(Received for publication, August 3, 1994; and in revised form, November 4, 1994)
From the
The cellular basis of down-regulation and desensitization in
phospholipase C-linked receptors is unclear. Recent studies with some
receptors suggest that elements in the carboxyl terminus of the
receptor are important in mediating these processes. Three mutant
gastrin-releasing peptide receptors (GRP-R) were studied: one whose
last 37 carboxyl-terminal amino acids were eliminated (construct
MGT346); one that replaced all of the carboxyl-terminal Ser and Thr
eliminated in MGT346 with Ala, Asn, or Gly (construct JF1); and one
that selectively replaced the Ser and Thr of the protein kinase C
consensus sequence (PKC-CS) located within the same region with alanine
(construct TS360AA). Desensitization was assessed by measuring the
ability to activate phospholipase C and increase cellular
[H]inositol phosphates, or increase
[Ca
]
, after
pre-exposure to 3 nM bombesin for 24 h. Wild-type GRP-R was
maximally desensitized and down-regulated after a 24-h exposure to 3
nM bombesin, and removal of the PKC-CS alone markedly
attenuated each process. Elimination of additional serines and
threonines by truncation (MGT346) or replacement (JF1) did not decrease
down-regulation or desensitization further. To confirm the necessity of
second messenger activation in mediating down-regulation, we further
investigated two additional mutant GRP-R that bound agonist with high
affinity but fail to activate phospholipase C (constructs R139G and
A263E). Neither construct underwent significant down-regulation.
Removal of all GRP-R carboxyl-terminal Ser or Thr, either by MGT346 or
JF1, reduced internalization by >80%, whereas elimination of the
PKC-CS in TS360AA only attenuated internalization by 21 ± 2%.
These data suggest that activation of the distal carboxyl-terminal
PKC-CS is essential for chronic desensitization and down-regulation of
the GRP-R, and provide no evidence for involvement of second
messenger-independent processes. In contrast, internalization is
equally regulated by both second messenger-dependent and independent
processes.
The mammalian bombesin-related peptides, gastrin-releasing
peptide (GRP) ()and neuromedin B, are thought to have
important roles in a number of diverse physiological and pathological
processes. These peptides act to regulate numerous central nervous
system functions including circadian rhythm (1) and body
temperature(2) , stimulate the release of many gastrointestinal
hormones(3, 4) , have profound effects on immune
function(5) , and are potent stimulators of gastrointestinal
tissues including the pancreas (6) , stomach(7) , and
muscle(8) . Importantly, these peptides also function as potent
growth factors both in normal tissues (9, 10) and
tumors(9, 11, 12, 13) , as well as
act as autocrine growth factors in some human small cell cancer cell
lines(9) . Bombesin-related peptides mediate their effects at
least in part by activating phospholipase C after binding to specific
GRP-preferring receptors (GRP-R) and neuromedin B-preferring
receptors(14, 15, 16) .
Consequent to
agonist exposure, bombesin receptors become refractory to further
stimulation by the same agonist (homologous desensitization). In
addition to desensitization, upon agonist exposure, bombesin receptors
also undergo internalization and down-regulation. Similar to other G
protein-coupled receptors, both acute desensitization (i.e. within minutes of agonist exposure) and chronic desensitization (i.e. agonist exposure for hours) have been reported with the
GRP-R(17, 18, 19, 20, 21, 22, 23) .
However, the inter-relationship between these multiple
receptor-modulatory processes (including receptor internalization,
down-regulation, and desensitization as well as their mediators) is
largely unknown not only for bombesin receptors but also for most
phospholipase C-linked receptors. In contradistinction, much is known,
especially for acute desensitization, about the mechanisms regulating
adenylate cyclase-activating receptors, such as the
-adrenergic receptor (
AR).
Acute
desensitization of the AR occurs as a consequence of
receptor phosphorylation driven by both second messenger-dependent and
second messenger-independent (i.e.
-adrenergic receptor
kinase I and II) kinases(24) , as well as due to receptor G
protein uncoupling; however, internalization is not
involved(25) . In contrast, with prolonged agonist exposure,
chronic desensitization of the
AR is completely
independent of any known second messenger-dependent or -independent
kinase and appears to involve receptor down-regulation(26) .
The role of such kinases in regulating the acute or chronic
desensitization of phospholipase C-linked receptors is largely unclear.
Although second messenger-independent kinases have been shown to
possess some degree of promiscuity, such that the
-adrenergic
receptor kinase can also phosphorylate the phospholipase C-activating
substance P receptor in vitro(27) , other
phospholipase C-linked receptors such as the
-adrenergic receptors are not so
phosphorylated(28) . Therefore, this cross-specificity may vary
for different phospholipase C-linked receptors. Similarly, the role of
second messenger-dependent kinases in regulating the desensitization of
phospholipase C-linked receptors likewise is unclear. Although
activation of both protein kinase C (PKC) and protein kinase A (PKA)
can induce acute desensitization of the
AR(29) , most phospholipase C-coupled
receptors do not activate this latter kinase, making it unlikely that
PKA regulates either the acute or chronic desensitization of these
receptors. Furthermore, the role of PKC in mediating either the acute
or chronic desensitization of phospholipase C-activating receptors is
unclear. For instance, although some studies have shown that
desensitization of phospholipase C-coupled muscarinic cholinergic
receptors is PKC-mediated and apparently involves receptor
phosphorylation(30) , other studies do not support these
conclusions (31, 32, 33) . Indeed, when
muscarinic cholinergic receptors, G-proteins, and PKC are reconstituted
in vesicles, agonist-induced receptor phosphorylation is not
observed(34) .
Some(22) , but not other (17, 23) studies, have demonstrated that PKC may play a role in mediating acute desensitization of the GRP-R. There is no data on the role of PKC in mediating chronic GRP receptor desensitization. It has been proposed that chronic desensitization may be mediated at least in part by receptor down-regulation and internalization(21) . In a recent study it was demonstrated that multiple serines and threonines located within the GRP-R carboxyl terminus, including a PKC consensus sequence, were important in regulating this receptor's internalization(35) . However, it is unknown whether these residues also are important in mediating chronic desensitization and down-regulation of the GRP-R. In this study, therefore, to gain insight into the possible importance of either the PKC consensus sequence or other serine/threonine residues in the GRP-R carboxyl terminus in mediating chronic desensitization and down-regulation, as well as into the possible inter-relationship of these processes, we have determined the ability of bombesin to induce these receptor changes in mutant GRP receptors.
The three mutant GRP-R cell lines demonstrated similar
dose-response curves for the ability of bombesin to inhibit binding of
[I-Tyr
]bombesin to that seen with
the unaltered wild-type receptor (35) (data not shown).
Specifically, half-maximal inhibition of binding (K
) of
[
I-Tyr
]bombesin with unlabeled
bombesin was observed at 0.9 ± 0.3 nM for wild-type
GRP-R, at 2.2 ± 1.0 nM for MGT-346, at 4.9 ± 0.8
nM for JF1, and at 1.0 ± 0.3 nM for TS360AA.
All cell lines expressed approximately similar numbers of receptors (B
, in fmol/10
cells), with
wild-type GRP-R expressing 672 ± 133, MGT346 expressing 609
± 115, JF1 expressing 897 ± 107, and TS360AA expressing
437 ± 88 (data not shown).
With agonist exposure, wild-type GRP-R was rapidly internalized(35) . The internalization of mutant-truncated receptor MGT346 and JF1 was significantly attenuated (Table 1). In contrast, removal of the protein kinase C consensus sequence in mutant TS360AA only partially attenuated internalization (Table 1).
We next determined the rate of GRP-R down-regulation. A previous study of the wild-type receptor revealed that GRP-R down-regulation was rapid, with exposure to 6 nM bombesin causing half-maximal decreases in receptor number by 3 h and maximal decreases by 6 h(18) . Similarly, in the present study, maximal GRP-R down-regulation was achieved after a 6-h exposure to 3 nM bombesin (Table 1). In contrast to wild-type GRP-R, mutant MGT346 underwent relatively little receptor down-regulation (Table 1, Fig. 1). That this attenuation of receptor down-regulation was not a nonspecific consequence of receptor truncation was demonstrated by mutant JF1. Replacement of the carboxyl terminus Ser and Thr in JF1 resulted in a similar attenuation of down-regulation as observed for the truncation mutant. In contrast to that which was observed for receptor internalization, removal of only the PKC consensus sequence in mutant TS360AA also markedly attenuated receptor down-regulation (Table 1, Fig. 1). These data suggest that receptor internalization and down-regulation are dissociable phenomena that are regulated by different carboxyl terminus residues in the GRP-R.
Figure 1:
Effect of 3
nM bombesin preincubation on receptor number (B) on cells expressing wild-type GRP-R, MGT346,
JF1, and TS360AA. Cells grown to confluence in F-175 flasks were
exposed to 3 nM bombesin for the indicated times and subjected
to bombesin competitive binding experiments with
[
I-Tyr
]bombesin. B
and K
were determined using the
least squares curve fitting program LIGAND(37) . There was no
significant change in K
for any of the
cell types with bombesin preincubation. Data are expressed as the
percentage of B
expressed in bombesin pretreated
cells as compared with untreated control cells processed in parallel.
Each data point represents the mean ± S.E. of at least three
separate experiments, with each value measured in duplicate in each
experiment. Top, membranes from cells expressing wild-type
GRP-R or mutant receptors TS360AA were incubated with 3 nM bombesin for the indicated times and were then subjected to
cross-linking with
I-GRP. Equal amounts of cell membrane
protein were loaded at each time point per cell type. This gel is
representative of two separate experiments.
To investigate further the potential role of second messenger-dependent kinase activation in GRP-R down-regulation as compared with its role in mediating internalization, we examined these processes in two mutant receptors that we have previously demonstrated fail to activate phospholipase C(41) . With substitution of arginine at position 139 with glycine (construct R139G) or alanine at position 263 with glutamic acid (construct A263E), the mutant GRP-Rs were found to retain high affinity binding but not to activate phospholipase C(41) . Internalization was markedly reduced in construct R139G and was partially reduced for construct A263E. Activation of second messenger-dependent kinases was clearly important for regulating down-regulation since this process was greatly attenuated for both of these mutant receptor constructs (Table 1).
The ability of the three mutant receptor types to
activate phospholipase C was similar to that observed with wild-type
GRP-R. Bombesin caused 10-fold increase in
[
H]IP formation in cells expressing the wild-type
receptor and each mutant (Table 2). Similarly, the concentration
of bombesin causing a half-maximal increase in
[
H]IP did not differ (i.e. 3-5
nM) for the wild-type receptor and the three mutant clones (Fig. 2; Table 2).
Figure 2:
Time course of the effect of 1
µM bombesin on total cellular
[H]inositol phosphate generation in desensitized
and control cells expressing wild-type GRP-R, MGT346, JF1, and TS360AA.
Confluent cells were incubated in DMEM supplemented with 2% fetal
bovine serum alone or additionally with 3 nM bombesin for 24
h. During the final 6 h of incubation, cells were further incubated
with 100 µCi/ml myo-[2-
H]inositol.
After washing in IP buffer, cells were then exposed to 1 µM bombesin for the indicated times. Isolation of
[
H]inositol phosphates was performed as described
under ``Experimental Procedures.'' Data is expressed as the
percent maximal response recorded at t = 120 min in
control cells. Each data point represents the mean ± S.E. of a
minimum of three separate experiments, with each value measured in
triplicate in each experiment.
We next determined whether there
were differences between the three mutant GRP-R and wild-type receptor
in undergoing desensitization in response to continued exposure to
agonist. Incubation of wild type GRP-R with 3 nM bombesin for
24 h significantly decreased the ability of 1 µM bombesin
to subsequently increase total cellular [H]IP (Fig. 3). After a 120-min exposure to 1 µM bombesin, control cells expressing wild-type GRP-R increased
[
H]IP over 10-fold, from 2076 ± 250 dpm to
22140 ± 410 dpm (Fig. 3). Preincubation with 3 nM bombesin, however, decreased the subsequent response to 1
µM bombesin by >70%, as cellular
[
H]IPs increased only 3-fold from 3380 ±
300 to 9040 ± 600 dpm (Fig. 3, topleftpanel; Table 2). In contrast, MGT346 was not
affected by preincubation with 3 nM bombesin, as control cells
increased [
H]IPs from 2890 ± 150 to 29560
± 680 dpm and bombesin-preincubated cells increased total
cellular [
H]IP from 3600 ± 310 to 27070
± 750 dpm (Fig. 3, toprightpanel; Table 1and Table 2). Removal of the
carboxyl terminus Ser and Thr similarly resulted in attenuation of
GRP-R desensitization. Cells expressing mutant receptor JF1 not
previously exposed to 3 nM bombesin increased total cellular
[
H]IPs when incubated with 1 µM bombesin for 120 min from 2640 ± 110 to 28260 ± 340
dpm, whereas cells expressing JF1 and previously exposed to 3 nM bombesin for 24 h increased [
H]IPs from 3620
± 220 to 26420 ± 280 dpm (89 ± 2% of control) (Fig. 3, bottomleftpanel; Table 1). That this attenuation in desensitization was
specifically due to the presence of the carboxyl terminus PKC consensus
sequence was revealed by the failure of cells expressing mutant
receptor TS360AA to undergo agonist-induced desensitization.
Specifically, control cells expressing TS360AA increased
[
H]IP from 3720 ± 270 to 32410 ±
440 dpm, whereas those preincubated with 3 nM bombesin
increased [
H]IP from 5500 ± 210 to 33330
± 420 dpm (97 ± 2% of control) (Fig. 3, bottomrightpanel; Table 1and Table 2).
These data support the contention that the distal carboxyl PKC
consensus sequence is needed for GRP-R desensitization.
Figure 3:
Effect of preincubation with 3 nM bombesin for 24 h on the dose-response curves for
[H]inositol phosphate generation in cells
expressing wild-type GRP-R, MGT346, JF1, and TS360AA. Confluent cells
were incubated in DMEM supplemented with 2% fetal bovine serum and 100
µCi/ml myo-[2-
H]inositol alone or
with 3 nM bombesin for 24 h. After washing in IP buffer, cells
were exposed to the indicated concentrations of bombesin for 60 min,
with data expressed as the -fold increase to total cellular
[
H]inositol phosphates. Each data point
represents the mean ± S.E. of a minimum of three separate
experiments, with each value determined in duplicate in each
experiment.
To determine
whether the alterations in receptor desensitization were accompanied by
changes in agonist affinity, [H]IP dose-response
curves in the presence and absence of 3 nM bombesin
preincubation were generated for all four cell types (Fig. 2).
There was no change in potency of bombesin in increasing
[
H]IP in either the wild-type or the three mutant
GRP receptors after desensitization (Fig. 2, Table 2).
These data demonstrate that desensitization is accomplished by a
reduction only in agonist efficacy without affecting agonist potency.
Since GRP-R-induced activation of phospholipase C increases
[Ca]
(6, 15, 16) ,
we next determined whether the agonist-induced desensitization observed
with [
H]IP formation also could be observed with
[Ca
]
. Cells expressing
wild-type GRP-R increased [Ca
]
from 103 ± 4 nM to 299 ± 4 nM when stimulated with 1 µM bombesin
(
[Ca
]
= 196
± 4; Table 2, Fig. 4). However, when these cells
were preincubated with 3 nM bombesin for 24 h, the ability of
1 µM bombesin to increase
[Ca
]
was attenuated by 64%
(
[Ca
]
= 71 ±
3 nM) (Fig. 4, topleftpanel; Table 2). In contrast, the ability of mutant
receptors MGT346, JF1, and TS360AA to increase
[Ca
]
was not significantly
attenuated by preincubation with 3 nM bombesin for 24 h (Fig. 4, Table 2). These data confirm that the distal
carboxyl-terminal PKC consensus sequence of the GRP-R specifically is
required for mediating agonist-induced desensitization of the increase
in [Ca
]
and
[
H]IP.
Figure 4:
Effect of preincubation with 3 nM bombesin for 24 h on the subsequent stimulation in
[Ca]
by 1 µM bombesin on cells expressing wild type GRP-R, MGT346, JF1, and
TS360AA. Cells were incubated in DMEM supplemented with 2% fetal bovine
serum alone or with 3 nM bombesin for 24 h and were then
incubated with 2 µM fura-2/AM for 30 min. Cells were
washed twice and change in fluorescence immediately determined
consequent to stimulation with 1 µM bombesin. These
tracings are representative of at least three separate
experiments.
In the present study, we investigated the possible role of
PKC or other kinases in mediating chronic desensitization and
down-regulation of the GRP-R by acting upon various serines and
threonines located within this receptor's carboxyl terminus. We
elected to investigate the GRP-R carboxyl terminus for a number of
reasons. Most importantly, studies involving the prototypical
AR have demonstrated the importance of this
receptor's carboxyl terminus in mediating desensitization through
both second messenger-dependent and independent kinases, which
phosphorylate multiple serines and threonines in this
region(24) . The few studies extant for phospholipase C-coupled
receptors, including those for epidermal growth factor (42, 43) and platelet-derived growth factor
(PDGF)(44) , demonstrate that these receptors also are
phosphorylated in the carboxyl terminus consequent to binding agonist
and that phosphorylation effects their ability to activate
phospholipase C. In contrast, a recent study of the seven
transmembrane-spanning phospholipase C-coupled M
muscarinic
cholinergic receptor revealed that although the threonine residues in
the carboxyl terminus were important for mediating down-regulation,
they were of minimal importance in regulating agonist-induced
desensitization(45) . This suggests that additional processes
or kinases may mediate desensitization, as opposed to down-regulation,
of the M
muscarinic cholinergic receptor. For many
phospholipase C-linked receptors, including those for epidermal growth
factor(42, 43) , platelet-derived growth
factor(44) , and the muscarinic cholinergic
receptors(34, 46, 47) , activation of PKC
appears to partially mediate receptor desensitization. However, other
studies of muscarinic cholinergic receptors (31, 32, 33) and studies of the phospholipase
C-activating thrombin(48) , somatostatin(49) , and
platelet-activating factor receptors (50) suggest that PKC may
not be important in mediating desensitization. Within the carboxyl
terminus of the GRP-R exists a PKC consensus
sequence(35, 51) . Although an earlier study of this
receptor suggested that multiple serines and threonines played a major
role in regulating GRP-R internalization, the PKC consensus sequence per se was only partially involved in regulating this
process(35) . Thus, while earlier investigations have
implicated the carboxyl terminus in regulating the desensitization of
some receptors, the specific role of PKC in mediating the
desensitization of phospholipase C-activating receptors in general is
not clear.
Most studies focusing on GRP-R desensitization have
examined the effects of incubations with agonists after relatively
short periods of exposure (acute desensitization, occurring within
minutes of added agonist). While some investigators have failed to
implicate a role for PKC in mediating acute
desensitization(17, 23) , others have concluded that
in at least certain cell types PKC does partially mediate
bombesin-induced acute desensitization(22) . In contrast,
however, relatively little is known about the potential mediators of
GRP-R desensitization consequent to long periods of exposure to
bombesin (chronic desensitization, occurring hours to days after the
addition of agonist). In this paper we demonstrate that the GRP-R
carboxyl terminus PKC consensus sequence is critical for mediating the
chronic desensitization seen with the GRP-R. When this consensus
sequence was mutated by changing the threonine at position 360 and the
serine at position 361 to alanines, the mutant receptor TS360AA
demonstrated minimal ability to undergo desensitization. This decreased
ability to undergo desensitization was not due to an impairment in
receptor affinity because this receptor had an identical affinity to
the wild type receptor. Furthermore, this decrease was not due to an
alteration in G protein-receptor coupling because the TS360AA mutant
had an identical EC for stimulating
[
H]IP as wild-type GRP-R, and maximally effective
concentrations of agonists caused a similar change in mobilization of
cellular calcium in both the mutant and the wild-type receptor. These
results stand in contrast to those of a similar study of the M
muscarinic cholinergic receptor where similar carboxyl-terminal
receptor alterations decreased receptor down-regulation but had minimal
effects on chronic desensitization(45) . These results also
demonstrate that the importance of the carboxyl terminus PKC consensus
sequences in mediating chronic desensitization varies with the
different phospholipase C-coupled receptors.
It has been suggested
that G protein-coupled receptor kinases may mediate the acute
desensitization of a number of different phospholipase C-coupled
receptors, similar to that shown to be the case for the
AR(52) . The role of G-protein receptor
kinases in causing chronic desensitization is unknown. One study (26) of the
AR concluded that neither second
messenger-dependent (PKA or PKC) nor -independent (
-adrenergic
receptor kinase) kinases were likely involved in mediating
agonist-induced chronic desensitization of this receptor because
ARs with the consensus sequences for these kinases
mutated demonstrated unaltered chronic desensitization. In contrast,
whereas our results support a role for PKC in mediating chronic GRP-R
desensitization, they do not provide support for the involvement of any
G-protein receptor kinase in mediating chronic desensitization of the
GRP-R. Removal of all of the GRP-R carboxyl terminus serines and
threonines, including the PKC consensus sequence, did not impair
desensitization more than removing the PKC consensus sequence alone.
Similarly, removal of most of the GRP-R carboxyl terminus did not
impair desensitization more than removing the PKC consensus sequence
alone. These results suggest that the carboxyl terminus PKC consensus
sequence is what is critical for regulating GRP-R desensitization and
provide no support for the involvement of G-protein receptor kinases in
regulating the desensitization of this receptor consequent to prolonged
agonist exposure. However, it remains possible that some G-protein
receptor kinase still could be involved in mediating a component of
this desensitization process under different experimental conditions.
Conversely, it is possible that the current mutants studied do not
allow for G-protein receptor kinase involvement to be detected.
Activation of the distal PKC consensus sequence, for example, could be
needed to activate the G-protein receptor kinases in this receptor.
Alternatively, potentiating interactions between activation of the
distal PKC consensus sequence and G-protein receptor kinases acting on
other serines and threonines in the carboxyl terminus, similar to the
effects observed between PKA and
-adrenergic receptor kinase in
causing acute desensitization of the
AR, might occur
but would not be detected using the GRP-R mutants discussed herein.
The current study also provides some important insights into the
possible importance of receptor down-regulation and internalization in
mediating chronic GRP-R desensitization. With the AR,
results obtained using mutant receptors and various specific inhibitors
of the different receptor-mediated processes have demonstrated that
acute desensitization is not mediated by down-regulation or
internalization(53) . Chronic
AR
desensitization also is independent of internalization, but likely
occurs by receptor down-regulation(26) . Although most studies
of desensitization have focused on acute phenomena, the few studies of
chronic desensitization of 7 transmembrane-spanning G-protein-coupled
receptors have provided conflicting data regarding the role of
down-regulation. For example, desensitization of the
adrenergic receptor consequent to 24 h of exposure to agonist was
not found to be due to receptor down-regulation(54) . In
contrast, chronic desensitization of the dopamine D1 receptor was
associated with its down-regulation(55) . In the present study
we found that with all mutant GRP-Rs that the effects on receptor
desensitization and down-regulation were identical. Although this
result does not establish that GRP-R desensitization is regulated via
its down-regulation, it does suggest that they are mediated by similar
processes and raises the possibility that they are coupled. Earlier
studies of the GRP-R, however, have not uniformly linked the chronic
desensitization of this receptor with its down-regulation. For the
GRP-R as well as for the closely related neuromedin B receptor, two
studies utilizing kinetic analyses of binding and changes in biological
activity have concluded that chronic desensitization is mediated by
receptor down-regulation(20, 56) . These observations
are in agreement with another study of GRP-R expressed by Swiss 3T3
cells where it was shown that chronic desensitization was mediated by
receptor down-regulation(21) . Yet these findings stand in
contrast to a study performed on GRP-R expressed by HIT-T15 cells (22) where stoichiometric and quantitative differences between
desensitization and down-regulation led the authors to conclude that
GRP-R desensitization was only partially mediated by receptor
down-regulation. Thus, our findings confirm the link between GRP-R
down-regulation and chronic desensitization and further provide
mechanistic evidence that these processes are likely mediated by PKC.
One study suggests that GRP-R internalization mediates chronic desensitization and down-regulation(20) . However, a recent study (57) using fluorescent-labeled antibodies against the GRP-R, as well as our results in the present and a previous study (18) using an acid stripping methodology, demonstrate that internalization is a rapid process. In contrast, the desensitization and down-regulation of the GRP-R observed in these studies takes hours, demonstrating that these processes are kinetically well separated from receptor internalization. Our study clearly demonstrates that internalization is at least partially mediated by different intracellular processes than those mediating chronic down-regulation or desensitization. The mutant receptor TS360AA, which lacks the distal carboxyl-terminal PKC consensus sequence, internalized 79% of receptors as compared with wild-type GRP-R, but underwent minimal chronic desensitization and down-regulation. Similarly, one mutant GRP receptor, which did not activate phospholipase C (A263E) yet underwent 50% of maximal internalization, failed to undergo down-regulation. These results suggest that PKC activation is involved in mediating only 50% of the internalization, but is essential for any down-regulation and chronic desensitization of the GRP-R. The mediation of these processes by different mechanisms is supported further by the effect of removing all serines and threonines in the GRP-R carboxyl terminus in addition to the PKC consensus sequence in mutant receptor JF1. This construct resulted in almost a complete loss of internalization, but failed to cause additional decreases in down-regulation or desensitization as compared with the PKC deletion mutant. This result supports the importance of additional sites besides the carboxyl-terminal PKC consensus sequence in mediating maximal internalization but not chronic desensitization or down-regulation.
In conclusion, this study provides definitive evidence that chronic desensitization of the GRP-R is regulated by a PKC consensus sequence located within the carboxyl terminus of this receptor and provides no evidence that other serines and threonines in this receptor's carboxyl terminus contribute to chronic desensitization. These data also suggests that chronic desensitization of the GRP-R secondary to prolonged agonist exposure is coupled to receptor down-regulation and that down-regulation is itself also regulated by PKC. In contrast, and in conjunction with our earlier study(35) , internalization and down-regulation or chronic desensitization of the GRP-R are not coupled but are mediated at least in part by different cellular mechanisms.