From the
C5a is a 74-amino-acid glycoprotein whose receptor is a member
of the rhodopsin superfamily. While antagonists have been generated to
many of these receptors, similar efforts directed at family members
whose natural ligands are proteins have met with little success. The
recent development of hexapeptide analogs of C5a has allowed us to
begin elucidation of the molecular events that lead to activation by
combining a structure/activity study of the ligand with receptor
mutagenesis. Removal of the hexapeptide's C-terminal arginine
reduces affinity by 100-fold and eliminates the ability of the ligand
to activate the receptor. Both the guanidino side chain and the free
carboxyl of the arginine participate in the interaction. The guanidino
group makes the energy-yielding contact with the receptor, while the
free carboxylate negates ``electrostatic'' interference with
Arg-206 of the receptor. It is the apparent movement Arg-206 induced by
this set of interactions that is responsible for activation, since
conversion of Arg-206 to alanine eliminates the agonist activity of the
hexapeptides. Surprisingly, activation is a nearly energy-neutral event
and may reflect the binding process rather than the final resting site
of the ligand.
The anaphylatoxin C5a is a 74-amino-acid glycoprotein generated
on activation of the complement cascade. The responses evoked by C5a
imply that it is an important mediator of
inflammation(1, 2, 3) . For example, it is a
potent chemotaxin and secretagogue for neutrophils, macrophages,
eosinophils, and mast cells and stimulates the generation and release
of histamine, prostaglandins, leukotrienes, interleukin-1,
interleukin-6, and tumor necrosis factor. While C5a plays an important
role in host defense, increased levels of C5a have been associated with
a number of immune and inflammatory disorders including rheumatoid
arthritis, systemic lupus erythematosus, psoriasis, and acute
respiratory distress
syndrome(4, 5, 6, 7, 8, 9, 10) ,
observations that suggest that a C5a antagonist might have significant
therapeutic utility.
The C5a receptor is a member of the rhodopsin
superfamily(11, 12) , and while useful antagonists have
been synthesized for many family members whose natural ligands are
small molecules, similar efforts directed at family members that
normally bind proteins have been largely unsuccessful. For example,
although many moderately potent low molecular weight agonists have been
developed for the C5a receptor, with one exception, attempts to develop
antagonists have failed(13) . Thus, knowledge of the
interactions between ligand and receptor is important not only for
elucidating the mechanism of receptor activation but also for the
design of antagonists.
The binding domain of the C5a receptor
consists of two subsites(14, 15) . Site 1, located in
the receptor's extracellular N terminus, interacts with the
globular core of C5a, while site 2 binds the C-terminal 8 amino acids
of C5a and appears to lie in the receptor's interhelical region.
Site 1 provides the energy necessary for high affinity binding and also
facilitates the interaction between the C terminus of C5a and site 2 of
the receptor. It is this interaction at site 2 that is primarily
responsible for receptor activation. Several laboratories have
described hexapeptide analogs of C5a, which bind with moderate affinity
at site 2(13, 16, 17) . Their small size makes
these ligands readily amenable to modification and therefore useful
probes for investigating the mechanisms of receptor activation. We have
taken advantage of these properties and have combined a
structure/activity study of the hexapeptide analogs with receptor
mutagenesis to begin dissection of the molecular events that can lead
to activation. In this communication we present evidence that movement
of Arg-206 of the recep-tor, as a result of interaction with the
ligands' C-terminal arginine, is a critical event in activation
of the receptor by the hexapeptides.
If the C-terminal carboxylate group of
the hexapeptide participates directly in an energy-yielding interaction
with the guanidino group of Arg-206, removal of either group will
eliminate that interaction resulting in a loss of binding energy. We
have just shown that removing or amidating the peptide's
C-terminal carboxylate results in a 40-200-fold loss of affinity
against the wild-type receptor. A similar loss of affinity for
carboxyl-containing peptides against the R206A receptor would be
diagnostic of a positive interaction. However, the R206A mutation has
little effect on the affinity of hexapeptides that terminate in
arginine
These data strongly suggest that the basic side chain of Arg-206 is
positioned so that it is an essential element of an electrostatic
barrier to interaction between the receptor and the C-terminal
guanidino moiety of the hexapeptides or C5a. The carboxylate group
neutralizes the barrier so that repulsion is only apparent when ligands
contain a positively charged C-terminal guanidino moiety but lack a
corresponding negative charge normally supplied by the acid
functionality. Under these conditions, Arg-206 impedes an
energy-yielding contact between the guanidino group of the ligand and
the wild-type receptor. Elimination of the barrier by removal of the
side chain of Arg-206 restores potency to peptides with missing or
blocked carboxyl groups. Ligands that contain a C-terminal arginine
bind more avidly to the wild-type receptor because the negatively
charged carboxylic acid moiety of arginine shields the opposing
positive charges between ligand and receptor. Thus, Arg-206 functions
as a gatekeeper denying access to positively charged ligands until
presentation of a password in the form of a shielding negative charge.
The
activation pathway used by C5a also involves Arg-206, although the
requirement is not absolute since C5a can induce functional responses
in cells transfected with the R206A receptor. The mechanistic details
that underlie the differences in activation pathways utilized by C5a
and the hexapeptides remain to be elucidated, but they must reflect
differences in the interaction domains between the receptor and the
ligands.
Activation of the C5a receptor is an almost energy-neutral
event. The R206A mutation has little effect on binding affinity but
eliminates the ability of the hexapeptides to initiate activation.
Similarly, a series of similar C5a hexapeptide analogs has been
described in which changes in position 5 (C terminus is position 6) led
to a progressive loss of agonism with little change in binding
affinity(13) . This property may, in part, underlie the
difficulties encountered in developing low molecular antagonists, as
opposed to agonists, for this
receptor(13, 16, 17, 25, 26) ,
since it suggests that the barrier to activation is low and readily
overcome. However, most of those efforts have focused on maintaining,
or mimicing, the interactions made by the C-terminal arginyl residue.
While the one low molecular antagonist that has been described does
contain a C-terminal arginine(13) , the current results suggest
that such a strategy is more likely to generate agonism than antagonism
and that a structure-based approach which avoids those contacts has a
greater probability of success. Finally, our observations demonstrate
that negation of deleterious interactions may be just as important as
direct energy-yielding contacts in determining binding affinity and
ligand-receptor specificity.
We thank Drs. Jerome Langer, William Moyle, and Linda
Wicker for helpful discussions and critical reading of this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Materials
Human C5a was purified and iodinated
as previously reported(18) . Peptides were synthesized as
reported previously, and their structures were confirmed by
electrospray ionization mass spectrometry.
Receptor Mutagenesis and Transfection
Mutagenesis
of the C5a receptor to replace Arg-206 with alanine was accomplished in
two rounds of polymerase chain reaction, which overlapped and extended
the receptor cDNA from the ScaI to NotI restriction
sites. The R206A mutant cDNA expression plasmid was sequenced in its
entirety prior to introduction of the plasmid into mammalian cells.
Stable transfectants of the wild-type and mutant C5a receptors were
generated as described previously(14) . To ensure that the
phenotype of the R206A line is a reflection of the single mutation, and
not of a single stable cell isolate, we demonstrated that binding
properties of membranes prepared from HEK-293 cells transiently
transfected with the R206A receptor were identical to those obtained
from the stable line (data not shown).
Binding Assays
All assays were carried out by
competition against I-C5a as described previously using
membranes prepared from human neutrophils (19) or RBL-2H3 cells
stably transfected with either the wild-type or R206A human C5a
receptors (14). The buffer used for these assays was 0.05 M
Hepes, pH 7.2, containing 0.1% bovine serum albumin, 5 mM MgCl
, 1 mM CaCl
, 1 mM phenylmethanesulfonyl fluoride, and 10 µg/ml each of
aprotinin, chymostatin, and leupeptin.
Functional Assays
Measurement of myeloperoxidase
release from human neutrophils (20) and ligand-induced
Ca fluxes in transfected RBL-2H3 cells (14) were carried out as described previously.
The Hexapeptide C-terminal Arginine Is Important for
Both Receptor Activation and High Affinity Binding
Removal of
the C-terminal arginine from C5a decreases its binding affinity for the
wild-type receptor by 100-fold(21) , and as shown in , similar decreases also occur for the hexapeptide analogs, 1 and 2. Both of these analogs are agonists as measured
by their ability to stimulate either degranulation (Fig. 1) or a
Ca flux from human neutrophils, or a Ca
flux from rat basophilic leukemia cells (RBL-2H3) stably
transfected with the wild-type human C5a receptor(14) . However,
these agonist activities are eliminated by removal of the C-terminal
arginine as illustrated for degranulation in Fig. 1. The decrease
in functional activity is not simply a reflection of decreased affinity
since the EC
values for peptides 1 and 2 are
about 0.2 µM (EC
degranulation/IC
binding
3), while the des-Arg peptides show little activity
at concentrations as high as 300 µM (EC
/IC
>30). Rather, the change
represents a true loss of the ability to activate the receptor. Thus
understanding the interaction of the C-terminal arginine with the
receptor should provide insight into the activation process.
Figure 1:
Removal of the C-terminal arginine
reduces the agonist activity of the hexapeptides. The abilities of the
peptides to activate the C5a receptor were evaluated by measuring the
stimulated release of the granule enzyme myeloperoxidase (mpo)
from human neutrophils. Data are shown for the agonists peptides 1
() and 2 (
) as well as des-Arg analogs 1d (
) and 2d
(
) (see Table I for sequences). Also shown are the data for
peptides 3 (
) and 4 (
) (see Table II for sequences). The
results are the averages of triplicate determinations. Similar results
were obtained from Ca
flux experiments on both
neutrophils and RBL-2H3 cells expressing the wild-type C5a receptor.
Although not shown, peptide 5 (Table II) is an agonist as measured by
both degranulation and Ca
flux.
The Guanidino and Carboxyl Groups Are Both Necessary for
High Affinity Binding
The hexapeptide's C-terminal
arginine contains two functionalities that may participate in receptor
binding, the side chain guanidino group and the free carboxylic acid.
To probe the relative contributions of the two groups we have
selectively eliminated them by making appropriate changes to the parent
hexapeptide, 2. Substitution of agmatine for arginine, which
deletes the carboxylate group while leaving the guanidino side chain,
reduces affinity 200-fold to a value (IC = 10
µM) equivalent to that of the des-Arg peptide (2d
and 3, ). Likewise, substitution of alanine for
arginine, which deletes the side chain while leaving the carboxylate,
reduces the affinity 40-fold (peptide 4, ). Thus,
both the guanidino and carboxyl groups contribute to binding and appear
to act in concert, as removal of either is equivalent or nearly
equivalent to removal of the entire arginine residue.
Relationship between Arg-206 of the Receptor and the
C-terminal Arginine of the Hexapeptides
The role the carboxyl
group plays in interaction with the receptor is likely to be related to
its electrostatic properties. This hypothesis is supported by the
observation that amidation of the group, to eliminate the negative
charge, produces a reduction in affinity similar to that caused by its
deletion (peptide 5, ). Inspection of the receptor
sequence suggested that Arg-206 at the top of transmembrane helix 5 (11, 12) was a likely candidate for interaction with the
carboxyl group. Further, Arg-206 aligns with Lys-199 of the angiotensin
II type 1 receptor, the residue proposed to interact with the
carboxylic acid moiety of angiotensin II (22) and with Ser-203
of the -receptor, which appears to be involved in catechol
binding(23) . Alanine substitution of Arg-206, to create the
mutant C5a receptor R206A, eliminates the potentially interactive side
chain while maintaining stereochemistry and allows us to test whether
this is the site of contact.
(
)or on C5a (), a result
that clearly shows the lack of a positive interaction between the
groups. Remarkably, however, peptides that contain a guanidino group
but have their carboxyl groups deleted (peptide 3, ) or amidated (peptide 5, ) exhibit
greatly increased affinity on the mutant receptor. In fact, the R206A
mutation almost precisely compensates for the affinity loss caused by
these carboxyl modifications when binding is measured against the
wild-type receptor (). Thus, while there is little direct,
energy-yielding interaction between the the peptides' C-terminal
carboxyl group and the guanidino group of Arg-206, the R206A mutation
eliminates the effect of, and the requirement for, the free carboxyl
group. The role of the carboxyl group seems to be to prevent a loss of
binding energy rather than as a positive contributor to binding.
Arg-206 Is Required for Receptor Activation by the
Hexapeptides
Since the hexapeptide's C-terminal arginine
is important for receptor activation and our data demonstrate an
interaction between this group and Arg-206 of the receptor, it seemed
likely that Arg-206 would play a key role in the activation process.
This is indeed the case. As shown in Fig. 2, the agonist peptides 1 and 2 both elicit a Ca flux from RBL
cells stably transfected with the wild-type receptor. In contrast, the
same peptides fail to generate a Ca
flux in cells
transfected with R206A receptor, even at concentrations as high as 100
µM (Fig. 2). The inability to stimulate the mutant
receptor is not due to global misfolding since the binding affinities
of the two hexapeptides are unchanged by the arginine to alanine
substitution. Similarly, hexapeptides 3 and 4, which are
agonists on the wild-type receptor, fail to activate the R206A mutant.
Thus Arg-206 plays an important role in receptor activation as well as
ligand recognition for the hexapeptides. While the effects are not as
dramatic, the R206A substitution alters activation by C5a as well.
Greater concentrations of C5a are required to generate a Ca
flux, and the profile of that flux has been changed by the
mutation (Fig. 2).
Figure 2:
The R206A mutation abolishes activation by
hexapeptide agonists but not by C5a. The ability of the ligands to
activate the receptors was determined by the induction of
Ca fluxes in RBL-2H3 cells transfected with either
the wild-type (A, C, E) or R206A (B, D, F) receptors. The hexapeptides were
used at concentrations of 1 nM (
), 10 nM (
), 100 nM (
), 1 µM (
),
10 µM (
), and 100 µM (▾).
Results with the agonist hexapeptide 2 are shown in A and B and in C and D for hexapeptide 1. The
effect of C5a, at concentrations of 1 pM (
), 10 pM (
), 100 pM (
), 1 nM (
), and 10
nM (
) are shown in E and F for the
wild-type and mutant receptors, respectively. Both transfected lines
expressed approximately 50,000 receptors/cell. Although not shown,
peptides 3 and 4 also fail to activate the R206A mutant receptor. All
of these hexapeptides are antagonists for the R206A receptor, since
they block activation stimulated by C5a.
Model for Hexapeptide Activation of the
Receptor
Our model for how the ligand recognition and activation
functions of Arg-206 are coupled is shown in Fig. 3. At some
point in the binding process the ligand's C-terminal arginine is
located so that it is subject, either directly or indirectly, to
substantial influence by Arg-206. This influence is bidirectional,
altering the position of Arg-206 and initiating the conformational
cascade that leads to receptor activation. More specifically, we
believe that a transient electrostatic interaction between the
C-terminal carboxylate of the ligand and the side chain of Arg-206
moves the latter, allowing the receptor to more readily accommodate the
ligand's guanidino group. It is the interaction between this
guanidino group and the receptor that accounts for most of the 2 kcal
of binding energy provided by the ligand's C-terminal arginine.
However, since the R206A substitution eliminates the ability of the
arginine-containing hexapeptides to activate the receptor without
altering their binding affinities, it is the movement of Arg-206 that
is responsible for activation. Such a change in position might induce
activation either because Arg-206, in its original location, acts as a
negative regulator holding the receptor in a quiescent state or because
its movement acts as a positive signal. Since the R206A receptor is not
constitutively active, relocation of Arg-206 is a positive signal.
Figure 3:
Model of receptor activation. Binding of
the hexapeptides to the receptor involves an interaction between
Arg-206 of the receptor and the ligand's C-terminal arginine. The
free carboxyl of the C-terminal arginine negates electrostatic
interference between the two guanidino groups, perhaps, as shown, by
moving the side chain of Arg-206. It is the change in position of
Arg-206 that appears to be responsible for hexapeptide-induced receptor
activation. See text for details.
Although the details of how movement of Arg-206 leads to activation
remain to be elucidated, two general classes of models can be proposed.
First, Arg-206 may make a new set of specific interactions following
ligand binding that initiate a conformational cascade in the receptor,
or alternatively, the induced movement of Arg-206 may, somewhat less
specifically, force a change in the relative orientations of the
transmembrane helices. The observation that both peptide 3,
which lacks the carboxyl group, and peptide 4, which lacks the
guanidino group, can, unlike the des-Arg peptides, activate the
receptor (Fig. 1) provides support for the second model. Although
possible, it seems unlikely, that peptides 3 and 4 would
force movement of Arg-206 to precisely the same location as the
arginine containing peptide 2. However, both peptides 3
and 4 are likely to induce a change in the position of Arg-206,
a movement that could alter the orientations of the helices. In this
regard several groups have proposed such reorientation as the generic
mechanism of activation for G protein-coupled receptors (24).
Table: Hexapeptides and the effect of removing the
C-terminal arginine on affinity
Table: 1634494836p4in
The IC
of the
peptides was determined by competition binding against
I-C5a on membranes prepared from RBL-2H3 cells into which
either the wild-type C5a receptor or the R206A receptor was stably
transfected. The values shown are averages determined from the number
of experiments given in parentheses.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.