(Received for publication, July 24, 1995; and in revised form, July 31, 1995)
From the
To identify the endogenous ligands for a cloned orphan receptor
that shares high degrees of sequence homology with opioid receptors,
this orphan receptor was expressed in Xenopus oocytes and in
mammalian cell lines CHO-K1 and HEK-293. The coupling of the receptor
to a G protein-activated K channel was used as a
functional assay in oocytes. Endogenous opioid peptide dynorphins were
found to activate the K
channel by stimulating the
orphan receptor. This activation was dose-dependent, with EC
values at 45 and 37 nM for dynorphin A and dynorphin
A-(1-13), respectively. The dynorphin effect was antagonized by
the non-selective opioid antagonist naloxone but at rather high
concentrations in the micromolar range. Naloxone also caused a
rightward shift of the dose-response curve for dynorphin A, suggesting
a competitive antagonism mechanism. In transiently transfected cells, 5
µM dynorphin A-(1-13) inhibited the
forskolin-stimulated cyclic AMP increase by 51 and 35% in CHO-K1 and
HEK-293 cells, respectively. Other classes of endogenous opioids, i.e. enkephalins and endorphins, caused very little activation
of this receptor. These results suggest that this orphan receptor is a
member of the opioid receptor family and that dynorphins are endogenous
ligands for this receptor.
After the cloning of all three major types of opioid receptors,
µ, , and
(1) , a novel receptor was cloned from
several species by using a homology screening
technique(2, 3, 4, 5, 6, 7, 8) .
The amino acid sequence of this receptor is similar to those of the
opioid receptors. However, whereas the three opioid receptors share
about 70% amino acid sequence similarity among themselves, there is a
reduced homology level at about 65% between this receptor and any of
the opioid receptors(4) . This suggests that this novel
receptor may be a member of the opioid receptor family, different from
the other three receptors, and was thus designated various names
including XOR1(
)(4) . In vitro and in
vivo assay systems have been used to find its ligands. A synthetic
non-selective opioid agonist etorphine was shown to inhibit adenylyl
cyclase in CHO-K1 cells transfected with this receptor clone, and
diprenorphine and naloxone antagonized the inhibitory action of
etorphine(2) . However, since no endogenous ligands have been
found for this novel receptor, it remains an ``orphan''
receptor.
To identify endogenous ligands for an orphan receptor, one
could perform receptor binding with radiolabeled compounds. This
approach has been used for the identification of 5HT receptor(9) . However, this approach is limited in its
scope, since many endogenous ligands are not available in radiolabeled
form. An alternative approach is to use a functional assay, in which
the orphan receptor is expressed in cells and a measurable cell
function is used as a readout of receptor activation, such as changes
in second messenger levels or membrane currents. In this way compounds
can be tested in unlabeled form and, if a proper cellular function is
chosen that the orphan receptor does couple to, there is an opportunity
to identify the endogenous ligands.
Xenopus oocytes have
been used in many functional studies for membrane receptors and ion
channels(10, 11) . In particular, opioid receptors
have been shown to couple to a cloned G protein-activated K channel (KGA) in
oocytes(12, 13, 14, 15) . Because of
the high degree of homology of this orphan receptor with the opioid
receptors, it may also be capable of functionally coupling to KGA in Xenopus oocytes, thus constituting an assay system for
identifying endogenous ligands that can activate this receptor. We took
such an approach, using XOR1 cloned from rat brain (4) for
oocyte expression. Here, we report the results of this study.
Figure 1:
Coupling of the opioid receptor-like
orphan receptor (XOR1) to a G protein-activated K channel (KGA). In vitro transcribed cRNAs of XOR1 and
KGA were coinjected into oocytes. Functional coupling of the receptor
to the K
channel was detected by two-electrode voltage
clamp. A, membrane current traces recorded at a holding
potential of -80 mV. Oocytes were superfused with hK solution in
the presence or absence of 300 nM dyn A (lefttrace) or 300 nM dyn A-(1-13) (righttrace) as indicated. B, bar graph of the
membrane currents evoked by different endogenous opioid ligands at a
concentration of 1 µM. Data are presented as mean ±
S.E., with n of 3-8.
Among
dynorphins, dyn A and dyn A-(1-13) are the most potent ones. Fig. 1A shows representative traces of K currents induced by dyn A or dyn A-(1-13), a major
metabolite of dyn A with physiological activity(18) . The bargraph in Fig. 1B summarizes the
ability of different endogenous opioid ligands to activate the orphan
receptor. For dyn A and its metabolite fragments, the potency decreased
with the decrease in the peptide length. Also, when the first amino
acid tyrosine was missing, such as in dyn A-(2-17) or dyn
A-(2-13), there was no receptor activation (data not shown).
Figure 2:
Dose-response curves of XOR1-KGA coupling
activated by dyn A and dyn A-(1-13). A, an example of
membrane current traces showing the calculation method for the
ligand-evoked response. Oocytes were superfused with different
solutions as indicated. Ligands were dissolved in hK solution and
applied as indicated by the bar above the current trace.
Spontaneous current (I) is the current
when the K
concentration is increased by switching the
bath solution from ND96 to hK. Receptor-activated current (I
) is the one when a ligand is applied
to activate the receptor. The ratio of I
/I
represents the extent of receptor activation by the ligand. B, dose-response curve of dyn A-evoked receptor activation.
The results are presented as the percentage of the maximum activation.
Data are mean ± S.E. (n = 4-5). Each
oocyte was used only once to avoid desensitization. The smoothline represents a computer-aided curve fitting to the
data using a simple Michaelis-Menten model. The EC
calculated from the curve was 45 ± 6 nM (mean
± S.E., n = 2). C, dose-response curve
of dyn A-(1-13)-evoked activation. The EC
calculated
from the curve was 37 ± 9 nM (mean ± S.E., n = 2).
Opioid receptors are
capable of regulating membrane conductance in neurons, leading to
membrane hyperpolarization and a decrease in the neuronal firing rate
or inhibition of neurotransmitter release(20) . The KGA has
been shown to exist in the brain (12, 19) and was
suggested to be the K channel mediating the neuronal
effects of neurotransmitters including opioids. The functional coupling
of XOR1 to KGA in Xenopus oocytes suggests that this receptor
may mediate the activation of the KGA in the central nervous system and
function in the neuronal regulation.
Figure 3:
Effect of naloxone on the activation of
XOR1 by dynorphins. A, two representative current traces
recorded with a holding potential at -80 mV. The dottedline represents a current trace induced by 300 nM dyn A-(1-13) in hK solution (dashedline below the trace). The solidline was recorded
with the sequential perfusion of solutions hK, 300 nM dyn
A-(1-13) in hK, and 300 nM dyn A-(1-13) plus 1
mM naloxone in hK. B, change of response evoked by
300 nM dyn A-(1-13) in the presence of different
concentrations of naloxone. The data are presented as the percentage of
the maximum I/I
(see Fig. 2legend), shown as mean ± S.E. (n = 4-5). C, naloxone produces a
rightward shift of dose-response curve for dyn A. The dose-response
curves were generated the same way as in Fig. 2, in the absence
(
) or presence (
) of 10 µM naloxone in the dyn
A-containing hK solutions. The EC
value for dyn A was
changed from 45 to 372 nM by
naloxone.
Does naloxone antagonize dynorphin effects on this
receptor in a competitive manner, as for the other opioid receptors? By
using naloxone with different concentrations of dyn A to perform
dose-response experiments, we found that 10 µM naloxone
caused a parallel rightward shift of the dose-response curve for the
dyn A-activated response (Fig. 3C). The parallel shift
of the dose-response curve suggests that the antagonism by naloxone at
the XOR1 is competitive in nature. In this case, the EC value of dyn A was shifted from 45 nM without naloxone
to 372 nM with 10 µM naloxone. These data gave
the apparent dissociation constant K
of naloxone
for the receptor at about 1.37 µM using the Tallarida
variation of Schild analysis(22) . Compared with the nanomolar
affinity values of naloxone for µ,
, and
opioid
receptors(23) , this value is 2-3 orders of magnitude
higher, thus making naloxone a low potency antagonist at this novel
receptor.
Figure 4: Inhibition of forskolin-stimulated cyclic AMP increase by dyn A-(1-13). CHO-K1 or HEK-293 cells transiently transfected with the XOR1 clone were treated with 10 µM forskolin with or without 5 µM dyn A-(1-13). The control cells were transfected with the plasmid vector and underwent the same treatment. Intracellular cyclic AMP content was determined using a radioimmunoassay kit (DuPont NEN). Data are shown as mean ± S.E. (n = 2). Asterisk indicates a significant difference from the forskolin-only treated cells (Student's t test, p < 0.01).
It is
interesting to note that, while the overall sequence homology between
this orphan receptor and each of the cloned µ, , and
opioid receptors is similar, there is apparent resemblance of the
highly negative charges in the second extracellular loop between this
receptor and the
opioid receptor. There are seven negatively
charged amino acid residues in this region for both the
receptor
and this receptor, whereas there are only two negatively charged
residues in either the µ or
receptor(4) . In opioid
receptors, this loop is the longest among the three extracellular loops
with a low level of sequence homology(26) , suggesting the
possibility that it may be involved in ligand binding specificity for
the receptors. Indeed, this region in the
receptor has been shown
to be critical for high affinity binding of dynorphin
peptides(27) , which are basic peptides with five positively
charged amino acid residues for both dyn A and dyn
A-(1-13)(18, 28) . The highly negative charges
in this region of the orphan receptor may contribute to the interaction
between dynorphins and the receptor.
What might be the physiological
role of this novel receptor? Reports in the literature have provided
certain clues. In vivo studies showed that dynorphins caused
certain physiological effects that may not be mediated entirely through
the opioid receptor, such as biphasic anti-nociception effect,
motor effects, immunomodulation, inflammation response, and modulation
on respiration and temperature
control(29, 30, 31, 32) . Our
results suggest the possibility that this novel opioid receptor may
play a role in mediating some of the dynorphin effects that are not
contributed by the
opioid receptor.
In conclusion, the data presented in this report indicate that this opioid receptor-like orphan receptor is indeed a novel member of the opioid receptor family, because it can be activated by the endogenous ligand dynorphins. Similar to the other opioid receptors, this receptor also inhibits adenylyl cyclase activity. Unlike the other opioid receptors, however, naloxone does not effectively block this receptor, suggesting that it may mediate some of the ``non-opioid'' effects of dynorphins. Endorphins or enkephalins are rather ineffective at this receptor, whereas several of the dynorphin peptides can activate it, suggesting that there may be other endogenous ligands for this receptor.