(Received for publication, November 3, 1994)
From the
Desensitization of a chemotactic receptor is an adaptive process
that terminates inflammation. Although homologous desensitization can
be well explained by the action of specific receptor kinases, the
mechanisms of heterologous desensitization remain elusive. As an
approach to evaluate the roles of G pathway in
desensitization of calcium signaling, we expressed a constitutively
active G
mutant (G
Q-L) together with
platelet-activating factor (PAF) receptor in Xenopus laevis oocytes. G
Q-L expression completely attenuated
the calcium-sensitive chloride current and the
Ca release
elicited by PAF. The G
-mediated desensitization could not
be ascribed to G protein/receptor uncoupling via receptor
phosphorylation, because (i) PAF-induced inositol 1,4,5-trisphosphate
(IP
) synthesis was only partially suppressed and (ii) a
mutated PAF receptor devoid of all Ser and Thr in the third cytoplasmic
loop and in the C-terminal tail was also completely desensitized by
G
Q-L. In G
Q-L expressing oocytes,
microinjection of IP
failed to elicit the calcium response,
and the IP
receptor, detected by a specific antibody,
disappeared. Thus, the G
-mediated desensitization can be
most likely explained by IP
receptor down-regulation. These
novel mechanisms may explain in part heterologous desensitization in
chemotactic factor-stimulated inflammatory cells.
Cloning of chemotactic factor receptor cDNAs had broadened
understanding of activation-desensitization processes in
agonist-stimulated inflammatory cells. Platelet-activating factor
(PAF)()(1, 2) , C5a(3) , and
thromboxane A
(4) receptors were found to possess
the seven-transmembranous structure characteristic of G protein-coupled
receptors. Subsequent studies revealed that these receptors could
transduce calcium signaling via newly emerged pertussis
toxin-insensitive G protein
subunits, G
and
G
(5, 6, 7, 8, 9, 10) .
Once a G protein-coupled receptor is activated with the first
stimulus, cells often become unresponsive to the same agonist or even
to others. Homologous and heterologous desensitizations are fundamental
adaptive processes that terminate inflammatory reactions. A widely
accepted mechanism of the homologous desensitization is a
phosphorylation of an agonist-occupied receptor by specific receptor
kinases, such as -adrenergic receptor
kinases(11, 12) , and subsequent receptor/G protein
uncoupling. We recently obtained evidence that phosphorylation of Ser
and Thr residues in the C-terminal cytoplasmic tail by
-adrenergic
receptor kinase-like kinases plays crucial roles in the homologous
desensitization of the cloned PAF receptor(13) . A recent study
using an artificial yeast system revealed that a pheromone receptor,
which coupled to tripartite G proteins, causes a homologous
desensitization even in cells devoid of G proteins(14) . These
observations suggest that signals downstream of G proteins may not be
indispensable for homologous desensitization in some systems.
In
contrast to homologous desensitization, heterologous desensitization
occurs in a previously unoccupied receptor (or in its signaling
pathway) and rationally requires signals downstream of the primarily
activated G proteins. Thus, to better understand heterologous
desensitization, it is crucial to know if G protein activation itself
will induce desensitization. In an attempt to address this question and
to elucidate the underlying mechanisms, we expressed wild and mutated
PAF receptors together with a constitutively active G
mutant(15, 16) . We used a gene expression system in Xenopus oocytes, since this system allows us (i) to co-express
multiple genes in a single cell, (ii) to sensitively detect calcium
response by electrophysiological methods, and (iii) to microinject
second messengers. We now report evidence that G
activation
attenuates subsequent chemotactic receptor-mediated calcium signaling
and that the main event related to this desensitization is
down-regulation of the inositol 1,4,5-trisphosphate (IP
)
receptor.
Figure 1:
PAF-induced membrane
current was inhibited by the co-expression of G Q-L
but not by G
. A, typical recordings of
PAF-induced calcium-sensitive chloride current. Oocytes injected with
cRNAs in combination (25 ng of each cRNA/oocyte) were voltage-clamped,
and the membrane currents elicited by the superfusion of 10 nM PAF (denoted by horizontalbars) were assayed.
Oocytes injected with PAFR cRNA (control) or with PAFR +
G
cRNAs (+G
)
displayed typical biphasic responses, whereas those injected with PAFR
+ G
Q-L cRNAs (+G
Q-L) failed to
respond to PAF. B, schematic transmembrane structures of PAFR
and PAFR-mut, and PAF-induced response in oocytes. (The C-terminal side
from the fifth membrane-spanning domain is represented. In the
C-terminal cytoplasmic tail, 4 Ser (S
)
and 5 Thr (T
) residues are present.) The bar graph summarizes the 10 nM PAF-induced membrane
currents. Oocytes expressing PAFR or PAFR + G
responded to PAF (640 ± 200 nA, n = 6, and 700
± 150 nA, n = 6, respectively; mean ±
S.E.), whereas co-expression of G
Q-L suppressed the
response (5 ± 10 nA, n = 7; mean ± S.E.).
Oocytes injected with PAFR-mut + G
cRNAs
responded to PAF (540 ± 130 nA, n = 4; mean
± SE), but those injected with PAFR-mut +
G
Q-L did not (0 ± 0 nA, n = 4;
mean ± SE). C, oocytes were injected with 25 ng/oocyte
of PAFR cRNA and various amounts of G
Q-L cRNA, and 10
nM PAF-induced response was examined. G
Q-L
cRNA dose-dependently suppressed the current. The vertical bars denote S.E. (n =
4-6).
Ca efflux from oocytes was
assayed essentially as described previously(18) . 20 oocytes
were labeled with
Ca (the final concentration, 1.85
MBq/ml) in 500 µl of calcium-free solution A for 3 h at room
temperature and then were thoroughly washed with solution A and
incubated in 500 µl of the same medium. 500 µl of the medium
was collected before and after the addition of PAF, at defined
intervals, and replaced with 500 µl of fresh solution A.
Radioactivity in the medium collected at each interval was counted.
Measurement of IP content in oocytes was performed as
follows. 10 oocytes in 200 µl of solution A were stimulated with
PAF for the indicated time, and the reaction was quenched by the
addition of 70 µl of 20% perchloric acid followed by vigorous
vortexing. The sample were centrifuged at 10,000
g for
5 min at 4 °C, and then the supernatant was neutralized with 10 M KOH. Precipitated salt was again removed by centrifugation,
and the supernatant was examined for IP
content by a
competitive radioreceptor assay using the D-myo-inositol 1,4,5-trisphosphate assay system
(Amersham Corp.).
The present study was designed to address two interrelated
questions. First, are downstream events of G activation
sufficient to desensitize the chemoattractant-induced calcium
signaling? And, second, if G
activation attenuates
subsequent calcium signaling, what is the mechanism? As an approach to
answer the questions, we expressed a GTPase-deficient mutant of guinea
pig G
(G
Q-L) (15, 16) that has been demonstrated to activate
downstream signals, including phospholipase C and MAP
kinases(19) . (
)As a model of a potential target of
G
-dependent heterologous desensitization, we used the
guinea pig PAF receptor (PAFR) and its mutant (PAFR-mut). In PAFR-mut,
Ser/Thr residues in the C-terminal tail, which play a crucial role in
homologous desensitization(13) , were totally removed by
deletion. In addition, a single Thr in the 3rd cytoplasmic loop was
substituted with Ala (see the schematic membrane structures in Fig. 1B). cRNAs of these constructs were prepared in vitro and injected in combination (25 ng of each
cRNA/oocyte, if not otherwise stated) into Xenopus oocytes.
We first examined the PAF (10 nM) induced calcium response
by monitoring the calcium-operated chloride
current(1, 2) . As shown in Fig. 1, in oocytes
expressing PAFR alone, there was a considerable response to 10 nM PAF, and co-expression of G Q-L with PAFR
completely suppressed this response. Co-expression of native
G
was without effect (Fig. 1). Viability of the
oocytes, as checked by resting membrane potential, was not affected by
G
Q-L expression (-40 ± 12 mV versus -38 ± 15 mV, in G
and
G
Q-L expressing oocytes, respectively; mean ±
S.D., n = 4). The G
Q-L-induced loss of PAF sensitivity could not be ascribed to
failure of PAFR expression, since PAF-induced IP
synthesis
was only partially suppressed in G
Q-L expressing
oocytes (see below and Fig. 2B). As shown in Fig. 1C, the effect depended on the amount of
microinjected G
Q-L cRNA. G
Q-L also
desensitized PAFR-mut; the PAFR-mut-mediated membrane current
completely disappeared in G
Q-L-expressing cells but
not in G
-expressing cells (Fig. 1B).
Thus, in contrast to desensitization by the homoligand(13) ,
the G
-mediated desensitization could not be fully explained
by the phosphorylation of Ser/Thr in the 3rd cytoplasmic loop and in
the C-terminal tail.
Figure 2:
G Q-L expression
abolished PAF-induced
Ca efflux but only partially
inhibited IP
synthesis. A, 20 oocytes injected
with PAFR+G
(opencircle) or
PAFR + G
Q-L (closedcircle)
cRNAs (25 ng of each cRNA/oocyte) were labeled with
Ca,
and
Ca efflux elicited by 10 nM PAF (horizontalbar) was measured. Co-expression of
G
Q-L suppressed
Ca efflux. These results
were a representative of two independent examinations. B, 10
oocytes injected with PAFR+G
(opencolumns) or with PAFR + G
Q-L (closedcolumns) cRNAs (25 ng of each/oocyte) were
challenged with 10 nM PAF, and IP
accumulation was
measured. Co-expression of G
Q-L partially inhibited
IP
synthesis by 40% at 1 min and by 26% at 5 min. The verticalbars denote S.E., n = 3.
These results were a representative of three independent
experiments.
To analyze the machinery needed for the
G-mediated desensitization, we dissected the signaling
pathway by measuring changes in key second messengers, calcium and
IP
. Oocytes were labeled with
Ca, and the
PAF-induced efflux of radioactivity was followed. PAF elicited a rapid
and significant
Ca efflux from oocytes injected with PAFR
and G
cRNAs. In the oocytes expressing
G
Q-L together with PAFR, this response disappeared, (Fig. 2A), thereby indicating that G
Q-L-suppressed elevation of intracellular calcium.
We next measured
change in the IP level by IP
-specific
radioreceptor assay (Fig. 2B). Basal IP
levels were below the detection limit in oocytes injected with
G
Q-L/PAFR cRNAs as well as those injected with
G
/PAFR cRNAs (detection limit; 0.25 pmol/10 oocytes),
probably reflecting the rapid metabolism of
IP
(20) . In the oocytes expressing
G
together with PAFR, PAF elicited a time-dependent
increase in IP
level. In contrast to complete loss of the
calcium efflux and the calcium-dependent chloride current,
PAFR-mediated IP
elevation was only partially inhibited by
the co-expression of G
Q-L, by 40% at 1 min, and by
26% at 5 min. These observations led to the notion that prolonged
activation of G
attenuated the PAFR-mediated calcium
response by modifying the IP
-induced calcium release, which
is mediated by IP
-sensitive calcium channel (tetrameric
complex of IP
receptor)(21) .
To determine
whether or not the IP-induced calcium release was
suppressed in oocytes expressing active G
, we
microinjected IP
into oocytes under the recording of the
calcium-dependent chloride current. As shown in Fig. 3,
microinjection of 40 pmol of IP
elicited a large inward
current in oocytes expressing G
, whereas the response
was almost completely suppressed in G
Q-L-expressing
oocytes. As G
Q-L did not suppress the current
directly activated by microinjection of 50 pmol CaCl
(Fig. 3A), the unresponsiveness to IP
was not likely to be due to altered channel functions.
Figure 3:
G Q-L suppressed
IP
-induced calcium response, and down-regulated IP
receptor. A, oocytes injected with G
or
G
Q-L cRNA (25 ng/oocyte) were microinjected with 40
pmol of IP
or with 50 pmol of CaCl
under
condition of recording of the membrane current. IP
elicited
a distinct inward current in oocytes injected with G
cRNA but not in those injected with G
Q-L cRNA.
CaCl
elicited almost comparable responses in the both
groups of oocytes. B, the bargraph summarizes IP
-induced membrane currents. The
amplitudes of the currents induced by 40 pmol of IP
were
960 ± 150 nA in G
-expressing cells and 20
± 40 nA in G
Q-L-expressing cells
(mean ± S.E., n = 6). C, crude
membrane fraction of oocytes was subjected to Western blotting using
anti-Xenopus IP
receptor antibody. In Exp.1, 50 ng of G
or
G
Q-L cRNA was injected, and 40 µg of crude
membrane was subjected to Western blotting. IP
receptor was
detected in G
-expressing oocytes, whereas the
immunoreactivity disappeared in G
Q-L expressing
oocytes. In Exp. 2, G
Q-L cRNA was
diluted (1:10, 5 ng/oocyte; 1:100, 0.5 ng/oocyte), and injected. Water,
or G
cRNA (50 ng/oocyte) injected oocytes were used as
controls. 20 µg of the membrane fraction was used.
G
Q-L cRNA dose dependently down-regulated the
IP
receptor; G
Q-L cRNA more than 5
ng/oocyte abolished IP
receptor-immunoreactivity.
We next
asked whether the loss of IP responsiveness is accompanied
by change in the level of IP
receptor molecule. In control
oocytes injected with G
cRNA, a single immunoreactive
protein that corresponds to Xenopus IP
receptor (17) was detected by a specific polyclonal antibody raised
against the cloned Xenopus IP
receptor (17) (Fig. 3C, Exp.1). In
G
Q-L expressing cells, this imunoreactivity almost
completely disappeared (Fig. 3C, Exp.1).
Decrease in the immunoreactivity depended on the amount of
G
Q-L cRNA; oocytes injected with G
Q-L cRNA at 0.5 ng/oocyte contained an almost comparable
immunoreactivity to that in oocytes injected with water or with
G
cRNA (control), whereas the immunoreactive band
disappeared in oocytes injected with G
Q-L cRNA at
more than 5 ng/oocyte (Fig. 3C, Exp.2).
These model experiments using a heterologous expression system
revealed two novel findings concerning desensitization mechanisms of
chemoattractant receptors. First, the continuous activation of
downstream signals of G was sufficient to desensitize
subsequent chemoattractant-induced calcium signaling. Second, one of
the major mechanisms of the G
-induced desensitization was
down-regulation of the IP
receptor molecule. The second
point readily explains the heterologous desensitization of calcium
signaling. Recently Quian et al.(22) found that
expression of a GTPase-deficient G
, a member of
G
family, desensitized both bombesin- and
PDGF-stimulated calcium mobilizations in Swiss3T3 cells. Since IP
receptor is the first convergent point of the calcium signaling
pathways originated from these different classes of receptors, the
observed IP
receptor desensitization/down-regulation may
explain the heterologous desensitization in Swiss3T3 cells.
Heretofore, several mechanisms for heterologous desensitization of G
protein-coupled receptors have been proposed, including receptor
phosphorylation by signal-dependent kinases, such as protein kinase C (20) , and down-regulation of G proteins(23) . These
scenarios, which affect receptor-mediated phospholipase C activation,
could not fully explain the G-dependent desensitization.
However, these mechanisms may explain the observed partial inhibition
of IP
production. This point should be studied further.
Recently, IP receptor down-regulation was noted in rat
cerebellar granular cells and in SH-SY5Y human neuroblastoma cells
continuously exposed to acetylcholine(24, 25) . Using
an active G
mutant, we obtained evidence that the
down-regulation is not a ligand-specific phenomenon but that rather
activation of G
pathway is sufficient for the
down-regulation. The loss of IP
sensitivity due to the
down-regulation was found to be the main reason for the
G
-induced heterologous desensitization of the PAF
receptor-originated calcium signaling. These observations also imply
that chemotactic factor receptors coupling to the G
-family
potentially elicit the same desensitization mechanisms. The next
important problem is to determine if the proposed mechanisms can
explain impaired leukocyte functions in inflammatory or autoimmune
disorders(26, 27, 28) . The precise
mechanisms of down-regulation of the IP
receptor remain
unclear. Elevation of intracellular levels of IP
and
calcium ion appear to be required for both the reversible inactivation
of the channel function (29) and the degradation (25) of IP
receptor/calcium channel. In an in
vitro study, IP
receptor was found to be a good
substrate for a calcium-activable neutral protease,
calpain(30) . However, the role of calpain in vivo is
still controversial(25) . The Xenopus oocyte system,
in which these second messengers or a calpain-specific inhibitor,
calpastatin (31) can be readily introduced intracellularly, may
serve as a useful system for studying the IP
receptor
turn-over.