Download Structural analysis of the D2 dopamine receptor

146s Biochemical SocietyTransactions ( 1992) 20
Structural analysis of tho q d o p u i n o rocaptor.
U R S U U M. D'SOUZA
and PHILIP G. STRANGE
Biological Laboratory, The University, Canterbury,
Kent CT2 7NJ, U.K.
The D, dopamine receptor binds to dopamine an
important
neurotransmitter
in the brain
and
periphery, and it is also a key site of action of
antiparkinsonian and antischizophrenic drugs. This
receptor belongs t o the family of receptors coupled
t o GTP binding proteins (G proteins) and has seven
putative transmembrane alpha helices. Recently two
forms of the D, dopamine receptor (D-,
and D-)
were identified by gene cloning and are produced by
alternative splicing [I]. The two subtypes differ by
a 29 amino acid insert in the third intracellular
loop.
Amino acid residues that are important for binding
ligands t o the D, dopamine receptor can be identified
from site directed mutagenesis studies and, from the
effects of pH changes and chemical modification. The
information will be useful f o r the design of more
selective drugs that bind t o the receptor.
It has been previously shown [ 2 J that a shift in pH
from 7.4 t o 6.0 affects the binding of antagonists
such as the substituted benzamide drugs t o a greater
extent than the classical antagonists such as
spiperone in binding to bovine caudate nucleus
membranes, a rich source of D, dopamine receptors.
Recently, data have been published on the pH
dependency of ( - ) sulpiride binding t o D, dopamine
receptors in bovine brain 131, where it was shown
that the binding of this substituted benzamide drug
involved an ionising group of the receptor whose pK,
was 7.3.
Similar studies were carried out here with a range
of substituted benzamide drugs D0710, clebopride,
raclopride and YM 09151-2, and also with the
classical antagonists of the D, dopamine receptor,
spiperone and haloperidol.
This was performed t o
compare the binding of the classical antagonists with
the substituted benzamide drugs which appear t o
behave differently.
The pH dependency of [%I] spiperone binding was
determined by performing saturation experiments at
different pH values (8.0 t o 5.5).
This involved
incubating approximately 1OOpg of bovine caudate
nucleus membranes
with a range of [%IJspiperone
concentrations and determining the amount of ligand
bound at each concentration. Incubation was achieved
at 25OC for 45 minutes before the membranes were
harvested by filtration using GFfB filters.
Non
specific binding was defined by the addition of 3pM
(+) butaclamol and mianserin was also added t o block
serotonin 5HT, receptors. The pH dependency of the
substituted benzamides and haloperidol was determined
by performing Competition assays with ['HJspiperone
at the different pH values.
In this case, the
membranes were incubated with a range of drug
concentrations and a single concentration o f
['HJspiperona (ligand). As the concentration of drug
increases, the amount of ligand bound decreases and
the Ki (inhibition constant) value is obtained from
The pK, of the ionising
the competition curve [2-3 J
groups is determined by usingthe following equation:
Ki,ob, = Ki ( 1 + [H+J/K, )
[21
The results indicate that the substituted benzamide
drugs D0710, clebopride, raclopride and YM 09151-2
bind t o bovine caudate nucleus membranes, where the
interaction involves an ionising group whose pK, is
7.02, 6.98, 6.74 and 6.88 respectively
The binding
of
the
classical
antagonists
spiperone
and
haloperidol t o the same membranes involved an
ionising group whose pK.
is 6.17
and
6.33
respectively
In summary, the binding of substituted benzamide
drugs to the D, dopamine receptor involves an
ionising group whose pK, is approximately 7.0,
whereas the binding of the classical antagonists,
spiperone and haloperidol t o t h e receptor involves an
ionising group whose pK, is about 6.0.
From site directed mutagenesis studies of the padrenergic [ 4 ] and ml muscarinic acetylcholine
receptors 151, conserved aspartic acid residues have
been shown to be involved in ligand binding.
In
particular an aspartic acid residue in the third
putative transmembrane region has been found t o be
important for ligand binding. This would correspond
.
.
.
t o aspartic acid 114 (asp 114) of the D, dopamine
receptor, and may be a candidate for interacting
electrostatically with the classical antagonists, and
may correspond to the ionising group of pK, about
6.0.
The nature of the group of pK, of about 7.0
that seems to affect the binding of the substituted
benzamide drugs is unclear.
However, mutation of
aspartic acid 80 (asp 80) in the second transmembrane
region of the D, dopamine receptor decreases the
regulation of the affinity of the D, receptors for
substituted benzamide antagonists by sodium and pH
161. This suggests that aspartic acid 80 may
correspond t o the ionising group.
We acknowledge financial support from the Medical
Research Council.
1. Giros, B., Sokoloff, P., Martres, M . , Riou, J.,
Emorine, L.J. & Schwartz, J. (1989) Nature. 341.
923-926
2. Williamson, R.A. & Strange, P.G. (1990)
J.Neurochem. 55, 1357-1365
3. Presland, J.P. & Strange, P.G. (1991) Biochem.
Pharmacol. 41, R9-Rl2
4. Strader, C.D., Sigal, I.S. & Dixon, R.A.F. (1989)
FASEB J. 3, 1825-1832
5. Fraser. C.M., Wang, C., Robinson, D.A., Gocayne,
J.D. & Venter, J.C. (1989) M o l . Pharmacol. 36,
840-847
6. Neve, K.A., Cox, B.A., Henningsen, R.A.,
Spanoyannis, A. h Neve, R.L. (1991) M o l .
Pharmacol. 39, 733-739