(Received for publication, March 30, 1995; and in revised form, May 18, 1995)
From the
The C5a anaphylatoxin ligand-receptor interaction on
polymorphonuclear granulocytes stimulates chemotaxis, degranulation,
and the oxidative burst. The receptor is a member of the large
G-protein-coupled family. The ligand is a cationic peptide of 72 amino
acids derived from the C5 component of complement and has been shown to
have a number of structural requirements for interaction with the
receptor. In order to probe the potential interaction sites between
ligand and receptor, we constructed a series of mutated receptor
molecules, targeting cysteines, prolines, and additional amino acids of
interest because of combinations of charge or hydrophobicity and
putative location with respect to the membrane. Transfected mutant
receptors were analyzed for cell surface expression, ligand binding,
and ligand-activated phospholipase C activity. The receptors created
can be placed generally in four distinct classes: those which bind and
signal like the natural receptor; those which bind but fail to
transduce signals; those which are expressed but neither bind nor
transduce signal; and those which are not expressed at the cell
surface. The human complement 5a (C5a) anaphylatoxin receptor is a member
of the large family of G-protein-coupled receptors
(GPCRs) Classical studies of the C5a
ligand demonstrate two regions of the protein that are required for
high affinity binding and potent signaling via heterotrimeric
G-proteins(2, 7, 8, 9, 10) .
The conformationally critical disulfide bonds present in C5a knit a
core domain essential for high affinity receptor binding. Chemical and
exopeptidase cleavages demonstrate that the ligand COOH-terminal region
encodes the receptor activating sequence,
MQLGR(11, 12) . Further studies using site-directed
mutagenesis have located distinct receptor contact points within the
ligand NH More recently, evidence for distinct binding and
activation sites in the C5a receptor has been derived from mutagenesis
and monoclonal antibody studies showing the importance of the receptor
NH Signal transduction by the
C5a/C5a receptor pair in COS cells results in minimal activation of PI
kinase and undetectable activation of PLC (1) . Previous
studies indicate coupling of the C5a receptor to G These findings suggest that C5a like other
peptidergic ligands that utilize GPCRs conform to the message:address
model of Schwyzer(19, 20) , where the
``address'' domain binds ``recognition'' sequences
in the receptor and presents the ``message'' domain to the
agonist site in the receptor. Previous studies demonstrate that
human C5a can bind and activate the murine C5a receptor with potency
almost identical to the human receptor, suggesting that elements in
common for both species provide the agonist site. Comparison of the
murine C5a receptor to the human molecule demonstrates that the
transmembrane segments are well conserved, while extracellular
sequences are quite divergent, thus providing a dramatic
``mutagenesis'' result(1, 21) . Since data
to date suggest that the address site for C5a is largely extracellular,
we undertook site-directed mutagenesis of hydrophilic and charged
residues within the transmembrane segments to investigate potential
message sequences.
Human embryonic kidney 293 cells were maintained as described for
COS cells and transfected with cationic liposomes as described
previously(23) .
COS
cells were plated on fibronectin-coated glass slide dishes (Nunc,
Naperville, IL) and transfected as described above. Cells were washed
with PBS, and incubated in PBS containing 3% BSA for 10 min at room
temperature to block nonspecific sites. Monoclonal anti-C5a receptor
antibody S5/1 (13) (generously provided by Prof. Otto Gotze,
Department of Immunology, University of Gottingen, Germany) was added
at 10 µg/ml and incubated 30-60 min at room temperature.
Cells were washed three times with PBS and subsequently stained with
biotinylated goat anti-mouse IgG, followed by avidin-biotin complex,
hydrogen peroxide and diaminobenzidine, and examined by light
microscopy (Olympus BH-2 microscope). Controls for nonspecific staining
included cells transfected with vector alone, and the staining pattern
compared to that obtained with cells fixed in 2% paraformaldehyde prior
to any antibody incubation, to confirm cell membrane expression.
Figure 1:
Schematic
diagram of the human C5a receptor. The location of each mutation
generated in the human C5a receptor (1) is shown as a filled circle, except Gln
Within the transmembrane segments, the C5a receptor contains only
two acidic residues (Asp Among the cysteine
residues mutated, three are required for binding of C5a
(Cys Cys
Figure 2:
Characterization of expression of C5a
receptor mutants. Indirect immunoperoxidase staining of unpermeabilized
transfected COS cells using a monoclonal anti-C5a receptor antibody was
performed on all mutants which showed abnormal binding (see Tables
I-III). Three patterns of staining were observed in all mutants and
examples of each are shown. Panels A and B show the
normal strong expression of C5a receptors on the cell surface (A, wild-type C5aR; B, Cys
Mutation of the remaining transmembrane
cysteines had differing effects on function of the receptor.
Cys
G-protein-coupled receptors appear to have evolved a common
strategy for the recognition of large peptide ligands. Ligand binding
to elements in the NH
In mutagenesis
experiments of other receptors, the aspartic acid residues in the
canonical tripeptide ``DRY'' and the TMS-2 motif
``LAVAD'' have been implicated in G-protein interaction and
conformational changes during activation(31) . The C5a receptor
also encodes an asparagine residue (Asn The results from
mutagenesis of all transmembrane cysteine residues show that three are
required for binding (Cys The Cys The remaining cysteine mutants all bind
ligand with wild-type characteristics and activate PLC. Two
(Cys
Additional mutations
(Leu
Using site-directed mutagenesis, and subsequent
binding and signaling studies of the mutant C5a receptors, we have
identified amino acids that are involved in the activity of the message
site. These amino acids located in the transmembrane segments are
Asp The
message:address model suggests that a unique site in the receptor
provides the message sequence. These studies do not distinguish direct
effects on the message sequence from effects on other aspects of the
change in state from inactive to activated receptor. As agonists and
antagonists are developed for the C5a receptor, these mutants will
provide additional insights to C5a receptor function and aid in the
identification of the second message site.
(
)(1, 2) . A paradigm
for receptor-ligand interactions among low molecular weight agonists
such as retinal and catecholamines has the ligands binding to their
receptors at a single site within the pocket formed by transmembrane
segments(3, 4) . In contrast, peptidergic
agonist-receptor systems such as those for C5a, interleukin-8, and
pituitary glycoprotein hormones appear to display two regions of
interaction: one receptor domain largely confers binding affinity,
while a second site mediates ligand biological
activity(5, 6) .
-terminal region and the disulfide
core(10) .
terminus for high affinity ligand
binding(13, 14, 15) . Treatment with a spider
venom metalloproteinase suggests that specific cleavage of the receptor
blocks binding of intact C5a, but not COOH-terminal C5a agonist
peptides(16) . Similarly to C5a, the cationic chemokines
interleukin-8, and MGSA/GRO
also recognize acidic
NH
-terminal receptor sequences, and selectivity in ligand
binding for these highly homologous receptors can be transferred with
molecules chimeric at the NH
terminus(17, 18) .
in
polymorphonuclear leukocytes to activate endogenous PLC
2, by
release of the
subunits(45, 46) . As the
PLC
2 isoform is absent in COS cells, signaling through the C5a
receptor can be assessed by taking advantage of the ability of
G
to substitute for the G
subunit,
which, following activation by ligand, stimulates the endogenous COS
cell PLC(27) .
Materials
Human C5a anaphylatoxin was expressed
in Escherichia coli(22) or obtained from Sigma. COS
cells (CRL 1651) and 293 cells (CRL 1573) were obtained from The
American Type Culture Collection, Bethesda, MD. I-Human
C5a and
H-inositol were from DuPont NEN. DEAE-dextran
(average molecular weight 500,000) and chloroquine were from Sigma.
Anti-FLAG M2 monoclonal antibody was obtained from IBI (New Haven, CT).
Fluorescein isothiocyanate goat anti-mouse IgG was from Becton
Dickinson (Lincoln Park, NJ), biotinylated anti-mouse IgG, horseradish
peroxidase-labeled avidin-biotin conjugates from Vector Labs
(Burlingame, CA), and mouse anti-horseradish peroxidase IgG1 was
obtained from Zymed (South San Francisco, CA).
C5aR Mutagenesis
The human C5aR cDNA was modified
to express an amino-terminal epitope by adding the FLAG epitope
sequence (MDYKDDDDKEF) and converting the initiating methionine (ATG)
to leucine (CTG)(23) . As described previously, this sequence
has no significant effect on binding or expression(23) . The EcoRI/XbaI cassette encoding the FLAG-wild-type C5aR
cDNA was subcloned into the mutagenesis vector pSELECT-1 (Promega
Corp., Madison, WI). Oligonucleotide-directed mutagenesis was carried
out using the Altered Sites mutagenesis protocols (Promega Corp.). All
mutations were verified by DNA sequencing (Sequenase, U.S.
Biotechnology Inc., Cleveland, OH)(24) . Mutant cDNAs were
subcloned into the mammalian expression vector, pCDM8, for functional
studies (25) .Cell Cultures and Transfection
COS cells were
maintained and transfected using DEAE-dextran as described
previously(1) . Cells were subcultured at 8
10
/10-cm plate 16-24 h before transfection. At
transfection, media was replaced with DMEM containing 2% fetal bovine
serum or Nu-serum (Collaborative Research, Bedford, MA), 1 mM chloroquine, 400 µg/ml DEAE-dextran, and 1 µg/ml plasmid
DNA. Cells were incubated at 37 °C for 3.5-4 h, transfection
medium aspirated and replaced with 10% Me
SO in PBS for
2 min at room temperature, and finally replaced with culture
medium. All functional studies were carried out 60-72 h later.
Binding Studies
Binding studies were conducted on
intact 293 cells or on crude membrane preparations from COS cells.
Membranes were prepared as follows: 60-75 h after transfection
cells were washed twice with PBS, scraped into TEN (40 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl), and
ruptured by one cycle of freeze-thaw in liquid N. Membranes
were pelleted by centrifugation (4 °C, 36,000
g for 15 min), resuspended in TEN, and protein concentration
determined by Coomassie Blue staining compared with BSA standards.
10
intact 293 cells or 100-200 µg of COS cell
membrane protein were used in binding experiments; in Hank's
balanced salt solution, 25 mM HEPES, 0.1% BSA, incubating with
0.1 nM
I-C5a and varying amounts of
unlabeled C5a on ice for 45 min. Unbound ligand was separated by
filtration on glass fiber filters (Whatman GF/F) that were soaked
overnight in 0.1% polyethyleneimine. Filters were washed three times
with ice-cold binding buffer. Bound ligand was quantitated by liquid
scintillation counting. Each binding experiment was conducted three
times in triplicate. All binding data presented in Table 1were
determined using membranes from transfected COS cells. Binding
affinities (K
) and sites/cell (B
) were determined using the Ligand program (26) . The relative counts/minute bound at 0.1 nM
I-C5a for each mutant compared to wild-type is also
presented.
Cell Surface Expression
Initial expression studies
were performed using FLAG-C5a receptor plasmids transfected in 293
cells. Three days after transfection, cells were suspended by
trituration in PBS. Approximately 10 cells were incubated
in PBS containing 3% BSA and 10 µg/ml anti-FLAG M2 antibody or
isotype matched anti-horseradish peroxidase for 1 h on ice. Following
staining with fluorescein isothiocyanate anti-mouse IgG, cells were
fixed in 2% paraformaldehyde and analyzed by flow cytometry.
Signal Transduction by Transfected
Receptors
Ligand-stimulated activation of phosphatidyl
inositol-specific phospholipase C was assessed as described previously (27) . Cells were co-transfected with C5a receptor or mutant
and a plasmid encoding G,
48 h later they were
washed with inositol-free DMEM (Life Technologies, Inc.), and incubated
another
24 h in this medium containing 10 µCi/ml
[
H]inositol and 10% fetal bovine serum. Labeling
medium was removed, and cell layers were incubated in triplicate in
inositol-free DMEM, 10 mM LiCl, and 0-150 nM C5a for 30 min at 37 °C. Reactions were terminated by addition
of an equal volume of 10% HClO
containing 4 mg/ml phytic
acid followed by incubation at 0 or -20 °C for at least 30
min. Inositol phosphates were purified by chromatography on Dowex-1
(formate; Bio-Rad) (28) and quantitated by liquid scintillation
counting. C5a-induced PLC activation was compared with controls in the
absence of ligand and untransfected cells. COS cells transfected with
vector alone or with unrelated receptor cDNAs were not activated by
C5a. All experiments were carried out in triplicate; values are
expressed as the mean ± S.E. percent stimulation above base line
and at least two independent experiments were conducted for each mutant
receptor.
Mutations of Acidic and Cysteine Residues
As the
carboxyl terminus of the C5a (arginine 74) is crucial for the full
potency of signaling activity of the protein (9) our strategy
utilized for this first phase of mutagenesis was to mutate residues
that are negatively charged or can provide a partial negative charge to
neutralize the guanidinium group. The mutants were made of the aspartic
acid and cysteine residues within the transmembrane segments (Fig. 1). As shown in Table 1, the binding, signaling, and
expression pattern of each mutant was investigated by transient
expression in COS cells. Analysis of binding and cell surface
expression were performed with transfection of the receptor cDNA alone,
while signal transduction for phospholipase C activation was evaluated
in cells co-transfected with G. Previous
investigations demonstrated no change in binding or expression in the
presence or absence of this G
subunit(27) .
Stop which is
shown as a filled square. Cys
and Cys
form a disulfide bond (indicated by stippled line). A
single consensus asparagine-linked glycosylation site is indicated. The
seven transmembrane segments are shown boxed and bordered
approximately by the dashed horizontal lines. The boundaries
of each of the predicted transmembrane domains conform to the
three-dimensional model proposed for
rhodopsin(31) .
and Asp
). These
amino acids, as well as Asn
, were each mutated
conservatively (Table 1). While the Asp
Asn
mutant did not express efficiently on the cell surface,
Asp
Asn and Asn
Gln mutants were
expressed on the cell surface and bound ligand with slightly lower K
than wild-type. These latter two
mutations had some effect on the ability to express the receptors at
the membrane, as the B
for each is lower than
wild-type. The Asp
Asn mutant is completely defective
at stimulating PLC activity, while the Asn
Gln
mutant is only 35% as effective as wild-type.
, Cys
, and Cys
).
Cys
and Cys
are positioned at the
homologous positions to the amino acids in rhodopsin and the
2-adrenergic receptor that form an intramolecular disulfide
bond(29, 30) . The mutations on the cysteines in the
first and second extracellular loops are not expressed on the cell
surface, as indicated by flow cytometry (Table 1). The binding
deficit for these two mutations is expected.
is
predicted to lie deep within TMS-5 and oriented toward TMS-4. To
determine if mutants were properly expressed on the cell surface,
indirect immunohistochemical staining of transfected COS cells was
performed using the C5a receptor-specific monoclonal antibody
S5/1(13) . Fig. 2shows typical results with wild-type
receptor (Fig. 2A) and the Cys
Ser (Fig. 2B) where both are expressed at high levels on
the cell membrane of unpermeabilized cells. These results indicate that
Cys
is required only for the binding of C5a. Random
mutagenesis at this site may reveal the spectrum of residues capable of
substituting for cysteine.
Ser). Panel C shows an example of mutations with aberrant staining
(mutant Pro
Ala). Panel D shows the
expression pattern of mutant receptors with very low levels of staining
(mutant Pro
Ala). Panel E shows the
staining of untransfected COS cells. Panels A-D are shown at 2
the magnification of panel
E.
Ser in TMS-2 resulted in a receptor with normal
binding affinity but only
30% of signal transduction in response
to 150 nM C5a (Table 1). Cys
Ser in
TMS-4 resulted in a receptor that binds ligand almost as well as
natural C5aR but produces no detectable PLC activation. Cys
is positioned at approximately the same depth as Cys
and is predicted to be oriented toward TMS-5(31) . The
Cys
Ser mutation binds ligand with wild-type
affinity and
43% of wild-type activation of PLC. In contrast,
mutation of both Cys
and Cys
yielded a
functionally normal receptor for binding and signal transduction. The B
for each of these mutants is close to that of
wild-type, except for Cys
Ser, which shows 4-fold
greater number of sites.
Mutation of Transmembrane Proline Residues
The
second strategy utilized in our mutagenesis experiments was to probe
the role of the structural rigidity of the receptor in ligand binding
by mutating transmembrane proline residues. The C5a receptor contains
16 proline residues, 7 of which are within or directly adjacent to
transmembrane segments. Of these, 4 (prolines 170, 214, 257, 297) are
well conserved among other members of the GPCR family and have been
mutated in the muscarinic M3 receptor(32) . The pattern of
functional importance of these ProAla mutations in the C5a
receptor differs substantially from that of the M3 muscarinic receptor (Table 2). Among the 7 proline TMS residues investigated, three
mutants (Pro
Ala, Pro
Ala, and
Pro
Ala) bind ligand at 0.1 nM
I-C5a comparable to wild-type receptor, despite the
observation that the B
determined from Scatchard
analysis for each of these mutants is substantially less than
wild-type. The other four mutants (Pro
Ala,
Pro
Ala, Pro
Ala, and
Pro
Ala) are unable to bind ligand at wild-type
levels. The expression patterns for these mutations were determined as
described for the cysteine mutations above. Only one of the
binding-impaired mutants expressed robustly
(Pro
Ala); the others were either expressed at very
low levels or in aberrant patterns of staining in cells ( Table 2and Fig. 2C). The ability of mutants that
bind ligand to stimulate PLC was determined. With the exception of
Pro
Ala, all other mutations were only partially
effective (Pro
Ala, Pro
Ala, and
Pro
Ala) for generating intracellular signals.
Mutation of Third Extracellular Loop Proline
Residues
Data from other receptors suggest that TMS6 and 7 may
be a separate functional domain(33, 34) . A mutation
was generated converting the LeuPro, yielding a
non-functional receptor that demonstrates a normal immunostaining
pattern (Table 2). This prompted us to produce the neighboring
mutation Pro
Leu, generating a protein that binds
minimally compared to untransfected cells and does not signal (Table 2).
Intracellular Loop Mutations
The third strategy
utilized to determine elements of functional importance in the C5a
receptor involved mutagenesis of intracellular loops. A recurrent theme
in receptor function is the interaction of heterotrimeric G-proteins
with intracellular loop residues, specifically in the third
intracellular loop and the proximal COOH-terminal tail. Seven point
mutations were generated and expressed (Table 3). Binding studies
with each of these mutants show that three have no effect on binding
(TrpPhe, Gly
Ala,
Ser
Ala) but have modest to severe effects on
levels of stimulation of PLC. The four remaining mutants showed
diminished levels of ligand binding at 0.1 nM
I-C5a, but rather dramatic effects on PLC stimulation,
indicating a perturbation in the interaction with G
.
A single point mutation in the first intracellular loop
(Arg
Glu), that disrupts both cell surface expression
and signal transduction, is unusual as most reported mutations of this
loop have little or no effect on expression or G-protein
coupling(35) . The B
for this construct
is very low, but other mutants with similar B
values (e.g. Pro
Ala) have normal
signaling capacity.
-terminal and extracellular loop
regions, constituting a high affinity binding site, leads to the
presentation of ligand sequences which activate receptors. The
identification of the latter sites has been approached in the present
work by site-directed mutagenesis of transmembrane sequences. As the
activating sequence of C5a is highly polar, we limited our mutagenesis
to residues with polar or structural characteristics.
Role of Hydrophilic and Cysteine Residues in the C5a
Receptor
Results from other GPCRs have demonstrated that charged
and other polar side chains are crucial for maintaining receptor
conformation and ligand binding. In the 2-adrenergic receptor, the
conserved aspartic acid residue in TMS-3 is required to provide a
counter ion to the catecholamine charge. In rhodopsin retinal is linked
via a Schiff base to Lys
, and the resulting charge is
thought to be balanced by Glu
((36) and reviewed in (37) ). Some mutations at these sites in rhodopsin are active
in the dark. Thus, charged residues within the TMS can be involved in
direct interaction with the ligand and/or play a role in the
equilibrium between active and inactive forms.
) at the position
where the amino group of catecholamine drugs binds to Asp
of
2-adrenergic receptors(38) . Conservative
mutations at these sites demonstrate that Asp
is
essential for the proper expression and trafficking of the C5a
receptor. Asp
and Asn
bind ligand nearly as
well as wild-type, but both are defective at stimulation of PLC via
G
(Table 1). This indicates that these residues
are crucial components for the generation of the active form of the
receptor. Because of differences in PLC activation and because both
provide wild-type-like binding, they may be different than similar
sites in the
2-adrenergic receptor.
, Cys
, and
Cys
) (Table 1). It has been previously determined
for other GPCRs, such as rhodopsin, that cysteines in extracellular
loops one and two are linked in a disulfide bond which stabilizes the
structure(39, 40) . The data from
Cys
Ser and Cys
Ser mutants are
consistent with these residues participating in a similar disulfide
bond in the C5a receptor. The Cys
Ser mutant does
not bind or signal and is expressed well at the surface of COS cells (Fig. 2B). Predictions of the structure of rhodopsin
suggest that a cysteine residue, present at the homologous position
(Cys
), is oriented within the transmembrane pocket toward
TMS-4(31) . In bovine rhodopsin Cys
can be
replaced by serine with little or no effect on function(29) .
It is unclear whether the carboxyl-terminal arginine of C5a directly
contacts this cysteine residue, as it is predicted to lie deep within
the plane of the transmembrane segments. Alternatively, Cys
may be important for structural changes associated with agonist
binding and activation.
Ser mutant binds
C5a almost as well as the wild-type receptor, but when co-transfected
with G
, fails to activate PLC. This cysteine is
predicted to be almost at the same depth within the membrane as
Cys
and is oriented toward Cys
in
structural models of GPCRs.
Ser and Cys
Ser) have
diminished ability to stimulate PLC.
The Role of Proline Residues in the C5a
Receptor
Structural studies of bacteriorhodopsin show that
proline residues within the transmembrane segments introduce kinks in
the helices, as the amine hydrogen is not available for helical
stabilization(41) . The data presented in Table 2show
that mutation of helical proline residues yields a number of changes in
receptor function. Among the prolines mutated, several are required for
the proper trafficking to the cell surface as demonstrated by binding
and immunohistochemistry experiments. Three fail to express well at the
cell surface (ProAla, Pro
Ala,
and Pro
Ala), and the expression of
Pro
Ala appears to cause dramatic changes in the
morphology of COS cells (Fig. 2C). Pro
,
unlike Pro
and Pro
, is not widely conserved
in other GPCRs but is present in other chemotactic receptors
(formylmethionylleucylphenylalanine and interleukin-8 receptors). The
effects of these mutations are likely the result of misfolding of the
receptors in the endoplasmic reticulum and regulation of this process
by chaperonins. A general defect in the processing of opsin in
Drosophila (ninaA) results from mutations in a cis-trans-prolyl isomerase, indicating that proline residues
in the TMS play a specific role in the ability of these receptors to
form proper structures(42) . Mutations of proline residues
corresponding to Pro
, Pro
,
Pro
, and Pro
in the C5a receptor have been
made in the rat M3 muscarinic receptor (Pro
,
Pro
, Pro
, and Pro
in the rat
M3 muscarinic receptor, respectively)(32) . The rat
Pro
Ala mutations at positions 242, 505, and 540 all affect the B
for N-methylscopolamine binding, and
the affinity of the Pro
mutant is two orders of magnitude
higher than wild-type. As shown in Table 2, the
Pro
Ala and Pro
Ala mutations in
the C5a receptor behave in a similar fashion to those in the muscarinic
receptor, but Pro
Ala provides 76% of the wild-type
binding and is diminished in its activation of PLC.
Pro
Ala is expressed on the cell surface and binds
ligand weakly, analogous to Pro
Ala in the
muscarinic receptor.
Pro and Pro
Leu) were made
in the extracellular loop connecting TMS6-7 (Table 2). Both
of these mutants are expressed on the cell surface.
Pro
Ala binds ligand (36% of wild-type), and both
fail to activate PLC. Studies of rhodopsin and the
2-adrenergic
receptor indicate that TMS6-7 are tightly associated in the
membrane and are able to function in ``split'' receptor
experiments caused by cleavage of rhodopsin with trypsin (43) or by co-expression of partial receptors(34) . Our
results indicate that the structure of the extracellular loop is
critical for adopting the necessary structure for activating
G-proteins.
Other Mutations
Mutations in the intracellular
loops of the receptor were constructed and characterized (Table 3). These mutations are unlikely to affect the direct
interaction of C5a with the receptor, but may shed light on the contact
points of G-proteins with the C5a receptor. In vivo the C5a
receptor has been shown to interact directly with
G(44, 45) , but due to the lack of
expression of G
-responsive PLC isotypes within COS
cells, we utilize co-transfections with G
in order to
study the receptor's ability to interact with
G-protein(27) . Among 7 intracellular loop residues mutated,
three (Trp
Phe, Gly
Ala,
Ser
Ala) have no effect on maximal binding of
C5a relative to the wild-type receptor, indicating that these sites are
not essential for precoupling of G-protein to the receptor. The
stimulation of PLC in each of these is diminished relative to
wild-type. Two additional point mutants (Thr
Ala and
Thr
Arg) are somewhat diminished in their ability to
bind ligand, and the ability of these receptors to stimulate PLC is
completely abrogated. This indicates this region of the third
intracellular loop is important for efficient coupling to G-proteins.
Truncation of the C5a receptor at Gly
diminishes binding
to 30% of wild-type, and as expected abolishes the ability to activate
PLC (Table 3).
, Asn
, Cys
, and
Cys
. Identification of the specific role of each of these
amino acids is beyond the scope of this work, but the nature of these
functional groups (acidic, thiol, or amide) suggests that hydrogen
bonding or ionic interactions within the 7TMS bundle are crucial for
providing ligand binding and G-protein activation. Proline mutations
and intracellular loop mutations of the receptor have distinct
behavior, for when they are defective in function, the number of
binding sites is also decreased, suggesting a processing defect.
SO,
dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's
medium; IP, inositol phosphate; PLC, phospholipase C; TMS,
transmembrane segment.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.