From the Department of Cellular and Molecular
Pharmacology, University of California, San Francisco, California
94143 and ** Cadus Pharmaceutical Corporation, Tarrytown, New York
10591-6705
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ABSTRACT |
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Hormones and sensory stimuli activate serpentine
receptors, transmembrane switches that relay signals to heterotrimeric
guanine nucleotide-binding proteins (G proteins). To understand the
switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random
saturation mutagenesis. A yeast selection identified 121 functioning
mutant receptors, containing a total of 523 amino acid substitutions.
Conserved hydrophobic residues are located on helix surfaces that face
other helices in a modeled seven-helix bundle (Baldwin, J. M.,
Schertler, G. F., and Unger, V. M. (1997) J. Mol.
Biol. 272, 144-164), whereas surfaces predicted to contact the
surrounding lipid tolerate many substitutions. Our analysis identified
25 amino acid positions resistant to nonconservative substitutions.
These appear to comprise two distinct components of the receptor
switch, a surface at or near the extracellular membrane interface and a
core cluster in the cytoplasmic half of the bundle. Twenty-one of the
121 mutant receptors exhibit constitutive activity. Amino acids
substitutions in these activated receptors predominate in helices III
and VI; other activating mutations truncate the receptor near the
extracellular end of helix VI. These results identify key elements of a
general mechanism for the serpentine receptor switch.
Serpentine receptors serve as ligand-activated molecular switches,
relaying signals from extracellular ligands to heterotrimeric ( A static model of the three-dimensional structure of the helix bundle
is beginning to take shape. A low resolution (6 Å) electron cryomicroscopic structure of rhodopsin (14), the retinal light receptor, reveals relative positions and tilts of seven transmembrane helices in the plane of the membrane. Based on many mutations, the
rhodopsin structure, and an analysis of the primary structures of more
than 500 rhodopsin-like serpentine receptors, Baldwin and co-workers
(15) constructed an How does the switch work? It is difficult to infer a conserved switch
mechanism from the functional effects of site-directed mutations
reported in a large number of different receptors, because relatively
few positions have been mutated in any one receptor (17, 18).
Accordingly, we undertook a systematic genetic analysis of a single
serpentine receptor, with the goal of identifying functionally
important residues and sites of helix-helix interactions that relay the
ligand signal to G protein activation. We selected functional receptors
after random saturation mutagenesis of the four transmembrane helices
(III, V, VI, and VII) most consistently implicated in ligand binding or
G protein activation by various serpentine receptors (3-6, 17, 18).
This comprehensive approach determines the relative importance of side
chains in each Library Construction--
To construct mutagenized C5a
receptors, we engineered restriction endonuclease sites (which did not
alter the amino acid sequence) into the C5a receptor gene at the
approximate boundaries of helices III (SphI and
PstI), V (BssHII), VI (BglII and
XhoI), or VII (HindIII). A NcoI site
at the start methionine introduced an aspartic acid in place of an
asparagine at amino acid position 2. To ensure efficient library
construction and eliminate the possibility of contamination by
wild-type receptor DNA, for each helix library we created a subcloning
vector that contained a piece of nonreceptor DNA substituted between
the flanking restriction sites. For example, in the Helix III
subcloning vector, a 383-base pair fragment of G Yeast Strains--
Strain BY1142, engineered by standard yeast
strategies, has the genotype far1 Yeast Transformation and Receptor Selection--
Using
electroporation, we cotransformed BY1142 yeast cells with plasmid
libraries of mutated C5a receptors and the C5a ligand plasmid, p1297.
Colonies bearing functioning receptors were selected by replica-plating
on histidine-deficient medium containing 1 mM
3-aminotriazole (AT)1
(Sigma). We recovered the plasmids encoding functioning C5a receptors and confirmed the phenotypes by retransforming each candidate C5a
receptor plasmid and the C5a ligand plasmid (or without C5a ligand, to
assess constitutive signaling) into the parental yeast strain, BY1142.
Growth after 3 days at 30 °C in the presence of different
concentrations of AT (0, 0.5, 1, 2, 5, and 10 mM) served to
quantitate the relative intensities of signals mediated by mutant
receptors. Each mutant receptor was tested at least twice and the
results were averaged. In each case, the wild-type C5a receptor
expressed in parallel yeast served as a positive control. The results
of ten independent transformations of wild-type C5a receptor were
growth at 5, 5, 5, 5, 5, 5, 2, 5, 2, and 5 mM AT. The
mutant receptor sequences were obtained by DNA sequencing with
Thermosequenase cycle sequencing (Amersham Pharmacia Biotech).
Selection of Functional Receptors--
We studied the human
chemoattractant C5a receptor, a member of the rhodopsin family of
serpentine receptors. This receptor activates Gi and
mediates chemotaxis of neutrophils toward the C5a ligand, a 74-residue
polypeptide (22, 23). To select functioning receptors from populations
of mutant receptors we took advantage of the G protein-mediated
response of Saccharomyces cerevisiae to mating pheromones
(24-26). We constructed a yeast strain, BY1142, in which activation of
the C5a receptor induces expression of the HIS3 gene,
thereby allowing cells with functioning receptors to survive and
proliferate in growth medium lacking histidine. In this selection
procedure, the relative intensities of signals mediated by mutant
receptors can be quantitated by assessing growth in the presence of
different concentrations of AT, a competitive inhibitor of the HIS3
gene product (27). Expression of both C5a and the C5a receptor allows
BY1142 cells to grow in selective medium lacking histidine and
containing up to 5 mM AT (Fig.
1).
Helix by helix, we subjected stretches of amino acid sequence to random
saturation mutagenesis, for a total of 93 positions in helices III, V,
VI and VII. Quality of the libraries was assessed in two ways. First,
DNA sequencing of 15-20 independent recombinants from each library
before pooling revealed oligonucleotide mutagenesis rates between
19-22%, resulting in amino substitution rates of 36-42% (Table
I). Second, we determined the
substitution rate at the third nucleotide position of degenerate codons
for amino acid positions at which few substitutions were observed in
functioning receptors (see below). The observed substitution rate of
17% closely matches the predicted rate of 20%, demonstrating that
little or no bias occurred in the randomization of the libraries.
We then selected recombinants from four separate libraries of mutant
receptors, each library containing mutations in a single helix, for
ability to support growth of BY1142 cells in the absence of histidine.
At an average mutation rate of 8-10 amino acid substitutions per
helix, fewer than one in 103 BY1142 cells transformed with
a mutated C5a receptor survived the
selection.2 In the four
scanned helices, the yeast selection identified 121 functioning mutant
receptors (at least 25 from each helix library, Figs.
2, 3,
4, and 5),
providing a data set of 523 amino acid substitutions (summarized in
Table I). The receptor helices tolerate an average of 4.3 amino acid
substitutions per mutated helix. We assume that most amino acid
substitutions occur at less important parts of the receptor switch;
some mutations, however, may compensate for functional defects caused
by other mutations in the same helix.
Patterns of Preserved Residues Confirm the Structural
Model--
We analyzed the mutant C5a receptors, amino acid position
by position, looking for physical properties of side chain character and hydrophobicity that were present or "preserved" in every
functional receptor. For this analysis, we defined side chain character
as preserved if we observed at that position no more than one amino acid substitution or if all substitutions at that position involved closely related amino acids (log odds score of 1.0 or greater in the
PAM 250 scoring matrix (28)). Single amino acid substitutions were
included to allow for the rare mutation that might have occurred at a
critical position, but only in the setting of other compensatory substitutions. For example, the Tyr-222 position is conserved in 91%
of serpentine receptors (15) and is preserved in 39 of the 40 mutated
receptors in our study. The single Y222H substitution obtained in
Arg-42 occurs in the setting of substitutions in the two neighboring
residues, C221S and T223N (Fig. 3). Hydrophobicity is considered
preserved at positions where only hydrophobic amino acids with free
energy transferoctanol of >0.1 kcal/mol (29) are observed.
The functioning mutant receptors preserve side chain character at 25 of
the 93 positions mutated (Figs. 2-5). Thirty of the 93 mutated
positions preserve their hydrophobic character in functioning
receptors; this group includes 11 of the 25 positions at which side
chain character was preserved (Figs. 2-5).
We expected that in functioning mutant receptors the side chains that
face other helices would tolerate fewer mutations than side chains that
face the surrounding lipid, because association of transmembrane
helices with one another depends upon preservation of complementary
shapes that maximize van der Waals interactions (30). Moreover, the
established role of hydrophobic interactions in stabilizing the
tertiary structure of soluble proteins (31, 32) suggests that
hydrophobicity should be preserved at positions that point toward other helices.
The results of our analysis meet both expectations; the patterns of
preserved side chain character and hydrophobicity strongly confirm the
helix orientations specified in the Baldwin model. In helical wheel
plots based on the Baldwin model, both sets of residues
(character-preserving and preserved hydrophobics) point toward other
helices or toward the center of the helix bundle, rather than toward
the surrounding lipid (Fig. 6). The
argument is circular, however, because the Baldwin model itself (15, 16) is based on patterns of side chain character and hydrophobicity conserved through evolution. Nonetheless, the patterns observed in
mutant receptors constitute experimental validation of the model. A
quite different approach, based on substituting cysteines at many
positions in helices of the D2-dopamine receptor, also furnishes a comprehensive confirmation of the helix orientations specified by the Baldwin model. In these experiments, cysteines that
proved accessible to a water-soluble chemical probe were located on the
inner faces of the four helices tested (III, IV, VI, and VII) (33-36).
In contrast to regions of the transmembrane helices involved in
helix-helix interactions, hydrophobicity is not strictly preserved at
positions predicted by the Baldwin model to face the surrounding lipid.
12 of the 29 mutated positions that tolerate substitution of a polar
residue are predicted to contact the lipid. This striking tolerance of
polar side chains in lipid-contacting faces of transmembrane helices
confirms a previous analysis of random mutations in three transmembrane
helices of a bacterial diacylglycerol kinase (37).
To assess the reliability of the mutation and selection procedures for
identifying side chains important for receptor function, we tested the
effect of a single amino acid substitution at many of the preserved
positions (Table II). Fourteen of 17 such
point mutations tested do impair receptor function (0 or + growth
versus +++ for the unmutated C5a receptor; see Fig. 2
legend), indicating functional importance of the corresponding
preserved residue. Three of the 17 preserved positions tolerate amino
acid substitution; side chains at these positions are presumably
important in a context of multiple additional mutations but are not
necessary for function in an otherwise wild-type receptor.
Preserved Residues Identify Working Parts of the Switch--
The
first clues to specific roles of preserved residues came from comparing
positions preserved in the functioning mutant C5a receptors with
positions deemed evolutionarily important among serpentine receptors.
We applied an evolutionary trace analysis method (38) to 62 serpentine
receptors closely related to the human C5a receptor. The evolutionary
trace method assesses patterns of sequence conservation in alignments
of genes for related proteins and maps the conserved positions onto a
shared backbone structure. The method successfully identified contact
surfaces in SH2 and SH3 domains (38) and G
Fig. 7 compares the evolutionary trace to
the pattern of preserved residues in mutant functioning C5a receptors,
as mapped onto the
We propose that the cluster of yellow residues disposed toward the
extracellular fluid defines a receptor surface that interacts with the
C5a ligand (Fig. 8A).
Mutations at two of these positions (Arg-206 in helix V and Asp-282 in
helix VII) alter binding affinity for C5a (40). In accord with the
two-site binding model proposed for C5a binding (41-44), the
carboxyl-terminal tail of C5a probably inserts into the interhelical
crevice depicted in Fig. 8A, whereas other portions of the
C5a ligand interact with the amino terminus of the C5a receptor.
Moreover, as shown in Fig. 8A, positions of five of the six
preserved residues near the extracellular termini of helices III and V
coincide precisely with positions assigned by the Baldwin model to
residues that bind ligands in receptors for biogenic amines (45-47).
Glu-113 of rhodopsin, which corresponds to the preserved Leu-112 of the
C5a receptor, serves as a counterion for the Schiff's base formed
between retinal and Lys-296 in helix VII (48-50). Thus small amines, a
retinal chromophore, and a 74-residue polypeptide probably interact
with amino acids at many of the same positions in the helix bundle
(albeit with different side chains) to activate serpentine receptors.
This suggests that serpentine receptors that bind very different
agonists share a common activation mechanism.
In the Baldwin model, the
Some positions (blue in Fig. 7) that are highly important in many
serpentine receptors throughout evolution are nonetheless sites of
frequent substitution in functioning C5a receptor mutants. They may
mediate a specific function subject to a selective pressure in
evolution that was absent in our yeast screen. One possibility is that
these blue residues, located in the extracellular half of the helix
bundle, participate in interhelical interactions responsible for
maintaining the receptor switch in the off state in the absence of
ligand. We have not tested whether single mutations at the blue
positions systematically produce constitutive activation of the receptor.
Activating Mutations and Truncations--
Although selected in
yeast cells expressing the C5a ligand, a substantial number of
functioning mutant receptors remains active when expressed in cells
lacking C5a (Table I, Figs. 2 and 4); several of these activated
receptors are truncated by stop codons in helix VI (Fig. 4). Taken
together, these mutations and the truncated receptors point to helices
III and VI as key elements of the turn-on switch and to a possible role
for helix VII as a ligand-sensitive inhibitor of activation.
Ligand-independent receptor activity resulted from point mutations in
helices III (five of 30 functional receptors) and VI (16 of 25 functional receptors) but not helices V and VII (Table I). We infer
that receptor activation involves release of constraints on movement of
helix VI relative to the rest of the transmembrane helix bundle. This
inference is compatible with the tolerance of helix VI for amino acid
substitutions (found in helix VI at almost twice the rates observed for
the three other helices tested, Table I) and with its remarkable
predilection for activating mutations. This susceptibility of helix VI
to activating mutations appears to be shared by receptors for biogenic
amines, luteinizing hormone, and thyrotropin (55, 56). The importance
of helix VII for constraining mobility of helix VI and preventing
constitutive activation was a principal conclusion of a computational
modeling analysis of activating mutations (predominantly in helix
VI) in the luteinizing hormone receptor (57).
A "hot spot" for activating mutations in helix III is also
compatible with a relation between receptor activation and movement of
helix VI in the helix bundle. The potential hot spot (Ile-124 and
Leu-127) was identified by inspecting the sequences of constitutively active helix III mutants (Fig. 2); it is confirmed by the
constitutively activated phenotype of a receptor with combined point
mutations that substitute polar residues (Asn and Gln, respectively)
for these two hydrophobic residues in helix III (Table II). Receptors containing individual substitutions of either I124N or L127Q show weak
constitutive activity (Table II). Other amino acid substitutions in
helix III may contribute to an activated phenotype; Arg-37 displays a
constitutively activated phenotype despite only a single mutation at
the Ile-124 and Leu-127 positions. In the Baldwin model, side chains at
these positions face helix VI (Figs. 6 and 8), suggesting that
introduction of polar groups may activate the receptor switch by
decreasing hydrophobic interactions and releasing helix VI from a
constraint that keeps it close to helix III. In contrast to helix III,
C5a receptors mutated in helix VI do not display an obvious consensus
for mutations that confer an activated phenotype. However, a point
mutation in helix VI, F251A, did generate a constitutively active
receptor (Table II). This was unexpected because the Phe-251 was
preserved in all of the full-length, functioning C5a receptors from the
helix VI screen (Fig. 4). Recently, a random saturation study of helix
VI of the m5 muscarinic receptor demonstrated that mutation of the
phenylalanine at the cognate position to Phe-251 caused constitutive
activity (58). In the Baldwin model, the phenylalanine side chain
potentially points toward the activating positions in helix III,
Ile-124 and Leu-127. Thus mutations that decrease the hydrophobicity of
closely adjacent surfaces of either III (I124N, L127Q) or VI (F251A)
activate the receptor, supporting the hypothesis that hydrophobic
interactions between these surfaces promote the inactive state of the receptor.
The idea that receptor activation depends upon changes in the
orientations of helices III and VI is supported by biochemical experiments based on fluorescence spectroscopy, chemical modification, and cross-linking the two helices (51, 59-63). An elegant study using
site-directed spin labels in retinal rhodopsin showed that activation
by light causes the cytoplasmic end of helix VI to turn by 30° on its
axis and to move away from helix III by ~12 Å (64). In addition,
salt bridges (63) or disulfide links (64) connect these two helices in
or near the cytoplasm block activation of the G protein.
Nine of the 21 constitutively activated C5a receptors are truncated in
the extracellular half of helix VI (Fig. 4). These truncated receptors
lack a significant fraction of the receptor polypeptide, including
extracellular loop 3, helix VII, and the C-terminal cytoplasmic tail.
Removal of DNA sequences 3' to the stop codon did not alter the
phenotypes of truncated C5a receptors, ruling out the possibility of
suppressed stop codons.2 To our knowledge, these mutants
are the only serpentine receptors with fewer than seven transmembrane
helices known to activate a G protein. Numerous studies describe
truncated versions of serpentine receptors arising from alternative
splicing; these include a GHRH receptor lacking helix VII, found in
human pituitary adenomas (65); an opsin lacking helix VI, detected in
human retina (66); and a thyrotropin receptor truncated in the middle
of helix V (67). In general, most alternatively spliced receptors do
not appear to be functional; however, a deletion of 14 amino acids in
the cytoplasmic half of helix VII in an isoform of the calcitonin receptor alters ligand specificity and selectively abolishes coupling to phospholipase C (68). The apparent dispensability of the C-terminal
tail of the C5a receptor in our experiments contrasts with evidence
that the C-terminal tails of other receptors contact the G protein
(perhaps the
The truncated C5a receptors demonstrate that helix VII and the
C-terminal tail of the receptor are not absolutely required for G
protein activation. This conclusion is especially surprising because
helix VII in our mutant screen tolerated the fewest average number of
mutations per helix in functioning receptors (3.2%, Table I) and
showed a high proportion of strongly preserved side chains (7 of 21 tested; Table I and Fig. 2). Moreover, helix VII mutations in
functioning receptors do not truncate or cause constitutive activation.
The dispensability of helix VII for activating the G protein can be
reconciled with its resistance to mutations if we postulate that helix
VII normally maintains the helix bundle in an inactive state. This
interpretation is in keeping with the observation that mutations that
interrupt a salt bridge between helices VII and III activate retinal
rhodopsin (72). Helix VII probably plays a finely tuned structural role
in the receptor, so that helix VII mutations that activate the receptor
drastically reduce its stability (57); this might be anticipated from
the Baldwin model, which places helix VII in close proximity to helices I, II, III, and VI.
In summary, we have performed a comprehensive genetic analysis of four
of the seven helices in the C5a receptor. The results identify 25 residues, located in two clusters, that are critically required for C5a
receptor function. One cluster includes residues, at or near the
extracellular face of the receptor, that probably constitute a binding
pocket for interaction with the C5a ligand; strikingly, this cluster
shares a very similar "footprint" with residues in distantly
related receptors that interact with rhodopsin and biogenic amines
(Fig. 8A). The second cluster, at the core of the helix
bundle, consists of residues that are conserved in most serpentine
receptors. Both clusters thus argue strongly for an activation
mechanism that is conserved in all or most serpentine receptors. We
propose that proper orientation of ligands
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
) G proteins on the cytoplasmic face of the plasma membrane. These receptors catalyze ligand-dependent exchange of
guanosine triphosphate (GTP) for guanosine diphosphate (GDP) on the
subunit of the heterotrimer, causing dissociation of
·GTP from the
dimer;
·GTP and free
subsequently activate effector
enzymes and ion channels (1, 2). More than 1,000 serpentine receptors of mammals share with their counterparts in yeast and plants a conserved three-dimensional architecture, comprising seven
-helices in a transmembrane bundle (3-6). The switch mechanism is also conserved, as indicated by the abilities of mammalian receptors to
activate G protein trimers in yeast (7-9). The switch clearly resides
in the seven-helix bundle: swapping of extra- or intracellular loops
preserves the ability of ligands to activate G proteins while
transferring specificity of ligand binding or G protein activation,
respectively, from one receptor to another (10-13).
-carbon template of the helix bundle, hereafter
termed the Baldwin model. In this model, transmembrane helices I-VII
bundle together in clockwise order as viewed from the cytoplasm. The
probable arrangement of helices and the positions of specific amino
acids in the model are inferred from patterns of conserved hydrophobic
and hydrophilic residues in many receptors. The Baldwin model specifies
which helix corresponds to which density in the electron projection
map, approximate orientations of cognate amino acids around the helical
axes, and the cytoplasmic and extracellular limits of each
transmembrane sequence (16).
-helix by identifying those that cannot be altered in
mutated receptors selected for maintenance of function. The results
suggest a hypothesis to explain the conserved switch mechanism of
serpentine receptors.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
12
digested with SphI and PstI was subcloned into
the C5a receptor DNA containing silent SphI and
PstI sites. The following oligonucleotides were used
(Genemed, S. San Francisco, CA; underlines denote bases doped with 20%
nonwild-type nucleotides): Helix III,
5'-CCCGCATGCTCTATTCTACCATCTCTAATTCTACTAAACATGTACGCTTCTATTCTACTACTAGCTACTATTTCTGCAGAA-3'; Helix V,
5'-TTTGCGCGCTGTTGCTATTGTTAGACTAGTTCTAGGTTTCCTATGGCCTCTACTAACTCTAACTATTTGTTACACTTTCATTCTGCTCCGGAGC-3'; Helix VI (two oligonucleotides);
5'-ATAAGATCTACCAAAACACTCAAAGTTGTTGTTGCAGTTGTTGCAAGCTTCTTTATCTTCTGGTTA-3', and
5'-AAACTCGAGAAAAGACATCATTATCCCCGTCACCTGGTATGGTAACCAGAAGAT-3'; Helix VII,
5'-CCAAAGCTTGATTCTCTATCTGTTTCTTTTGCTTACATTAATTGTTGTATTAATCCAATTATTTACGTTGTGGCCGGCCA-3'. Oligonucleotides encoding Helices III, V, or VII were mutually primed
via extended palindromic sequences at their 3' termini (19, 20), and
the complementary strand synthesized using Klenow DNA polymerase. The
products were digested with the appropriate restriction enzymes and
subcloned into the C5a receptor gene in pBS-SK Bluescript vector.
Because of technical difficulties in synthesizing doped
oligonucleotides greater than 100 base pairs, we annealed two
oligonucleotides encoding either the N- or C-terminal half of helix VI
via a 12-base pair overlap at their 3' termini, synthesized the
complementary strand using Klenow polymerase, and subcloned the
BglII and XhoI digested products into the C5a receptor gene in pBS-SK Bluescript vector. After determining the complexity and the quality of the library (by DNA sequencing and restriction mapping), we subcloned NcoI and FseI
fragments of each mutated helix library into the yeast shuttle vector,
p1303ADE2 (derived from p1303, Cadus Pharmaceuticals, New Jersey). The
number of recombinants obtained equaled or exceeded the initial size of
the mutated helix library.
1442 tbt1-1 FUS1-HIS3 can1
ste14:: trp1::LYS2 ste3
1156
gpa1(41)-G
i2 lys2 ura3 leu2 trp1 his3
ade2. BY1142 expresses a yeast/human G
chimera in which the
N-terminal 41 residues of yeast GPA1 (a region predicted to interact
with G
based on the crystal structure of the Gi
heterotrimer (21)) replaces the first 33 residues of human
G
i2. A SacI restriction site introduces a
point mutation that substitutes leucine for valine at position 34 of
G
i2. The chimera is designed for optimal ability to
sequester yeast G
and release it upon receptor activation, thereby triggering the mating response pathway. Expression of the
FUS1/HIS3 reporter enzyme, stimulated by the mating response pathway,
allows BY1142 cells (his3) to grow in histidine-deficient medium. The far1 deletion blocks cell cycle arrest that is
induced by pheromone. The tbt1-1 mutation increases
transformation efficiency by electroporation and ste14, a
carboxymethylase deletion, reduces basal signaling of the pheromone
response pathway. Inclusion of an ADE2 gene in the C5a
receptor plasmid, p1303 (p1303ADE2, PGK-hC5aRADE2 REP3 2 µm-ori AmpR f1ori), allowed us easily to infer from the color of a colony whether its growth on histidine-deficient medium depends on the C5a receptor (red colonies lack ADE2 and, by
implication, the receptor plasmid). A separate plasmid, p1297
(ADH1-mf
1-hC5aURA3 REP3 2µm-ori AmpR f1ori,
Cadus Pharmaceuticals), allowed autocrine expression of the C5a ligand
as an
-factor prepro/C5a ligand fusion protein; autocrine expression
was necessary because C5a cannot traverse the yeast cell wall.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
C5a and the C5a receptor allow growth of
yeast strain BY1142. Yeast BY1142 cells (11) were transformed with
ADE2 plasmid DNA encoding the C5a receptor (rows
1 and 3) or, as a negative control, a C5a receptor
containing a stop codon in helix III (STOP, rows
2 and 4), and URA3 plasmid DNA encoding
vector alone (rows 1 and 2) or C5a ligand
(rows 3 and 4). After selection on uracil- and
adenine-deficient medium (UA), colonies were grown to near
stationary phase and aliquots spotted onto nonselective UA medium or
histidine-deficient medium containing the indicated concentrations of
the HIS3 inhibitor, AT, and incubated at 30 °C for 3 days.
Characteristics of mutated C5a receptors
, amino acid substitution rate; avg, average per
scanned transmembrane segment.
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Fig. 2.
Functioning C5a receptors selected from a
library containing random substitutions in helix III. Mutated C5a
receptors and the C5a ligand were expressed in BY1142 yeast and
functioning receptors selected by the ability of yeast to grow in the
absence of histidine and in 1 mM AT. C5a receptor sequences
are shown from the extracellular (top) to the cytoplasmic
surface (bottom). Wild-type sequence of the C5a receptor and
the amino acid position numbers are indicated at the left
and right of each figure. Position numbers in the notation
of Baldwin (15, 16) are indicated at the left of the
boxed columns. Horizontal lines indicate proposed
borders of the lipid bilayer (16). Columns representing individual
mutant receptors (each designated by R and a
number, top) display their individual sequences
(dot = unchanged from wild type). Receptor signaling
strength, indicated below each mutant receptor sequence, was
quantitated by growth on histidine-deficient media in the presence of
AT. ++++, growth on 10 mM AT; +++, 5 mM AT; ++,
2 mM AT; +, 0.5 or 1 mM AT; 0, no growth on 0.5 mM AT. Constitutively active receptors are grouped
separately. Genetic code refers to amino acid substitutions
that were possible at the corresponding position by substituting only a
single nucleotide base. Standard single-letter abbreviations indicate
amino acids at each position; bold letters indicate amino
acids that are not conserved with respect to the wild-type C5a receptor
sequence. Characteristics of mutated residues in functional receptors
(indicated by X in the boxed columns) are presented in four
classes, as follows: Preserved, amino acid positions where
side chain character is preserved (see text); Evol.
Conserved, residues identified by the evolutionary trace method
(38); Hydrophobic, positions at which only hydrophobic amino
acids are observed (see text); Tolerates Polar, aspartate,
glutamate, asparagine, glutamine, lysine, or arginine tolerated; @,
stop codons observed. Numbers in parentheses indicate percent identity
in 199 rhodopsin-family serpentine receptors (16).
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Fig. 3.
Functioning C5a receptors selected from a
library containing random substitutions in helix V. Mutated C5a
receptors and the C5a ligand were expressed in BY1142 yeast and
functioning receptors selected by the ability of yeast to grow in the
absence of histidine and in 1 mM AT. For details, please
refer to Fig. 2 legend.
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Fig. 4.
Functioning C5a receptors selected from a
library containing random substitutions in helix VI. Mutated C5a
receptors and the C5a ligand were expressed in BY1142 yeast and
functioning receptors selected by the ability of yeast to grow in the
absence of histidine and in 1 mM AT. For details, please
refer to Fig. 2 legend. @, stop codons observed. Note that four
positions in helix VI (Ile-253, Phe-254, Trp-255, Leu-256) were not
subjected to mutation; the corresponding nucleotides were kept constant
to anneal two sets of randomized oligonucleotides.
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Fig. 5.
Functioning C5a receptors selected from a
library containing random substitutions in helix VII. Mutated C5a
receptors and the C5a ligand were expressed in BY1142 yeast and
functioning receptors selected by the ability of yeast to grow in the
absence of histidine and in 1 mM AT. For details, please
refer to Fig. 2 legend.
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Fig. 6.
Side chains are preserved on faces of helices
that point toward other helices in the Baldwin model. Helical
wheel representations are based on the Baldwin model (16). Helices are
presented as viewed from the cytoplasm and in positions that correspond
to their relative positions at the middle of the bilayer (16).
Larger circles indicate residues closer to the cytoplasm.
Red letters and numbers indicate residues and
positions at which side chain character is preserved in mutant
functional receptors (see text). Dark blue circles indicate
positions at which hydrophobicity is preserved; white
indicates positions that tolerate polar substitutions (as defined in
the legend of Fig. 2); other positions are gray.
Blue, orange, and green letters
indicate positions (Ile-124, Leu-127, and Phe-251, respectively) at
which substitutions caused constitutive activation of the receptor (see
text and Table II).
Site-directed point mutations of key residues identified by random
saturation mutagenesis
C5a) or an
-factor prepro/C5a ligand fusion
protein (+C5a). Receptor signaling strength was quantitated by growth
on histidine-deficient media in the presence of AT: ++++, growth on 10 mM AT; +++, 5 mM AT; ++, 2 mM AT;
+, 0.5 or 1 mM AT; 0, no growth on 0.5 mM AT.
surfaces that interact
with
and the serpentine receptors
(39).3 Figs. 2-5 indicate
the top ranked positions (labeled Evol. Conserved). As
expected, the evolutionary trace approach identifies many of the same
residues found in these helices by Baldwin's analysis of 199 unique
serpentine receptor sequences (16). The latter analysis identified 16 positions at which residues are conserved in at least 60% of the
receptors (Figs. 2-5); 12 of these positions appear in the
evolutionary trace.
-carbon template of the Baldwin model (15).
Positions preserved in mutant C5a receptors form two distinct clusters
in the three-dimensional model, as indicated by the red and yellow balls in Fig. 7. Red indicates positions at which residues are both
preserved in mutant receptors and identified by the evolutionary trace
method; yellow indicates preserved positions that are not highly
conserved throughout evolution. (Blue indicates positions that are
deemed evolutionarily important but are not preserved in our genetic
screen, see below.) Yellow positions are located at or near the
extracellular face of the receptor, whereas red positions cluster
tightly in the cytoplasmic half of the transmembrane helices (Fig. 7).
The distributions indicate different functions for these important
residues.
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Fig. 7.
Comparison of the locations of preserved
amino acids in the mutant functional C5a receptors versus
highly conserved residues in evolution. Colored
tubes represent the -carbon backbones of the helices in the
Baldwin model. Portions of helices III (green), V
(blue), VI (pink), and VII (purple)
were subjected to random saturation mutagenesis; gray
indicates residues or helices not so tested. Yellow and
red spheres indicate
-carbon positions at which side
chain character is preserved in functioning C5a receptors;
blue and red spheres indicate positions that are
conserved, as assessed by the evolutionary trace method (38). Red
spheres indicate positions that are both preserved in the genetic
screen and also conserved in evolution of serpentine receptors. The
yellow grid (lines separated by 10 Å) indicates proposed
borders of the lipid core of the membrane (16).
View larger version (41K):
[in a new window]
Fig. 8.
Positions of functionally important residues
in the Baldwin model of serpentine receptors. Positions at which
side chain character is preserved are indicated by spheres.
A, the ligand-binding pocket, as viewed from the
extracellular fluid. Yellow indicates -carbon positions
of residues implicated in binding ligands for amine receptors and the
retinal chromophore of rhodopsin. Thus the yellow spheres
indicate
-carbon positions of residues preserved in functional
mutant C5a receptors that coincide with ligand-binding residues in
other receptors. Green or purple spheres indicate
positions at which amino acid character is preserved (but which are not
coincident with ligand-binding residues in amine receptors or
rhodopsin). Together the spheres near the extracellular fluid
constitute a probable ligand-binding pocket for C5a (see text).
Blue and orange (van der Waals representation)
indicate side chains of helix III at positions (Ile-124, Leu-127)
subject to activating mutations; light green (van der Waals
representation) indicates the side chain of Phe-251 in helix VI, where
an alanine substitution caused constitutive activation (see Table II).
The positions of these side chains are specified by their
-carbon
positions in the Baldwin model (16) and rotamer side chain angles
predicted by SCWRL program (73). B, putative G
protein-interacting pocket, viewed from the cytoplasm. Cyan
indicates
-carbon positions of residues (including the highly
conserved ERY/DRF motif in helix III) that are implicated by
biochemical and genetic evidence in other receptors as contact sites
for G proteins. Many of these positions were not tested in the C5a
receptor screen; partly for this reason, amino acid character is
preserved at only two of the putative G protein-interacting positions
(Tyr-222 and Leu-241, cyan spheres in helices V and VI,
respectively). In panels A and B, the
yellow grids (lines separated by 10 Å) indicate the
proposed extracellular and cytoplasmic borders, respectively, of the
lipid core of the membrane (16); the ribbon coloring scheme is the same
as in Fig. 6 (see legend).
-carbons of all ten clustered red
positions in Fig. 7 are located within 10 Å of the
-carbon of at
least one other highly conserved residue. (Some of these are conserved
residues in helices I, II, and IV; conserved is defined as identical in
at least 60% of 199 serpentine receptors assessed by Baldwin
et al. (15).) This cluster, highly preserved both in
evolution and in our genetic selection for functional C5a receptors, is
positioned to form an interacting network that is apparently crucial
for structure and function of serpentine receptors. We focused our
mutations on components of a transmembrane switch, rather than on
residues implicated in interactions with G proteins; for this reason
the red positions do not overlap significantly with regions in other
receptors that are thought to bind G proteins (51-54) (cyan in Fig.
8B). Instead, we propose that these positions form a
structural core that allows conformational changes induced by ligand
binding to be transmitted to the G protein interaction surface.
subunit) (69, 70) and sometimes play key roles in
determining receptor specificity for G protein (71). The contrast may
be more apparent than real, however, because none of these functions
was tested in our yeast selection procedure. Thus C-terminal tails of
different receptors play different functions, but these probably are
more or less dispensable in some receptors.
C5a, biogenic amines, and
probably others
in the binding pocket (yellow residues in Fig. 6)
induces a conformational change that is transmitted through the
conserved core of the helix bundle to G proteins. At present we can
only infer that locations of these critical residues indicate that they
perform specific functions. To define the roles of these residues more
precisely will require further biochemical and structural information.
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ACKNOWLEDGEMENTS |
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We thank Fred E. Cohen, Shaun Coughlin, Mark von Zastrow, Ira Herskowitz, and members of our laboratories for helpful discussions and reading the manuscript, Drew Murphy and Lauren Silverman, from Cadus Pharmaceuticals, for the C5a ligand encoding plasmid, Ken Boakye for an ADE2 disruption plasmid, and Helen Czerwonka for secretarial assistance.
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FOOTNOTES |
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* The research was supported in part by National Institutes of Health Grant GM-27800 (to H. R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A Howard Hughes Medical Institute Physician Postdoctoral Fellow. Present address: Depts. of Medicine and Molecular Biology and Pharmacology, Washington University School of Medicine, Box 8103, 660 S. Euclid Ave., St. Louis, MO 63110.
¶ Supported by funds from the American Heart Association. Present address: Dept. of Molecular and Human Genetics, Baylor College of Medicine, 1 Baylor Plaza, Rm. T921, Houston, TX 77030.
An Early Career Fellow of the Swiss National Science Foundation.
|| Supported by a Julius H. Comroe Jr. Award by the Cardiovascular Research Institute. Present address: Fourth Department of Internal Medicine, University of Tokyo School of Medicine, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan.
§§ Supported by the Danish Heart Association and by funds from Daiichi. Present address: Laboratry of Molecular Cardiology, University of Copenhagen, Rigshospitalet 9312, Juliane Mariesvej 20, DK-2100 Copenhagen, Denmark.
¶¶ To whom correspondence should be addressed: Dept. of Cellular and Molecular Pharmacology, University of California, Box 0450, 513 Parnassus Ave., San Francisco, CA 94143. Tel.: 415-476-8161; Fax: 415-476-5292; E-mail: h_bourne{at}quickmail.ucsf.edu.
2 T. J. Baranski and H. R. Bourne, unpublished data.
3 A more complete analysis of the evolutionary trace in lineages of serpentine receptors is in preparation, O. Lichtarge, H. R. Bourne, and F. E. Cohen.
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ABBREVIATIONS |
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The abbreviation used is: AT, 3-aminotriazole.
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REFERENCES |
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