From the Departments of Medicine and
§ Immunology, Duke University Medical Center,
Durham, North Carolina 27710
Leukocytes participate in host defense by
accumulating at local sites in response to inflammatory mediators where
they may engulf foreign material and/or release toxic products that can cause substantial tissue damage. Agents of diverse chemical nature (short peptides, proteins, and lipids) have been identified as chemoattractants and stimulate leukocytes through G-protein-coupled receptors (1). Responses of leukocytes can be mediated by
chemoattractants alone or modulated by other agents. For example,
leukocytes that are attached to adhesion molecules respond to
chemoattractants to elicit far greater cytotoxic responses than
non-adherent cells. Leukocyte chemoattractant receptors are also
subject to desensitization. Given that multiple mediators are present
at sites of inflammation and that leukocytes contain receptors for many
of them, their responses are likely to be cross-regulated. Although
much has been learned about cellular activation and regulation by
single receptors, mechanisms of receptor cross-regulation leading to priming or desensitization are only beginning to be unraveled.
Chemoattractants such as the formylpeptide
N-formylmethionylleucylphenylalanine
(fMLP),1 a complement
cleavage product (C5a), leukotriene B4 (LTB4),
and platelet-activating factor (PAF) were identified years ago (2). More recently, a superfamily of related chemotactic cytokines (chemokines) and their receptors have been recognized with
interleukin-8 (IL-8) being the best characterized among this group (3,
4). Of note, chemokine receptors CCR5 and CXCR4 participate with CD4 in
the entry of human immunodeficiency virus (HIV) into cells (5).
Chemoattractant receptors stimulate leukocytes via G-proteins that
activate phospholipase C (2, 6). Most of these receptors are coupled to
a pertussis-sensitive G-protein, presumably
Gi Cellular responses to chemoattractants can be up-regulated through
priming and down-regulated by desensitization. This review will focus
on the latter, although cellular models allow approaches to
understanding both (9). Two types of desensitization termed homologous
and heterologous have been described for G-protein-coupled receptors
(10, 11). Homologous desensitization occurs in receptors in the
agonist-occupied state and involves phosphorylation by G-protein-coupled receptor kinases. These phosphorylated receptors associate with members of the arrestin family of proteins resulting in
a decreased affinity of the receptor for G-proteins and
internalization. Heterologous desensitization occurs when a receptor
loses its responsiveness following phosphorylation by second
messenger-activated kinases (i.e. protein kinase A (PKA) or
protein kinase C (PKC)), which have been activated by different
receptors or signaling processes (10). Heterologous desensitization
does not require agonist occupancy and does not lead to
arrestin-mediated receptor internalization. Studies with leukocytes
have demonstrated an additional level of complexity and the description
of a new form of "heterologous" desensitization with selectivity
for groups of chemoattractant receptors.
Early studies suggested a complexity of receptor cross-regulation
beyond the classic concepts of homologous and heterologous desensitization (12-15). An approach to understanding
"cross-desensitization" among chemoattractant receptors was
provided by Didsbury et al. (16). They demonstrated that in
HEK293 cells transiently coexpressing receptors for fMLP and C5a,
activation of one receptor resulted in cross-desensitization of
Ca2+ mobilization stimulated by the other.
Cross-desensitization was specific for the chemoattractant receptors
that activate phospholipase C (PLC) via a pertussis toxin-sensitive
G-protein. Native
INTRODUCTION
Top
Introduction
References
Mechanism of Leukocyte Activation and Regulation
2 (6). Nonetheless, receptors for PAF and
LTB4 activate a Gq-like G-protein as well as
Gi. As chemoattractant receptors stimulate multiple
responses, it is not yet certain whether selective G-protein usage
mediates different responses. Although, by definition, all chemoattractants stimulate directed migration, at higher doses (about
20-fold) many also activate the opening of calcium channels and
activate phospholipase D. These activities correlate with the onset of
cytotoxic responses such as exocytosis and respiratory burst (7, 8).
There is a hierarchy among chemoattractants for stimulation of
cytotoxic responses with fMLP and C5a being more active than others,
i.e. IL-8, PAF, and LTB4. These differences are
likely related to the activation of shared pathways for chemotaxis and
a distinct pathway for cytotoxic activation requiring prolonged receptor signaling (2, 7).
Identification of a New Form of Chemoattractant Receptor
Regulation
1-adrenergic receptors that activate
PLC via a pertussis toxin-insensitive G-protein were not desensitized
by fMLP and C5a and viceversa. This discovery led to the extensive
characterization of specificity of this type of cross-regulation in
neutrophils (17). For these studies, the chemoattractants fMLP, C5a,
IL-8, PAF, and LTB4 and the purinoceptor agonist ATP
S
were evaluated for their ability to cross-desensitize each other as
measured by ligand-stimulated GTP
S binding to membranes or
intracellular Ca2+ mobilization. It was shown that all
receptors undergo effective homologous desensitization. In addition,
fMLP, C5a, and IL-8 cross-desensitized Ca2+ mobilization to
one another as well as to LTB4 and PAF (Table I) (17). PAF, LTB4, or
ATP
S did not, however, cross-desensitize the peptide chemoattractant
receptors. The strength of receptors to desensitize Ca2+
mobilization to one another was fMLP > C5a > IL-8. In
contrast, the susceptibility of peptide chemoattractant receptors to
undergo cross-desensitization was reversed with IL-8 > C5a > fMLP. The ability of fMLP to induce a greater desensitization of
Ca2+ mobilization by C5a and IL-8 was correlated with its
ability to block C5a and IL-8-stimulated G-protein activation at the
level of receptor/G-protein coupling. Surprisingly, neither C5a nor IL-8 inhibited fMLP-stimulated G-protein activation, although both
blocked Ca2+ mobilization. Based on these studies it was
postulated that chemoattractant receptor cross-regulation occurred at
two levels, one at the level of receptor/G-protein coupling and another
at a level distal to G-protein activation, resulting in a reduced
activation of phospholipase C.
Cross-desensitization of chemoattractant-stimulated Ca2+
mobilization in human neutrophils
S to inhibit
responses to themselves or others is indicated on a scale of
desensitization as follows: ++++,
85%; +++, 50-84%; ++, 26-49%;
+, 10-24%;
,
10%. Boxed area indicates the group of peptide
chemoattractants undergoing bi-directional cross-desensitization (17).
Blackwood et al. (18) demonstrated that fMLP and C5a
cross-regulate both chemotaxis and arachidonic acid release stimulated by each other. Although IL-8 desensitized chemotaxis stimulated by fMLP
and C5a, it was less efficient in blocking arachidonic acid release by
these chemoattractants. Campbell et al. (19), however, found
that neutrophils displayed normal chemotactic responses to fMLP even
after maximal stimulation with IL-8, but activation of neutrophils even
with low concentrations of fMLP abrogated these responses to IL-8.
Nonetheless, in a murine pre-B cell line coexpressing fMLP receptor
(FR) and an IL-8 receptor, CXCR2, both fMLP and IL-8 desensitized each
other's chemotactic responses although IL-8 was less effective in
desensitizing Ca2+ mobilization by fMLP (20). These
findings are consistent with a rank order of potency of chemoattractant
receptor cross-regulation for Ca2+ mobilization (17). This
further suggests that cross-regulation of chemoattractant-mediated
biological responses such as adhesion, chemotaxis, Ca2+
mobilization, degranulation, and PLA2 activation occur via
the modulation of multiple steps in the signal transduction pathways.
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Mechanism of Chemoattractant Receptor Cross-desensitization |
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Role of Receptor Phosphorylation-- Study of the molecular mechanisms of chemoattractant receptor cross-regulation was facilitated by the use of a basophilic cell line, RBL-2H3, which could be transfected to express receptors singly or multiply. This cell line possesses the same complement of G-proteins as found in neutrophils and responds to chemoattractants to elicit a number of biochemical and biological responses in common with neutrophils (21). As with other G-protein-coupled receptors, agonist-stimulated phosphorylation of FR, C5aR, CXCR1, CXCR2, PAFR, and LTB4 receptor are associated with homologous desensitization (22-24). For all chemoattractant receptors studied, with the notable exception of FR, ligand-stimulated receptor phosphorylation is mediated via the activation of both G-protein-coupled receptor kinase and PKC (22-24). Although fMLP does activate PKC, its receptor is resistant to phosphorylation by this protein kinase. These findings provided a foundation for delineating some of the mechanisms involved in chemoattractant receptor cross-regulation as well as for explaining hierarchies of responses.
In neutrophils and in RBL-2H3 cells expressing different combinations of chemoattractant receptors, peptide chemoattractants (fMLP, C5a, IL-8) desensitized Ca2+ mobilization to one another and to PAF (25, 26). In contrast, PAF did not desensitize Ca2+ mobilization stimulated by any of these peptide chemoattractants. Studies with RBL-2H3 provided an understanding for the unidirectional desensitization of PAF-mediated responses. It was shown that PAFR was cross-phosphorylated upon activation of FR, C5aR, or CXCR1. This correlated with cross-desensitization of G-protein activation in membranes as well as Ca2+ mobilization in intact cells. The demonstration that phorbol ester also caused phosphorylation of PAFR and that a PKC inhibitor blocked PAFR phosphorylation by fMLP, C5a, and IL-8 indicates that the susceptibility of PAFR to cross-desensitization is due at least in part to PKC-mediated phosphorylation of PAFR. This contention was extended by the finding that when a phosphorylation-deficient, truncated PAFR (mPAFR) was coexpressed in RBL-2H3 cells with either FR or CXCR1, neither fMLP nor IL-8 cross-desensitized PAF-mediated responses (26). Interestingly, mPAFR, which activates cellular responses of greater magnitude and for longer duration than PAFR, resulted in cross-phosphorylation and desensitization of CXCR1 but not FR (Table II). Also, mPAFR generated a signal downstream of R/G coupling to desensitize its own Ca2+ mobilization response but did not cross-desensitize the response to fMLP (27). These findings demonstrate that the ability of fMLP, C5a, and IL-8 to unidirectionally desensitize PAF-mediated responses is exclusively because of PKC-mediated phosphorylation of the PAFR, and the downstream component is not affected. The inability of PAFR to induce phosphorylation of C5aR and CXCR1 is likely because of its own rapid phosphorylation and desensitization. Although PAFR is resistant to regulation of its downstream component by FR, C5aR, or IL-8R, neither it nor the highly active mPAFR provides a signal for downstream modification to regulate the peptide chemoattractant receptors. Given the susceptibility of FR, C5aR, and CXCR1 and the resistance of PAFR and the phosphorylation-deficient mutant (mPAFR) to inhibition by pertussis toxin, the lack of downstream cross-regulation between these groups of receptors may reflect their distinct G-protein usage (Fig. 1).
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Receptor phosphorylation plays an important but not an exclusive role in desensitization among chemoattractant receptors. For example, activation of FR resulted in the cross-phosphorylation and cross-desensitization of G-protein activation and Ca2+ mobilization stimulated by C5a and IL-8 (25). C5a and IL-8 also cross-phosphorylated and cross-desensitized responses to each other. However, receptor phosphorylation cannot explain the ability of C5aR to desensitize Ca2+ mobilization and IP3 generation by FR and by a phosphorylation-deficient mutant of CXCR1 (M2-CXCR1) despite a lack of both cross-phosphorylation and suppression of G-protein activation (28). This indicates that the ability of peptide chemoattractant receptors to cross-desensitize Ca2+ mobilization to one another is mediated via two processes: a PKC-mediated receptor cross-phosphorylation, to which FR and M2-CXCR1 are resistant, and a downstream component, which is shared by some but not other receptors, resulting in decreased activation of PLC. The effects of these two modifications appear to be additive. Accordingly, the inability of FR to undergo receptor cross-phosphorylation probably results in its relative resistance to cross-desensitization by other chemoattractants in neutrophils. In contrast, the greater susceptibility of IL-8-induced response reflects a higher susceptibility of its receptor to cross-phosphorylation in addition to inhibition of the downstream component (Table II, Fig. 1). PAFR, which couples to a Gq-like G-protein, may have an independent downstream regulatory component. Evidence for this comes from homologous desensitization of calcium mobilization by mPAFR, which is resistant to receptor phosphorylation (27).
Evidence for the Role of PLC Modification in "Downstream"
Desensitization--
The downstream component whose modification
results in the cross-regulation of a select group of chemoattractant
receptors has not yet been identified, but clearly, it results in
decreased activation of PLC
as IP3 production is
depressed. The finding that PLC
2 is phosphorylated by
PKA and that this is associated with the inhibition of PLC-mediated
responses stimulated by G
but not G
14,
G
15, and G
16 suggested a role for PLC
phosphorylation on cross-desensitization (29). This idea is supported
by the finding that fMLP, C5a, and IL-8 but not PAF stimulate cAMP
formation in neutrophils and in transfected RBL cells (30, 31). It is possible that PKA-mediated phosphorylation of PLC
by a group of
chemoattractant receptors selectively inhibits activation by G
but not by G
14, G
15, or
G
16. This would provide a mechanism for cross-regulation
of chemoattractant receptors at the downstream level. Studies by Ali
et al. (27, 30) in RBL-2H3 cells showed that fMLP but not
PAF stimulated cAMP production. A membrane-permeable cAMP analog
resulted in inhibition of both phosphoinositide hydrolysis and
exocytosis stimulated by fMLP but not PAF. In addition, both phosphoinositide hydrolysis and exocytosis by fMLP but not PAF were
greatly enhanced by a PKA inhibitor. The inhibitory effect of cAMP on
fMLP-mediated responses likely involves phosphorylation of PLC
by
PKA. As evidence, both fMLP and a membrane-permeable cAMP analog caused
phosphorylation of PLC
3, the only PLC
isozyme expressed in this cell line. Furthermore, the purified catalytic subunit of PKA phosphorylated PLC
3 immunoprecipitated
from this cell line, and preincubation of cells with fMLP but not PAF
blocked in vitro phosphorylation of PLC
3 by
PKA. C5a also stimulates cAMP formation in RBL-2H3 cells, and cAMP
regulates the function of this receptor as it does for fMLP. These
findings are consistent with the hypothesis that receptor-stimulated
cAMP production and the subsequent phosphorylation of
PLC
3 by PKA cross-desensitize receptors that activate
PLC
by the same mechanism. This contention is supported by the
finding that
and
subunits of G-proteins activate PLC
by
interacting at distinct sites (32).
Evidence against the Role of PLC Modification in
"Downstream" Desensitization--
The role of PLC
phosphorylation in downstream peptide chemoattractant receptor
desensitization remains to be tested directly. Recent data raise doubts
that this is the sole mechanism for the downstream effect. A major site
for phosphorylation of PLC
3 by PKA has recently been
identified as Ser-1105 (33). Phosphorylation of PLC
3 at
this site by PKA blocked PAF and other Gq-coupled receptor-mediated responses in RBL-2H3 and COS cells coexpressing the
receptor and G
q. This is in contrast to neutrophils,
differentiated HL-60 cells, transfected RBL-2H3 cells, and COS cells
where PLC
phosphorylation by PKA leads to inhibition of fMLP but not
PAF-mediated phosphoinositide hydrolysis and Ca2+
mobilization (30). The reason for this discrepancy may be related to
the overexpression of G
q in the studies of Yue et
al. (33) because PKA did not effect PAF responses in RBL cells not
transfected with Gq (27). Recent studies by Richardson
et al. (34) also questioned the role PLC
phosphorylation
as the sole determinant of downstream chemoattractant receptor
cross-regulation. CXCR2, which stimulated Ca2+ mobilization
and caused PLC
3 phosphorylation similar in magnitudes to
those stimulated by CXCR1, did not cross-desensitize the response to
fMLP or C5a although CXCR1 did. However, a phosphorylation-deficient mutant of CXCR2 (331T), which induced a greater cell activation for a
longer duration, resulted in cross-desensitization of both fMLP and
C5a-induced responses, indicating that a downstream component was
inhibited by 331T-CXCR2. Nonetheless, wild-type and
phosphorylation-deficient receptors both phosphorylated
PLC
3 to a comparable extent, and Ca2+
mobilization to both was inhibited by exogenous cAMP (34). The findings
were taken as evidence that PLC
phosphorylation may be necessary but
not sufficient for chemoattractant receptor cross-desensitization.
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Role of Signal Length on Chemoattractant Receptor Cross-desensitization |
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Studies in neutrophils showed that IL-8 is not only the most
susceptible chemoattractant receptor to undergo cross-desensitization but it also provides the weakest signal for cross-desensitization of
other chemoattractant receptors (17, 19). In neutrophils, responses to
IL-8 are mediated via the activation of both CXCR1 and CXCR2 (35).
Although CXCR1 cross-desensitizes responses to other peptide
chemoattractants, CXCR2 did not. Richardson et al. (34)
suggested that IL-8 provides the weakest desensitizing signal because
of the brief receptor signaling. For example, CXCR2, which did not
produce a cross-desensitizing signal, is rapidly phosphorylated and
internalized upon ligand stimulation so that >95% of the surface
receptors were lost within 5 min (36, 37). In contrast, the
phosphorylation-deficient mutant 331T-CXCR2 was resistant to
internalization (<5% internalization after 30 min) and generated a
signal for cross-desensitization presumably because of greater
G-protein turnover (34, 38). The sustained production of second
messengers likely activates inhibitory pathways to cause both
phosphorylation of susceptible receptors and modification of downstream
components to diminish the activation of PLC by certain
chemoattractant receptors (Table III). As
receptor phosphorylation leads to G-protein uncoupling and
internalization the hierarchy of chemoattractant receptors to generate
cytotoxic signals as well as their susceptibility to
cross-desensitization and the ability to cross-desensitize other
receptors is likely regulated by receptor phosphorylation sites
and, as a consequence, signaling time.
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Role of G-protein Modification on Chemoattractant Receptor Cross-desensitization |
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The signal for Gi-coupled receptors is initially
mediated by G, and therefore, modification of these proteins may
regulate PLC
activation. Studies on isoprenylation and
carboxymethylation of the
subunit of G-protein (G
) indicate that
this regulates G
-mediated responses in neutrophils.
Isoprenylation and carboxymethylation of G
allow it to localize
to the plasma membrane where it activates PLC
(39). In
vitro reconstitution studies showed that decarboxymethylated G
was 10-fold less effective in activating PLC
(40). fMLP stimulated G
2 carboxymethylation in neutrophils (41).
Inhibition of carboxymethylation blocked the fMLP-induced respiratory
burst. Phosphorylation of G
12 by PKC substantially
blocked the ability of the G
1
12 to
activate effector enzymes (42). Thus, modification of G
by
carboxymethylation and/or phosphorylation could be involved in
cross-desensitization (Fig. 1). This may explain the finding of Pike
and Snyderman (43) that inhibition of carboxymethylation in leukocytes
depresses chemoattractant function.
A newly described family of proteins known as regulators of G-protein
signaling (RGS) reduces the length of G-protein signaling by enhancing
its GTPase activity, thus making less G available (44). RGS could
therefore play a role in chemoattractant receptor cross-desensitization
by regulating signal length. In this regard, transient overexpression
of RGS1, RGS3, and RGS4 but not RGS2 was found to inhibit
chemoattractant receptor-mediated motility in a transfected lymphoid
cell line (45).
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Concluding Comments |
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Prior exposure of cells to some signals enhances or depresses subsequent responses to others. Study of chemoattractant receptor cross-regulation has been facilitated by the development of cellular systems allowing genetic and biochemical manipulation. These investigations have disclosed a previously unrecognized complexity of receptor cross-regulation and indicate that this occurs by at least two distinct mechanisms. The first, at the level of receptors, is mediated through phosphorylation by protein kinases activated by second messengers. A second, downstream regulatory site also controls the activation of classes of chemoattractant receptors. Cross-regulation via this site inhibits the activation of PLC and appears to be shared by groups (classes) of receptors using the same G-protein. Chemoattractant receptors have a hierarchy in producing desensitizing signals for both sites, which is inversely correlated with their susceptibility to desensitization. This hierarchy appears to be related to the length of signaling, which in turn is regulated by receptor phosphorylation as well as rate of internalization. Of interest, chemoattractant receptors for the same or similar ligands (i.e. IL-8) appear to be differentially regulated solely by their signal length, which endows the receptor with different biological activities (i.e. migration versus cytotoxicity) and abilities to cross-desensitize other receptors.
The concepts currently being developed in leukocyte receptor
cross-regulation may well be of more general significance. For example,
receptors with both shared ligands and signal transduction pathways
mediate the biological effects of a large number of chemokines. Among
other activities, chemokines regulate the migration and homing of T and
B lymphocytes and also act as coreceptors for HIV infection (46, 47).
As these cells contain multiple receptors for chemokines, their
migration as well as their susceptibility to infection may be subject
to receptor cross-regulation. Recent studies indicated that defects in
host defense in opiate abuse may be because of cross-regulation and
inhibition of chemokine receptors (48). Although the molecular
mechanisms are not clear, there is already evidence that leukocyte
adhesion molecules and chemoattractant receptors cross-regulate each
other's function in coordinating the transmigration (49). Here again,
some of the general principles of chemoattractant receptor
cross-regulation appear significant as both receptor
phosphorylation-dependent and -independent mechanisms are
likely involved (50, 51). In mammals the sense of smell is mediated
through thousands of G-protein-coupled receptors that undergo rapid
desensitization (52). Selective receptor cross-regulation may form a
basis for rapid desensitization to similar odors without affecting the
sense of smell to others. The recognition that receptor
cross-regulation is an important way to fine tune the cellular
responses should allow greater attention to this area of research and a
more precise understanding of cross-regulatory mechanisms.
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FOOTNOTES |
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* This minireview will be reprinted in the 1999 Minireview Compendium, which will be available in December, 1999. This work was supported by National Institutes of Health Grants DE-037838 (to R. S.), HL-54166 (to H. A.), AI-38910 (to R. M. R.), and AI 43184 (to B. H.).
¶ To whom correspondence should be addressed: Depts. of Medicine and Immunology, Duke University Medical Center, Box 3701, Durham, NC 27710. Tel.: 919-684-2345; Fax: 919-681-7020; E-mail: snyde001{at}mc.duke.edu.
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ABBREVIATIONS |
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The abbreviations used are:
fMLP, formylmethionylleucylphenylalanine;
LTB4, leukotriene
B4;
PAF, platelet-activating factor;
IL-8, interleukin-8;
HIV, human immunodeficiency virus;
PKA, protein kinase A;
PKC, protein
kinase C;
PLC, phospholipase C;
ATPS, adenosine
5'-O-(thiotriphosphate);
GTP
S, guanosine
5'-3-O-(thio)triphosphate;
FR, fMLP receptor;
PAFR, PAF
receptor;
mPAFR, phosphorylation-deficient, truncated PAFR;
IP3, inositol trisphosphate;
RGS, regulators of G-protein
signaling.
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REFERENCES |
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