PDF hosted at the Radboud Repository of the Radboud University
Nijmegen
The following full text is a publisher's version.
For additional information about this publication click this link.
http://hdl.handle.net/2066/16758
Please be advised that this information was generated on 2015-01-23 and may be subject to
change.
Biochimica^*
et Biophysica Acta
ELSEV IER
Biochimica cl Biophysica Acta 1308 (1996) 1 7-22
Short sequence-paper
Cloning and sequence analysis of a hypothalamic cDNA encoding a D k
dopamine receptor in tilapia 1
Anne E. Lamers \ Diet Groneveld a, Dominique P.V. de Kleijn Felix C.G. Geeraedts \
Jack A.M. Leunissen b'*, Gert Flik \ Sjoerd E. Wendelaar Bonga d, Gerard J.M. Martens a
Department o f Animal Physiology, Faculty o f Science, University o f Nijmegen. Toernooivelcl, NL-6525 ED Nijmegen, The Netherlands
b CAOS / CAMM centre. University o f Nijmegen, Toernooiveld, NL-6525 ED Nijmegen, The Netherlands
Received 3 January 1995; revised 9 April 1996; accepted I I April 1996
Abstract
Physiological and pharmacological studies have indicated that during acid stress a D|-like dopamine receptor becomes functional on
intermediate pituitary melanocyte-stimulating hormone cells of tilapia ( Oreochromis mossambicus). As a first step towards physiological
expression studies we isolated a D ,-Iike dopamine receptor from a tilapia hypothalamus cDNA library. Construction of a phylogenetic
tree of most of the D,-like receptors known in human, rat, Xenopus, goldfish and Drosophila revealed that the here presented clone is
most likely the tilapia equivalent of the Xenopus D k. dopamine receptor.
Keywords: Cloning; Sequence analysis; Dopamine receptor D k ; (Hypothalamus); ( O. mossambicus)
types o f
dopam ine receptor activity w'as induced in the a -M S H -
dopam ine receptors, D ,-lik e and D^-like [1]. D ,-1 ike recep­
producing cells o f the tilapia NIL when the fish were
tors are coupled to a stimulatory G-protein to effect a
ex p o sed
stim ulation o f a secon d m essen g er system in the cell (see
receptor has a higher affinity for dopam ine than the D 2-like
review Ref. [2]). D^-like dopam ine receptors are generally
receptor present in the NIL. The activation o f these D ,-1 ike
coupled to an inhibitory G-protein enabling the inhibition
and D^-like receptors appears to result in stimulation o f
o f a secon d m essenger. The D ,-like and the D :-like recep­
a -M S H release at picom olar concentrations o f dopam ine
tors can be distinguished pharm acologically using sp ecific
but in inhibition at nano- to m icrom olar dopam ine c o n c e n ­
D , and D 2 agonists and antagonists. Receptor su b classes
trations. A s a first step towards a study o f dopam ine
o f the D ,-1 ike and D :-like receptors have been identified at
receptor expression in the tilapia pituitary gland, w e here
the m olecular level: D^-like receptors have been classified
describe the isolation and sequencing o f a hypothalam ic
into D : , D 3 and D4; D , - like into the subtypes D , / D la and
c D N A clone en cod in g a tilapia D ,-1 ike dopam ine receptor.
D 5/ D lb [2,3]. R ecently an additional D ,-lik e receptor sub-
C on stru ction o f the tila p ia h y p o th a la m ic cD N A lib rary.
D opam ine
signals are transduced
via tw o
type w'as lound in the X e n o p u s, termed D k. [4],
The regulation o f release o f a -m ela n o cy te-stim u la tin g
for 7 days to acid water (pH 4 .5 ) [7]. This
A tilapia c D N A library w as constructed from about 4 jxg
hypothalam ic p o ly (A )
R N A o f fish raised in fresh water
horm one (u - M S H ) from the pituitary neurointermediate
(pH 7.8), using the \ Z A P - c D N A synthesis kit (Stratagene,
lobe (N IL ) ot tilapia ( O re o c h ro m is m o s sa m b ic u s ) during
see also Ref. [ 8 ]). R N A w as isolated by the acid guani-
adaptation to acidified water has been described previously
d in iu m -th io c y a n a te /p h en o l-c h lo r o fo rm procedure [9] and
[5,6].
subsequently p o ly (A ) '
Pharm acological
studies
revealed
that a D ,-like
R N A was purified with an o lig o
(dT) cellu lo se colum n (Stratagene) according to the m anu­
facturers instructions. c D N A
Corresponding author.
O. mossambicus m R N A lor D, / D 5 dopamine receptor accession
number in E M B L Nuceotide Sequence Database: X81969.
0 16 7 -4 7 8 1 / 9 6 / $ 15.00 © 1996 Elsevier Science B.V. All rights reserved
PH SO I 6 7 - 4 7 8 I ( 9 6 ) 0 0 0 8 6 - 3
w as syn th esized using an
o lig o n u cleo tid e containing a poly(dT ) sequ en ce and an
X h o \ restriction site. E c o RI adaptors were ligated and the
c D N A was directionally cloned into E c o R \-X h o \ sites o f
18
A.E. Limiers et al. / Biochimica et Biophysica Acta 1308 ( 1996) 17-22
5 ' -----(* 1 5 5 0 ) GGATCCGCATCAGACACACCTGAGC
ATGAAGACCGCGGGAAACCTTCAGATTTCAGTCAAGAACGACACAGACGAATTACTGCCCAGCTGATCCCGnmft
GCGTCACGGTCATACAGGCAACCGAGACGACACTGAGAGACCCTCACGGCACAGACAGGTGTGGGCAGGCGCAGG
TCGTGACGTCTTTGGCACATTAAAAACATAAAGAAACTGAAGCAAACGAGCTCCGCTGGACTTTCTTCAGTCCAG
ATGTTCACTTCAGCCTGGAGGTGATTTTTGTTTGTTTTTTACAGACCGAACATTTGAGTGTGCGTTTCACGTGTG
CTATTAAGTGTTGGAGTGCTCTGAGCTCCCGTGGATTAAGTATACCCGAGGGAGGGAGGGACCCTCTTAGTGTCC
19
Met Glu Ile Phe Thr Thr Thr Arg Gly Thr Ser Ala Gly Pro Glu Pro Ala Pro Gly
ATG GAG ATT TTT ACA ACG ACA CGC GGA ACC AGC GCA GGA CCA GAG CCA GCA CCC GGT
Asp Leu Ser Leu Arg Ala Leu Thr
GAC CTC AGC CTC CGC GCG CTC ACC
57
Leu Leu Gly Asn Ala Leu Val Cys
CTT CTG GGG AAC GCG CTG GTG TGC
74
Lys Phe Arg His Leu Arg Ser Lys Val Thr Asn Ala Phe Val lie
AAG TTC CGC CAC CTG CGC TCC AAA GTC ACT AAT GCC TTC GTC ATC
95
TM
Ser Asp Leu Phe Val Ala Val Leu Val Met Pro Trp Arg Ala Val
TCC GAC CTG TTC GTG GCC GTG CTC GTG ATG CCG TGG AGG GCC GTG
.111
Gly Val Trp Leu Phe Gly Ala Phe Cys Asp Thr Trp Val Ala Phe
GGC GTC TGG CTG TTC GGC GCT TTC TGC GAC ACG TGG GTG GCT TTC
133
TM III
Ser Thr Ala Ser Ile Leu His Leu Cys lie lie Ser Met Asp Arg
TCC ACG GCC TCC ATC CTC CAC CTG TGC ATC ATC AGC ATG GAC CGC
152
Ser Ser Pro Phe Arg Tyr Glu Arg Arg Met Thr Pro Arg Phe Gly
TCC AGC CCG TTC CGC TAC GAG CGC AGG ATG ACG CCG AGG TTC GGC
111
TM[ IV
Gly Val Ala Trp Thr Leu Ser Val Leu lie Ser Phe lie Pro Val
GGC GTG GCG TGG ACG CTG TCT GTG CTT ATC TCC TTC ATC CCC GTG
190
His Ala Arg Gly Arg Glu Arg Thr Asp Pro Gly Asp Cys Asn Ala
CAC GCG CGC GGG CGA GAA CGC ACG GAC CCC GGG GAC TGC AAC GCG
209
__TM, V
Thr Tyr Ala lie Ser Ser Ser Leu lie Ser Phe Tyr lie Pro Val
ACC TAC GCC ATC TCC TCC TCC CTC ATC AGC TTC TAC ATC CCC GTC
228
Gly Thr Tyr Thr Arg lie Phe Arg lie Gly Arg Thr Gin lie Arg
GGC ACG TAC ACG CGC ATC TTC CGC ATC GGC CGC ACG CAG ATC CGG
247
Leu Glu Arg Ala Ala Pro Arg Ala Thr Arg Gly Pro Ala Leu Cys
TTG GAG AGG GCT GCG CCG CGC GCG ACA CGC GGC CCC GCG CTC TGC
266
Ser Leu Lys Thr Ser Phe Arg Arg Glu Thr Lys Val Leu Lys Thr
TCG CTG AAG ACT TCC TTC CGC CGC GAG ACC AAA GTG CTG AAG ACG
285
TM
Met Gly Val Phe Val Phe Cys Trp Leu Pro Phe Phe Val Leu Asn
ATG GGC GTG TTC GTG TTC TGC TGG CTG CCG TTC TTC GTG CTC AAC
304
Phe Cys Arg Leu Glu Pro Ala Ala Ala Pro Cys Val Ser Asp Thr
TTC TGC CGC CTG GAG CCT GCG GCC GCG CCG TGC GTC AGC GAC ACC
323
VII
Phe Val Trp Phe Gly Trp Ala Asn Ser Ser Leu Asn Pro Val lie
TTC GTG TGG TTC GGC TGG GCC AAC TCG TCC CTG AAC CCG GTC ATC
342
Ala Asp Phe Arg Lys Ala Phe Ser Thr H e leu Gly Cys Ser Arg
GCC GAC TTC CGG AAG GCC TTC TCC ACC ATC CTG GGC TGC AGC CGC
361
Ser Ala Val Glu Ala Val Asp Phe Ser Asn Glu Leu Ala Ser Tyr
TCG GCG GTG GAG GCG GTG GAC TTC AGC AAC GAG CTG GCG TCC TAC
380
Thr Leu Gin Lys Glu Ala Ser Ser Arg Gly Asn Ser Arg Gly Gly
ACC CTG CAG AAG GAA GCG TCG TCG CGC GGG AAC TCG AGG GGG GGC
Gly His Gly Gly Thr Asp Ser Pro Arg Thr Ser
GGT CAC GGC GGC ACG GAC AGC CCG CGC ACG AGC
__TM
Gly Cys Val Leu Cys lie Leu lie Val Ser Thr
GGA TGC GTC CTG TGC ATC CTG ATC GTG TCC ACG
Ala Ala Val lie
GCC GCC GTC ATC
Ser Leu Ala Val
TCT TTG GCC GTG
Ser Glu Val Ala
TCC GAG GTG GCC
•
Asp H e Met Cys
GAC ATC ATG TGC
Tyr Trp Ala lie
TAC TGG GCC ATC
Cys Val Met lie
TGC GTG ATG ATC
Gin Leu Asn Trp
CAG CTC AAC TGG
Ser Leu Asn Arg
AGC CTG AAC CGC
Leu lie Met Val
CTC ATC ATG GTG
Arg lie Ser Ser
CGG ATC TCC TCG
Asp Glu Glu Ser
GAC GAG GAG AGC
Leu Ser Val lie
CTG TCG GTC ATC
Cys Met Val Pro
TGC ATG GTT CCG
Thr Phe Ser Val
ACG TCC AGC GTG
Tyr Ala Phe Asn
TAC GCC TTC AAC
Tyr Cys Arg Thr
TAC TGT CGC ACC
His His Asp Thr
CAC CAT GAC ACC
Pro Tyr Gin Phe Ala Leu *t*
CCG TAC CAA TTC GCC CTA TAG TGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACT
GGGGAAAAACT ("950 bp) GGTAGAATAACATGTGATGTGATGGCCAAAAAAAAAAAA -3’
Fig. 1. Nucleotide sequence and deduced amino acid sequence o f hypothalamic c D N A clone p T D A l encoding the tilapia D k. dopamine receptor
Numbering starts at the putative initiation methionine and ends at the termination codon. The positions o f the transmembrane (TM ) regions are overlined
Arrowheads indicate putative glycosylation sites, the termination codon is indicated with asterisks and the polyadenylation signal is underlined.
A.E. Laniers et al. / Biochimica et Biophysica Acta 1308 ( I W6 ) 17-22
19
an U n i-Z A P XR vector. The resulting library contained
obtained with the programs
approx. 2 X 105 independent c lo n e s and w as am plified
[17,18].
according to standard procedures [ 10 ].
n eig h b o r
[16] and
protpars
Isolation a n d se q u e n c e a n a ly sis o f h y p o th a la m ic cD N A
S creen in g o f the cD N A lib ra ry. Approx. 1 0 0 0 0 0 recom ­
binants o f the tilapia hypothalam ic c D N A
library were
en co d in g a tila p ia D ,-lik e d o p a m in e re c e p to r. Screening
o f approx. 100 000 recom binants o f the am plified tilapia
screened using a human D, dopam ine receptor g en e probe
hypothalam ic c D N A
( h D ,3 z ) [I I ]. The probe w as " P -la b e lle d by random prim­
three
ing according to standard procedures [10]. After pre-hy­
analysis revealed that the three c lo n es contained the sam e
bridization
mM
4.7 kb insert. One clon e ( p T D A l ) w as used for further
sodium phosphate-buffered (pH 7.4), 25% form am ide, 6 X
analysis. A n alysis o f the nucleotide seq u en ce o f the pT D A 1
SSC [1 X
sodium
clon e revealed an open reading frame (O R F ) cod in g for a
citrate], 0.1% sodium dod ecyl sulfate (S D S ), 100 fJig/m l
protein o f 368 am ino acids (Fig. 1). Seven putative trans­
denatured herring sperm D N A , 0.1% polyvin ylp yrrolid on e,
m em brane regions, characteristic for G-protein coupled
1 mM E D T A and 2 X Denhardts solution [I X Denhardts
receptors,
solution is 0 . 1% p olyvinylpyrrolidone, 0 . 1% b ovin e serum
contained an extrem ely long 5' n on -cod in g region o f ap­
albumin and 0.1% Ficoll 400], filters were incubated with
prox. 2.2 kb and a 3' n on -cod in g region o f about 1.3 kb.
in
hybridization
SSC = 150 mM
buffer containing
NaCl and
40
15 mM
the probe in hybridization buffer at 42°C. A fter
library resulted in the isolation o f
hybridization-positive
may
phage
be assigned
plaques.
Restriction
to the protein. The clone
18 h,
The deduced am ino-acid sequ en ce o f p T D A l sh ow ed
filters were w ashed tw ice for 30 min with 2 X S S C at
high similarity to the D ,-lik e dopam ine receptors (Table
room temperature, 0.1% S D S and tw ice for 30 min with
I ). The degree o f am ino-acid seq u en ce identity betw een
2 X
the putative G-protein coupled receptor and the h u m a n /r a t
SSC,
0.1%
SDS,
at 56°C.
H ybridization-positive
clo n es were purified, and Bluescript D N A was prepared by
D , / D la, the h u m a n /r a t D 5/ D lh and the
in v iv o ex cisio n according to the Stratagene protocol.
receptors is about 66 %, 65% and 74%, respectively. The
DNA
se q u e n c e
a n a lysis.
DNA
seq u en cin g
of
both
am ino-acid
seq u en ces
of
the
tw o
X en o p u s D k
human
stimulatory
strands w as perform ed with T 7 D N A polym erase and the
dopam ine receptors (D , and D 5) sh o w about 65% identity,
d id eoxy chain termination m ethod [ 12 ], using su b clon es
w hereas the stimulatory human
and synthetic o lig o n u cleo tid es. S eq u en ce alignm ents were
human D , receptor sh ow only 29% identity [19]. From this
performed according to N eed lem an and W unsch [13] using
w e con clu d e that the tilapia clon e en co d es a stim ulator}'
the G A P program o f the G C G program package [14].
( D | -1 ik e ) dopam ine receptor, most likely o f the D k sub-
C on stru ction o f the p h y lo g e n e tic tree. The am ino-acid
sequence o f the tilapia D ,-1 ike receptor w as aligned with
D,
and the inhibitory
type. W e further term the here presented clo n e tilapia D k
GDic).
the
The 5'-untranslated region o f the t D k receptor contains
databases S W IS S P R O T and EM BL: gold fish D , (g fD ,;
several small O R Fs o f w hich the four m ost downstream
accession No. P 3 5 4 0 6 ), human D , ( h D l ; P 2 I 7 2 8 ), human
are show n (Fig.
D 5 (hD 5; P 2 1918), rat D lu (r D la ; P 18901), rat D lb (r D lb ;
bases and the fourth o f 24 bases. A lthough small O R Fs are
P 2 5 1 1 5 ) the X e n o p u s D hl ( X D l a ; X 1 0 7 8 6 3 ),
X enopus
generally rare in vertebrate m R N A , they are not u n com ­
D lb ( X D lb ; X I 0 7 8 6 4 ), X en o p u s D Ic ( X D I c ; X I 0 7 8 6 5 ),
mon in m R N A s o f receptor gen es, p roto-on cogen es and
D, 5
grow th-control g en es [20,21]. The function o f the small
( d m D ,/ 5 ; X 7 7 2 3 4 ). Invariant positions were deleted from
O R Fs is yet unknown, hut a role in the regulation o f
the alignm ent, 1000 bootstrap sam ples were created using
translational initiation o f the main O R F has been suggested
the
[22,23]. It is not clear whether the sm all upstream ORFs
the
and
fo llo w in g
the
dopam ine
translated
seqboot
receptors
D r o s o p h ila
obtained
from
m e la n o g a s te r
[15] program, and p h ylog en etic trees were
1). The first three O R Fs consist o f 66
Table 1
Amino-acid sequence identities in percentages, between the human D s (hD5). rat D jh (rD lb ), Xenopus D !t,. human D, ( h D , ). rat D, ( ( r D k ). Xenopus D u
( x D la ), goldfish D, ( g f D ( ), Xenopus D k ( x D lc ), tilapia D k ( t D l e ) and Drosophila D , / s ( d m D l / 5 )
hD5
hD5
rD lb
xD 1b
hDl
rD la
xD la
gIDI
xD lc
tD Ic
dm D 1/ 5
100
rD lb
87.3
100
xD lb
75.5
75.7
100
hDl
63.4
64.5
65.7
100
rD la
62.8
62.6
64.9
90.4
100
xD la
64.5
65.4
67.9
83.2
82.7
100
gIDI
xD lc
65.7
65.4
68.3
68.9
68.3
76.3
76.0
78.6
100
71.3
68.6
67.4
69.9
71.8
100
tD I c
65.0
65.1
64.6
65.9
65.8
67.6
69.4
74.2
100
d m D l/5
38.2
38.1
39.3
37.2
36.4
38.0
40.3
40.8
37.1
100
A.E. Limiers et a l . / Biochimica et Biophysica Acta 1308 ( 1996) 17-22
G
A
R
G
E
E
D
G
G
E
T
A
D
S
S
S
s
G
G
V
G
AG A
A T •
Q R R
L VV
L? A
L L T
F M •
S
S
S
S
S
S
S
S
S
A
DL
D L
DL
DL
DL
DL
DL
D L
DL
DL
F
F
L
L
L
L
L
F
F
F
V
V
V
V
V
V
V
V
V
V
ALLVMP
ALLVMP
ALLVMP
AVLVMP
AVLVMP
AVLVMP
AVLVMP
ALLVMP
AVLVMP
ASLVMT
P
P
P
P
P
P
P
P
P
P
I WT L L
V WT L L
F WT L F
L E T L L
L S T L L
L S T L L
L S T L L
L S T L L
L C I L I V S T L L
F L S VA
WKAVAEVAGY
WKAVAEVAGY
WKAVAEVAGH
WKAVAE I A G F
W K A V A E I AGF
WKAVAE I A G F
WKAVTEVAGF
WKAVTEVAGF
WRAVSEVAGV
FAGVNDLLGY
HPFGA
WP F G T
WP F G A
WP F G S
WP L G P
WP F G T
WP F G A
WVF G D
WLF G A
WI F GA
FRYKRKMTQR
FRYERKMTQR
F RYERKMT QR
F R Y E R K MT P K
F QYERKMTPK
F RYE RKMT P K
F R Y E R K MT P R
FRYERKMTQR
F R Y E R R M T P R
LRYGRWVTRR
MA
V A
V A
AA
AA
V A
VA
V A
F G
VA
L
L
L
F
F
F
F
F
C
V
V
V
L
I
I
I
V
I
V
I
MV
MV
MI
L I
Lï
MI
MI
MI
MI
T I
W
W
W
W
W
W
W
W
W
W
T
T
A
T
T
T
T
T
T
L
L S I L I
L S I L I
L S V L I
L S V L I
L S V L I
L S V L I
L S V L I
L S I L I
L S V L I
L A A F V
NLANWTPWEE
. L S N G T P W E E
.....................................E
D F WE P D V
G . WE L E G
D HL L S N H
E T
S L E DT
. L HD R
S R R D L
L NG
N
• .
G K
N
N
N
N
N
N
N
N
D
T
D
D
D
D
D
D
D
D
N
A
S
S
S
S
T
S
S
S
A
L
S L H RT Y
S L N R T Y
S L N R T Y
S L S R T Y
RL S R T Y
S L N R T Y
S L NR T Y
S L NR T Y
S L NR T Y
DL T P T Y
A
A
A
A
A
A
A
A
A
A
I S
I
I S
I
I S S S L I
I S S s V
I S s S L I
I S s S L I
I S s S L I
I S s S L I
I S s S L I
VV s S C I
Q
Q
0
Q
Q
R T Q
Q K H
IRR
IRR
I KR
IRR
IRR
IR R
IR R
IRR
IRR
V KS
IS S L E
IS S L E
IST LE
I AALE
ISALE
ISALE
ISALE
IS S LE
IS S L E
I KA V T
R A
RA
RA
RA
RA
RA
RA
RA
RA
R P
EHA . . . . . Q
E H A . . . . .Q
E B A . . . . .Q
VH A . . . . . K
V BA . . . . . K
V HA . . . . . K
E S A . . ... Q
V E H A . . ... Q
A P R A . . ... T
G E VAEKQRYK
S •• •• S
S •• • • R
S N RVDS
T T T G N G
T T AG N G
QN S T S N R
I R H D S MG S G
RC S S R L S N
R G P A LC DE
SIR R P K N Q P
VFV
VFV
VFV
VFV
VFV
VFV
VFV
V F V
VF V
V F L
CCWL
CCWL
CCWL
CCWL
CCWL
CCWL
CCWL
F C WL
FCWL
I C W V
P
P
P
P
P
P
P
P
P
P
F
F
F
F
F
F
F
F
F
F
I L N C M V P F C .
I L N C M V P F C S
I L N C M V P F C D
I L N C I L P F C G
I S N C M V P F C G
I LNC I V P F C D
I L N C M V P F C K
V L N C M I P F C H
VL NCMVP F CR
C V N I T A A F C K
s G HP E GP P AG
s G DA E G P KT G
F
F
F
F
F
F
F
F
F
F
NAD
NAD
NAD
NAD
NAD
NAD
NAD
NAD
NAD
NKE
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
F
AQ
AQ
S S
S T
S T
S N
A I
T T
S T
KR
L
L
L
L
L
L
L
I
I
I
L
L
L
L
L
L
L
L
L
L
G
G
G
G
G
G
G
G
G
T MR
A
A
V
N
N
N
V
V
V
L
L
L
G N R E V DN
G D R E V G E
•
•
•
•
•
•
I
L I P HAV G
L I P HAV G
L I P HA I I
D
E
D
S
S
C
E
E
D
S
S
P
E
E
N
E
E
B
. . . M L P P G S N
. G T A Y P G Q F A
LYQQLAQGNA
. . . MLPP GRN
. R T A Q P A R L G
LQRQLAQVDA
. . . MYQP F QH
LDSDQVASWQ
S P E ML MNKS V
...................................................................................................... M R T L N T S A M
........................................................................................................... H A P S T S T M
........................................................................................................... M T F N I T S M
.........................................................................................M A V L D L N L T T V
...................................... M E N F S I F N V T V
NVWHADLDVG
...................................... M B I F T T T R G T S
A G P E P A P G G H
Q L L T N G Y D G T
T L T S F Y N E S S
WT N A S E MD T I
hD5
rDlb
xDlb
hDl
rDla
xDla
g f DI
xDlc
tD lc
dmDl-5
G
G
G
G
G
G
G
G
G
G
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmDl-5
WVAFDI MCST
W V A F D I M C S T
W V A F D I M C S T
W V A F D I M C S T
WV A F D I MC S T
WV A F D I MC S T
W V A F D I M C S T
WV A F D I MC S T
WV A F D I MC S T
WVAFDVMCST
A
A
A
A
A
A
A
A
A
A
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmDl-5
VQLNWHRDQA
VQLNWHRDKA
V Q L S WH K S E T
VQLSWHKAKP
VQLSWBKAKP
VQLNWHKAKT
VQLKWHKAQP
VQLSWBKSH .
VQLNWHARG .
I S L G I H R P D Q
AS WGGLDLP N
GS QGQE GL .
P
P
F
F
S
L
F
L
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmD1 - 5
IPVAIMIVT Y
IPVAIMIVT Y
IPVAIMIVT Y
IPVAIMIVT Y
IPVAIMIVT Y
IPVAI MIVTY
IPV A IMIVTY
IPVVIMIGT Y
IPVLIMVGTY
FPCVVMIGI Y
TRI
TRI
TRI
TRI
T S I
TRI
T Q I
TRI
TRI
CR L
Y
Y
Y
Y
Y
Y
Y
Y
F
Y
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmDl-5
L
L
L
F
F
L
F
L
L
S
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmDl-5
VFVWFGWAN
I FVWFGWAN
I FVWFGWAN
VFVWFGWAN
VFVWFGWAN
VFVWFGWAN
VFVWFGWAN
I FVWFGWAN
VFVWFGWAN
I L T W L G Y S N
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmDl-5
Q
Q
Q
.
.
.
H D T T F Q K D I P
HDTTLQKE • .
AKNVVVMNSG
VT F NNS HL P K
VVDQDQEVLE
R S S A E L E Q V S
A I .
hD5
rDlb
xDlb
hDl
rDla
xDla
g f D1
xDlc
tD lc
dmD1 - 5
D
D
D
D
D
D
I
I
I
I
I
I
N
N
N
N
N
N
N
N
N
N
R
R
R
K
K
K
K
K
K
S
VL VC AAI V
V L V C A A I V
I L V C T A V M
T L V C A A V I
T L V C A A V I
T L V C A A V I
T L VC AAVT
T L V C L A V I
A L V C A A V I
I L V C L A I Y
AS
AS
T S
MS
MS
T S
L S
T S
T S
P Y
D
D
D
.
.
.
C
C
S
Y
Y
Y
I K
I K
I K
F K
F K
F K
F K
F R
F R
HV
K
K
K
R
R
R
R
K
R
S
E T
E T
B T
E T
E T
E T
E T
E T
E T
D H
L N L C V
L N L C I
L N L C V
L N L C V
L N L C V
L N L C V
L N L C V
L NL C I
L H L C I
L N L C A
D
D
D
E
E
R
P L I F E
T
T
T
I
S
W
S
G
I
I
I
I
I
I
I
I
I
I
R
R
R
R
R
R
R
R
R
C
G
G
L
V
A
E
D
I S
I S
I S
I S
I S
I S
I S
I S
IS
I S
NAT
N
N
N
D
R
R
F
I
.
E
T
T
T
A
E
D
IA
IA
IA
IA
IA
IA
IA
IA
IG
YA
Q
Q
Q
Q
Q
VQ
VQ
I Q
K
K
AK
Q K
Q T
K
K
K
K
K
K
K
K
K
K
V
V
V
V
V
V
V
V
V
A
L K T L S V I H G
F K T L S M I M G
L K T L S I I MG
L K T L S V I M G
L K T L S V I MG
L K T L S V I M G
L K T L S V I M G
L K T L S I I MG
L K T L S V I M G
A V T V G V I MG
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
L
L
L
L
L
I V F H K E I A
T V F H R E I A
T L F H K D I V
AMF S S HHE
V VF S S HHB
VVYSCQQE
E G E I S L D
B E E V S L G
E V D I S L H
D T D V S L E
D T D V S L E
D A u V S L E
R S R H L R A N MT
R S R K L R A K MT
R F R H L R S R V T
R F R H L R S K V T
R F R H L R S R V T
R F R H L R S K V T
K F R H L R S K V T
K F R H L R S K V T
K F R H L R S K V T
T D G S L . P R I G
S
S
S
S
S
S
S
S
S
S
K
K
K
K
K
K
E C E A E I T L E T
N
N
N
H
N
S LU
S
S
S
A
L
L
L
F
N
N
N
N
A
T
T
P
P
P
A
A
A
R
R
K
Y
Y
Y
G
G
G
P
P
P
P
P
P
P
P
P
P
V
I
I
I
I
I
I
V
V
I
I
I
I
I
I
I
I
I
I
I
I
V
V
S
S
H
H
N
I
I
I
MMP
MI P
MI P
S KE
S KD
P N E
s
V
P
S
D
D
D
I
N
G
V
G P S Q V V T A C L
GP AQVVT AGL
V A G Q I V T G S L
F S VR I L T A C F
F S F R I L T A C F
S S F R V L T G C F
R S V R V L T G C F
L B L S A L T G L L
L S L R A L T G C V
VSI . V V V G I F
hD5
rDlb
xDlb
h Dl
rDla
xDla
g f DI
xDlc
tD lc
dmDl-5
YA •
YA •
YA •
YA •
YA •
YA •
YA •
YA •
YA •
YS I
N
N
N
C
C
C
T P F T P N G F H
S P L T P N C F D
T P S M S N G I H
QP I TQNGQH
Q P V T H S G Q H
N P I T Q N G Q P
I T P F T S T G P L
N
N
N
N
H
N
N
N
H
N
V
I
I
F
F
F
F
F
A
L
F
F
F
F
F
F
F
F
F
F
I
I
I
V
V
V
V
V
V
L
V
V
V
I
I
I
I
I
I
A
S
S
S
S
S
S
S
S
S
S
LAV
LAV
LAV
LAV
LAV
LAV
LAV
LAV
LAV
L A I
V D R Y W A I S R
V D R Y W A I S R
V D R Ï t î A I S S
V D R Y W A I S S
V D R Y W A I S S
V D R Ï t î A I S S
V D R Y W A I S S
L D R Y W A I A S
MD R Y WA I S S
MDRYI HI KD
T
S
D
V
V
V
P
S
C
Y
Y
Y
Q
R
R
R
Q
R
R
R
R
R
K
K
K
K
K
K
R
K
K
D
V
V
V
A
A
A
A
A
A
A
F
F
F
F
F
F
F
F
F
F
P P
•
P
KE
E R
E R
E R
B S
S
G T D S P R T S
G E E P E P L s
SLA
NA E
RT E
S T G
I D
E DD
T MD
P T D
HT E
. P G
KY P
A
A
A
A
A
A
A
E
E
E
D
D
D
C
C
C
C
C
C
C
C
C
N
S
S
G
Y
Y
Y
Q
N
S
P
HF C
H F C
H WC
R L C
R L C
R L C
R L C
R F C
R YC
WC C
G P F D
GP F D
DA F D
•
•
L K
•
•
L K
E V L K
R
H
H
K
K
K
M
M
M
E
E
E
GT C F D KV S VL
A S S R
KT A
P T
S T
KT
E C L P Q L V A D E
L
L
L
D
D
E
D
D
D
L
DRHYTTKLY
C
C
C
C
C
C
C
C
C
C
S
S
S
N
N
C
C
C
C
C
NC
G
G
S
S
S
G
S
G
G
A
L
L
T
V
V
V
G
V
V
A
A
A
A
A
A
A
A
A
A
I
R
R
R
Q
Q
R
S
S
p SL
R . .
M NL
L EP
P G HP Q
G E
E E
T T
S G
S N
.
P GQ N E
A A . .
T
.P
.P
.P
NA
NA
NI
S .
NN
SA
GN
G
C
K
N
N
S
K
A
T
T
T
G
P
G
Q
Q
E
L
E
V E T
V QT
V E T
I E T
I E T
I E T
ME T
V E A
V E A
I HP
V
V
V
V
V
V
P
V
V
R
T
T
T
T
T
S
S
.
.
D
F
S
S
E
E
D
I Y Q T S P D G
I S P T T P DG
I S Q T SANN
AG I A RP L B
GG I A KP L E
S G L S KS L E
S T S H G T R S Q K
G N S R G G P Y Q F
477
475
457
446
446
451
363
465
386
511
T L L I
T L L I
L L L I
S L L I
S L L I
S VL I
S V L I
LSLLI
LSVLI
.
.
.
.
.
.
.
.
.
Q
SSL
SSL
F
F
F
F
F
F
F
F
F
F
C
C
C
C
C
C
C
C
C
C
DV
D I
D I
N I
N I
N I
D I
DT
DT
DT
175
173
176
158
157
157
159
163
170
278
s
s
s
1s
s
s
s
s
s
s
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
235
229
217
204
204
203
205
205
206
320
T S
283
277
269
25 9
25 9
258
257
251
252
380
F
F
F
F
F
F
F
F
F
F
PS
T s
Ss
Ss
Ss
s s
KF KV R NL HT H
P
P
P
F
F
F
.
P
P
.
NI
NI
NI
SI
SI
SI
S L
N F
D F
N S
D
D
E
K
K
K
C
C
C
C
C
C
C
C
C
C
V
V
V
I
I
I
I
V
V
I
NN
N K
S N
DR
115
113
116
98
97
97
99
103
110
218
P
P
P
P
P
P
P
P
P
P
A C A . . .P D
A Y E . . .P D
S R H . • . HQ
PV BC S Q PE
P V E C AQ S E
S L DC QQ PE
N MD . . . L E
F
F
L
P
P
P
P
P
A
56
54
57
39
38
38
40
44
51
159
S F I
S F I
S F I
S F I
S F I
S F I
S F I
S F I
S F I
S F V
. .E N s
. .E S s
S R
FR
S T
P A
P T
PT
P G
SS
RT
AQ
Q
Q
Q
A
A
E
.
.
.
N
N
N
I
.
.
V
A
L
L
L
L
L
L
L
S E T T F D
S E T T F D
S E T T F D
DS NT F D
D S I T F D
S S T T F D
S P T T F D
S E T T P N
S DT T F S
GG QT F K
342
337
328
314
314
317
312
311
307
429
E L I S
E L I S
E L I S
NGA .
NGA .
NGA .
N . . .
E L VS
BLAS
F I T D
397
392
383
368
368
371
363
366
362
489
YN
YN
YN
•
•
•
•
•
•
•
•
Y H
YH
Y A
P V A E S V WE
L A A E S V WE
L A T D S MC E
L S P A L S V I
L S P A L S V I
MS P AF S G I
L
L
L
L
L
L
N L HL P AGVQF
A L . , . .
457
452
437
424
424
429
363
426
386
511
A.E. Limiers el al. / Biochimica et Biophysica Acta 1308 ( 1996) 17-22
21
highest identity (7 4 .2 % ) with the recently discovered third
D r like recep to r o f the X e n o p u s ( X D l c ) [4]. P hylogenetic
trees o f m e m b e rs o f the D ,-1 ike d o p am in e receptor family
w ere co n stru cted to verify the identity o f the t D , c receptor.
T he topologies o b tain ed by the n e i g h b o r and p r o t p a r s
p ro g ram s were essentially identical. T he co n sen su s tree o f
1000 bootstrap sam ples o f the p r o t p a r s program is show n
in Fig. 3. T he co n sen su s tree indicates that the D r o s o p h ila
D 1/5 r e c e p to r, the h u m a n
D , / r a t D la/ X e n o p u s
D la/ g o l d f i s h D, receptors, the hum an D s/ r a t D ]h/ X e n o ­
p u s D , b receptors and the X e n o p u s D k. / t i l a p i a D lc are
four significantly diverg ent groups within the family o f
D ,-1 ike d o p am in e receptors ( P = 1.00). The sequence o f
e m erg en ce o f the three subtypes from the ancestral D,
gene cannot be co n clu d ed from this tree.
Fig. 3. Phylogcnetic tree o f the D ,-Iike dopamine receptor family.
Numbers in branches indicate the bootstrap values calculated with the
p ro tp a rs
program.
are translated, because none o f the A U G triplets o f the
small O R F s are in a favourable con tex t for translational
initiation ( A / G N N A T G G is c o n sid ered the ideal context
for translational initiation [24]).
C o m p a riso n o f the t D l( r e c e p to r with D ,-lik e r e c e p to r s
o f o th e r sp ecies. Fig. 2 sh o w s a alig n m en t o f the am in o-acid
sequence o f the t D k receptor with those o f hum an D s, rat
D lh, X e n o p u s D lh, hum an D ,, rat D la, X e n o p u s D la,
goldfish D |, X e n o p u s D k. and D r o s o p h ila m e l a n o g a s t e r
D, 3. The highest degree o f identity is located in the
tran sm em b ran e regions, the first and second intracellular
loops, the first extracellular loop and in the regions ad ja­
cent to the tra n sm e m b ra n e regions VI and VII o f the third
intracellular loop and the cy to p lasm ic tail. The length of
the cy to p lasm ic tail o f the t D , / D s recep to r is interm ediate
co m p ared to that o f the goldfish D, [25] and those o f the
m am m alian D la and D lh receptors and the a m p h ib ian D lc
receptor [4,19,26]. P resum ably, this has no c o n seq u e n c e
for binding to the G -protein, as recent studies have re­
vealed that only the region o f the cy to p lasm ic loop nearest
to the tra n sm e m b ra n e VII is involved in G -protein c o u ­
pling [27,28].
The percentages o f overall seq uen ce identity betw een
the t D , c d o p am in e receptors and h D ,, h D s, r D Ia, r D Ib,
x D Ia, X D lh, x D lc, g fD , and d m D , 5 receptors are show n
in T able 1. T he t D lc receptor is related sim ilarly the
m am m alian D ]/la and D 5/ l b receptors, and sh o w ed the
References
[I
Stoof, J.C. and Kebabian. J.W. (1984) Life Sci. 35, 2 2 8 1 - 2 2 % .
[2 Civclli. O., Bunzow, J.R. and Grandy, D.K. (1993) Annu. Rev.
Pharmacol. Toxicol. 32, 2 8 1 - 3 0 7 .
[3 Gingrich. J.A. and Caron. M.G. (1993) Annu. Rev. Neurosci. 16,
299-321.
[4 Sugamori, K.S.. Demchyshyn, L.L., Chung, M. and Niznik. H.B.
(1994) Proc. Natl. Acad. Sci. USA 91. 10536-10540.
[5 Lamers, A.E.. Flik. G., Atsma, W.. W endelaar Bonga. S.E. (1992) J.
Endocrinol. 135, 2 8 5 - 9 2 .
[6 Lamers, A.E., Flik. G. and W endelaar Bonga, S.E. (1994) Am. J.
Physiol, (in press).
[7 Lamers, A.E.. Ter Bruuce, P.J.. Flik, G. and Wendelaar Bonsza. S.E.
(1994b) Endocrinology, submitted.
[8 Groneveld, D.. Eckhardt, E.R.M., Coenen, A.J.M, Martens, G.J.M,
Balm. H.P.M. and W endelaar Bonga. S.E. (1994) .1. Mol. En­
docrinol., accepted for publication.
[9 Chomczynski, P. and Sacchi, N. (1987) Annu. Rev. Pharmacol.
[10
[II
Toxicol. 32, 2 8 1 - 3 0 7 .
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular
Cloning: A Laboratory Manual (2nd Ed.), Cold Spring Harbor
University Press, Cold Spring Harbor, NY.
Zhou, Q-Y., Grandy. D.K.. Thambi, L.. Kushner, J.A.. Van Tol,
H.H.M., Cone. R., Pribnow, D.. Salon, J.. Bunzow, J.R. and Civelli.
[13
O. (1990) Nature 347, 7 6 - 8 0 .
Sanger. F., Nicklen, S., and Coulson. A.R. (1977) Proc. Nall. Acad.
Sci. USA 74. 5 4 6 3 - 5 4 6 7 .
Needleman. S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48. 4 4 3 -
[14
453.
Devereux. J., Haeberli. P. and Smithies, O. (1984) Nucleic Acids
[12
[15
[16
[17
Res. 12, 3 8 7 - 3 9 5 .
Felsenstein, J. (1985) Evolution 39, 7 8 3 - 7 9 1 .
Saitou. N. and Nei, M. (1987) Mol. Biol. Evol. 4. 4 0 6 - 4 2 5 .
Eck. R.V. and Dayhoff. M.O. (1966) Atlas o f Protein Sequence and
Structure. National Biomedical Research Foundation. Silver Springs,
Maryland.
Fig. 2. Alignment o f the amino-acid sequences o f human D_s (hD5). rat D )h (rD Ib). Xenopus D , h ( x D lb ). human D, ( h D l) . rat D , a (rD Ia), Xenopus D la
(xD Ia), goldfish D, (gfD I), Xenopus D k. ( x D lc ). tilapia D k. ( t D , c ) and Drosophila D, 5 ( d m D l / 5 ) dopamine receptors. The one-letter amino-acid code
is used. Black boxes indicate identical amino acids in all receptors, hatched boxes indicate conservative substitutions and gaps ( • ) are introduced to achieve
maximum similarity.
22
A.E. Lamers ei al. / Biochimica et Biophysica Acta 1308 (1996) 17-22
[18] Fitch. W.M. (1971) System. Zool. 20. 4 0 6 - 4 1 6 .
[19] Sunahara, R.K.. Ni/.nik. Fl.B.. Weiner. D.M.. Stormann. T.M.. Brann.
M.R.. Kennedy, J.L.. Gelernter. J.E.. Rozmahcl, R., Yang. Y.. Israel,
Y., Sccman. P. and O ’D ow d (1990) Nature 347. 8 0 - 8 3 .
[20] Kozak, M. (1989) J. Cell. Biol. 108. 2 2 9 - 2 4 1 .
[21] Marth. J.D., Overell. R.W., Meier. K.E., Krebs, E.G. and Perlmutter,
R.M. (1988) Nature 332, 171-173.
[22] Hunt, T. (1985) Nature 316. 5 8 0 - 5 8 1 .
[23] Martens, G.J.M., Molhuizen, H.O.F.. Gröneveld, D. and Roubos,
E.W. (1991) FEBS Lett. 281, 8 5 - 8 9 .
[24] Kozak. M. (1987) Nucleic Acids Res. 15, 8 1 2 5 - 8 1 4 8 .
[25] Frail, D.E.. Manclli, A.M.. Witte, D.G., Lin, C.W., Steffey, M.E.
and Mackenzie, R.G. (1993) Mol. Pharmacol. 44, 1113-1118.
[26] Sunahara, R.K., Guan, H.-C., O 'D o w d , B.F., Sccman, P., Lauricr.
L.G., Ng, G., George, S.R.. Torchia, J., Van Toi, H.H.M. and
Niznik, H.B. (1991) Nature 350. 6 1 4 - 6 1 9 .
[27] Savarcsc. T.M. and Fraser, C.M. (1992) Biochem. J. 283, 1-19.
[28] König, B. and Grätzel, M. (1994) Biochim. Biophys. Acta 1223,
261-266.