing epitope-tagged D2L or D2S receptors, with a pKd value

of
10. Co-immunoprecipitation using antibodies specific

for the tags showed constitutive homo-oligomerization of

D2L and D2S receptors in Sf9 cells. When the FLAG-tagged

D2S and HIV-tagged D2L receptors were co-expressed,

co-immunoprecipitation showed that the two isoforms can

also form hetero-oligomers in Sf9 cells. Time-resolved

FRET with europium and XL665-labelled antibodies was

applied to whole Sf9 cells and to membranes from Sf9 cells

expressing epitope-tagged D2 receptors. In both cases, con￾stitutive homo-oligomers were revealed for D2L and D2S

isoforms. Time-resolved FRET also revealed constitutive

homo-oligomers in HEK293 cells expressing FLAG-tagged

D2S receptors. The D2 receptor ligands dopamine,

R-(–)propylnorapomorphine, and raclopride did not affect

oligomerization of D2L and D2S in Sf9 and HEK293 cells.

Human D2 dopamine receptors can therefore form consti￾tutive oligomers in Sf9 cells and in HEK293 cells that can be

detected by different approaches, and D2 oligomerization in

these cells is not regulated by ligands.

Keywords: G protein-coupled receptors; D2 dopamine

receptor; oligomerization; Sf9 cells; HEK293 cells.

The G protein-coupled receptors (GPCR) represent one of

the largest families of genes in the human genome. They are

responsible for the detection of a large variety of stimuli and

control many physiological processes, including neurotrans￾mission, cellular metabolism, secretion, differentiation, and

inflammatory and immune responses. Consequently, many

existing therapeutic agents act by either activating or

blocking GPCRs. There is now an increasing body of

evidence showing that GPCRs can form oligomers and that,

in some cases, oligomerization of the receptors is required

for their function [1,2]. The diversity of the receptors

described suggests that the phenomenon of oligomerization

may be general to the whole GPCR family, rather than

being restricted to some subgroups of receptors. Hence,

oligomerization of receptors belonging to the same family

(homo-oligomerization) or between receptors belonging to

different families (hetero-oligomerization) has been repor￾ted. These include the b2-adrenoceptor [3,4], the chemokine

receptor CCR5 [5,6], the M3 muscarinic acetylcholine

receptor [7], the M2 muscarinic cholinergic receptor [8],

the melatonin MT1 and MT2 receptors [9], the V2

vasopressin receptor [10], the 5-HT1A, 5-HT1B and

5-HT1D receptors [11], the d and j opioid receptors [12–14],

the histamine H2 receptor [15], the somatostatin sst2A and

sst3 receptors [16], the yeast Ste2 receptor [17] and the D2

dopamine receptor [18,19]. Hetero-oligomerization between

c-aminobutyric acid GABABR1 and GABABR2 receptors

was shown to be a requirement for the expression of

functional receptors at the cell surface [20]. Other examples

of hetero-oligomerization include b2-adrenoceptor and the d

or j opioid receptors [13], dopamine D2 receptor and

somatostatin sst5 receptor [21], a2-adrenoceptor and M3

muscarinic receptors [22], dopamine D2 and D3 receptors

[23]. More recently, Salim et al. described the hetero￾oligomerization of 5HT1A receptors with a large number of

diverse receptor subtypes, including EDG1, EDG3, GPR26

and GABABR2 receptors [11]. All these data strongly

Correspondence to P. G. Strange, School of Animal and Microbial

Sciences, University of Reading, Whiteknights, Reading, RG6 6AJ,

UK. Fax: + 44 118 378 6537, Tel.: + 44 118 378 8015,

E-mail: [email protected]

Abbreviations: BRET, bioluminescence resonance energy transfer;

D2L, D2Long; D2S, D2Short; Eu3+, europium; FRET, fluorescence

resonance energy transfer; GPCR, G protein-coupled receptor;

HIV, human immunodeficiency virus; m.o.i., multiplicity of infection;

NPA, R-(–)propylnorapomorphine; Sf9, Spodoptera frugiperda 9.

*Present address: Molecular Pharmacology Group, Division of Bio￾chemistry and Molecular Biology, Institute of Biomedical and Life

Sciences, University of Glasgow, Glasgow G12 8QQ, UK.

Authors who contributed equally to this work.

(Received 9 April 2003, revised 8 July 2003, accepted 30 July 2003)

Eur. J. Biochem. 270, 3928–3938 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03773.x