(Received for publication, August 16, 1996, and in revised form, October 21, 1996)
From the Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205
cAMP receptor 1 (cAR1) of
Dictyostelium couples to the G protein G2 to mediate
activation of adenylyl and guanylyl cyclases, chemotaxis, and cell
aggregation. Other cAR1-dependent events, including
receptor phosphorylation and influx of extracellular Ca2+, do not require G proteins. To further characterize
signal transduction through cAR1, we performed random
mutagenesis of the third intracellular loop (24 amino acids),
since the corresponding region of other seven helix receptors has been
implicated in the coupling to G proteins. Mutant receptors were
expressed in car1 cells and were
characterized for G protein-dependent and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third intracellular loop.
Of the 21 positions where amino acid substitutions were observed, one
or more replacements were found that retained full biological function.
However, certain alterations resulted in receptors with reduced ability
to bind cAMP and/or transduce signals. There were specific signal
transduction mutants that could undergo cAMP-dependent cAR1
phosphorylation but were impaired either in coupling to G proteins, in
G protein-independent Ca2+ influx, or in both pathways. In
addition, there were general activation mutants that failed to restore
aggregation to car1
cells and displayed
severe defects in all signal transduction events, including the most
robust response, cAMP-dependent cAR1 phosphorylation.
Certain of these mutant phenotypes were obtained in a complementary
study, where the entire region of cAR1 from the third to the seventh
transmembrane helices was randomly mutagenized. Considered together,
these studies indicate that the activation cycle of cAR1 may involve a
number of distinct receptor intermediates. A model of G
protein-dependent and -independent signal transduction through cAR1 is discussed.
G protein-coupled receptors mediate diverse cellular functions in
eukaryotic cells. Several hundred of these receptors, which possess
four extracellular domains, three intracellular loops, an intracellular
C-terminal domain, and seven transmembrane helices, have been
identified. Agonist association with the receptor triggers the exchange
of GTP for GDP on the -subunit of the associated heterotrimeric G
protein, inducing dissociation of the activated G
-subunit from the
G
-complex (for reviews, see Refs. 1 and 2). These both modulate
the activity of a number of effectors including adenylyl cyclases (3),
phospholipases (4, 5), MAP1 kinases (6),
and ion channels (7).
A number of receptor domains are required for the activation of G proteins. In a variety of receptors, the three cytoplasmic loops act together with the membrane proximal region of the C-terminal domain during this process (8-13). Of these, the third intracellular loop has been most thoroughly characterized. Mutational analysis, use of synthetic peptides, and chimeric receptor studies suggest that the ends of this loop adjacent to the fifth and sixth transmembrane helices play a role in the formation of specific G protein-receptor complexes and in subsequent events required for the activation of G proteins (14-20). In support of this, the central portion of the third intracellular loops of several receptors can be deleted, without adverse effects on the coupling to G proteins (16, 21).
Recent evidence, however, suggests that the third loop domain may not couple directly to G proteins through specific amino acid side chain interactions, but may act as a hinge, which facilitates the exposure of binding domains for G proteins and kinases once the receptor is activated (22). First, synthetic peptides corresponding to the second intracellular loop or the tail domain of the N-formyl peptide receptor, but not the third intracellular loop, inhibited the association of G proteins with the receptor (23). Second, mutations in the human muscarinic acetylcholine receptor, subtype M1 (Hm1) in an amino acid motif that is thought to interact with G proteins, BBXXB or BBXB (where B is a basic amino acid, X is a nonbasic amino acid) (24) had minimal effects on receptor coupling to G proteins (25). Third, two distinct point mutations proximal to the sixth helix of Hm1 severely inhibited function and a third point mutation gave rise to a constitutively active receptor but the triple mutant was considerably less impaired (22). Fourth, a number of constitutively active G protein-coupled receptors resulting from amino acid substitutions within the third intracellular loop adjacent to the sixth membrane helix have been identified (26-30). Together, these findings suggest that receptor conformational changes can occur within the same domain thought to interact with G proteins, making it difficult to interpret how previously identified amino acid substitutions and deletions in the third loop influence coupling of G proteins to receptors.
In the social ameba Dictyostelium, chemotaxis and
differentiation are regulated by a family of cell surface cAMP
receptors (cARs), which have seven transmembrane helices and are
analogous to mammalian G protein-coupled receptors such as rhodopsin
(31-33). cAR1 is maximally expressed in aggregating cells and
interacts with the G protein -subunit G
2 to activate adenylyl and
guanylyl cyclases, phospholipase C, and changes in cytoskeletal
components required for chemotaxis (for review, see Ref. 34). cAR2 and cAR3 can substitute for cAR1 for many of these
events2 (35). A remarkable feature of cARs
is that they also activate a number of signaling events in cells
lacking functional G proteins. These include a stimulated
Ca2+ entry (36, 37), activation of a MAP kinase (38), and
regulation of several gene expression events occurring during
development (39, 40). G proteins are also not required for
cAMP-dependent cAR1 desensitization (37), where cAR1 is
phosphorylated on several serine residues present in its C-terminal
domain (41) and undergoes a reduction in affinity for cAMP (42).
To explore the functional role of the third intracellular loop of cAR1
in G protein-dependent and -independent signal
transduction, we extensively mutagenized the entire loop region. In
another study, the entire region from transmembrane III through
transmembrane VII was randomly mutagenized (77). Importantly,
Dictyostelium provides a useful system to screen for random
mutations in the receptor; car1 cells have
been constructed and these fail to aggregate, a phenotype that is
reversed when the cells are transformed with an extrachromosomal vector
containing the gene encoding cAR1 (43). The presence of both G
protein-dependent and -independent signal transduction pathways mediated by cAR1 provides a unique opportunity to determine whether mutants defective in coupling to G proteins are, in fact, activation mutants defective in both signal transduction pathways. In
this study, 22 individual mutants were characterized for their ability
to carry out G protein-dependent and -independent signal transduction. Thirteen mutants previously characterized for G protein-independent signal transduction (44) were also
characterized for their ability to couple to G proteins. Analysis of
both sets of mutants has led to the identification of affinity mutants, general activation mutants, and selective signal transduction mutants
that decouple G protein-dependent signaling events, G protein-independent Ca2+ influx, and cAR1
phosphorylation.
96-well polyvinylidene difluoride-bottomed
filtration plates (0.65 µm) were from Millipore, silicon oil was from
Wacker Silicones Corporation, MI, GTPS was from Boehringer Mannheim,
and Renaissance Western blot chemiluminescence reagent was from Dupont
NEN. Other materials used were of analytical grade and purchased from
the suppliers indicated in Milne and Devreotes (36).
In this
study, the car1 G418-sensitive strain JB4 (44)
was transformed with plasmids containing wild-type or mutant versions of cAR. Transformants were grown in HL5 (45) supplemented with 20 µg
of Geneticin/ml of HL5. JB4 was grown in HL5. Cells were maintained in
Petri dishes. For biochemical experiments to screen many transformants
([32P]cAMP binding, Ca2+ influx, receptor
phosphorylation, and development), cells were grown in shaking
suspension in 5-ml cultures to a density of ~5 × 106 cells/ml in sterile 50-ml Corning tubes. For all other
experiments, cells were grown in Erlenmeyer flasks. To initiate
development, cells were washed in developmental buffer, resuspended to
1 × 107 cells/ml, and plated on non-nutrient agar as
described previously (46).
Two partially degenerate
oligonucleotides were synthesized. The first, corresponding to
nucleotides 649-681 of the cAR1 gene, was comprised of the sequence
TTCTCTTTatta
tca
cta
aca
cca
tta
tga
atAACAACAT (region B). The
second, corresponding to the nucleotides 673-713 (region C) had
the sequence AATTTGAAt
tgg
tat
gtt
a
aa
tgt
ttc
tct
ttATTATCAC. Capital
letters represent a homogeneous position. Positions without a prime
contain 93% of the indicated nucleotide and 2.3% of the other
nucleotides. Positions with a prime are as follows: a
= 92% A, 8% G;
t
= 92% T, 4% A, 4% G; g
= 92% G, 8% C; c
= 92% C, 4% A, 4%
G. The cAR1 cDNA, subcloned into bacteriophage M13, was randomly
mutagenized using these degenerate oligonucleotides as described
elsewhere (44). A BamHI-BstXI fragment from the replicative form DNA was subcloned as described into pMC34, an extrachromosomal Dictyostelium expression vector carrying a
neomycin resistance gene, to generate a library of mutant cAR1
plasmids. In this construct, cAR1 DNA is flanked by the actin-15
promoter, which is active during growth and aggregation, and the 2H3
terminator. Two mutant cAR1 sequences identified from sequencing of
phage clones, IIIa-1 and I-11, were individually subcloned into
pMC34.
JB4 cells were transformed with the degenerate cAR1 libraries by electroporation as described elsewhere (47). After 12-16 h in HL5, cells were resuspended in HL5 containing 20 µg of Geneticin/ml and divided into 96-well plates. Viable cells were streak-plated on SM agar in association with Klebsiella aerogenes to obtain isolated clones, which were reselected into 24-well plates containing selective media. For plasmid rescue, total DNA was recovered from 4 × 107 cells as described previously (48) and used to transform MC1061 bacteria. Plasmids were isolated and sequenced using standard techniques. Examination of the sequences of mutant cARs (B and C region clones, Table I) indicates that the mutagenesis procedure introduced, by unknown means, mutations throughout the entire third intracellular loop, rather than in the expected central one-third or the C-terminal one-third of the third cytoplasmic loop. In contrast, mutagenesis of the N-terminal one-third of the third intracellular loop yielded mutants present in the anticipated region (A region clones) (44). While we have not ruled out the possibility of mutations elsewhere in every B and C region clone, full-length sequencing of mutants wild-type-like (wtl)-4, IIIa-1, I-14, and IV-8 did not reveal the presence of additional mutations. No unexpected mutations were seen in partial sequencing of mutants wtl-9 (nucleotides 350-800), wtl-12 (nucleotides 350-890), I-8 (nucleotides 333-530 and 780-900), and IV-6 (nucleotides 333-901).
In order to maintain a consistent mutant nomenclature between this study and that described in a companion study (77) and to allow that the names convey the general properties of the mutants, the following mutant cARs identified previously (44) were renamed as follows (old name = new name): A2 = wtl-1; A22 = wtl-2; A62 = IIIa-2; A5 = I-3; A16 = I-9; A60 = I-10; A42 = IV-1; A53 = IV-2; A3 = IV-3; A55 = IV-4; A81= IV-5.
cAMP Binding AssaysFor [32P]cAMP binding, transformants (4 × 105) washed once in phosphate buffer (PB; KH2PO4/Na2HPO4, pH 6.1) were resuspended in PB and loaded into wells of a 96-well filtration plate. Surface [32P]cAMP binding sites were assayed as described (44) in the presence of 3 M ammonium sulfate, which stabilizes the binding of cAMP to cAR1 (49).
For Scatchard analysis, cells were starved in shaking suspension for
6 h in the presence of 100 nM cAMP pulses, harvested by centrifugation, and resuspended in 50 ml of ice-cold PB. After shaking (22 °C, 250 rpm, 30 min) cells were harvested, washed once
in ice-cold PB, and resuspended to 1 × 108 cells/ml
in PB. After shaking 10 min on ice, [3H]cAMP binding was
performed in triplicate in the presence of either 1 × 109 M or 1 × 10
8 M
[3H]cAMP and various concentrations of nonradioactive
cAMP (0-10
6 M) (49). Scatchard plots were
generated and analyzed using the computer program LIGAND (50).
2 × 108 cells were starved in shaking suspension for 6 h
in the presence of 100 nM cAMP pulses to induce the
expression of the G protein G2, harvested by centrifugation, and
resuspended in 50 ml of ice-cold PB. After shaking (22 °C, 250 rpm,
30 min) cells were harvested, washed once in ice-cold PB, and
resuspended in 5 ml of the same buffer. After shaking 10 min on ice,
cells were lysed through 3-µm Nucleopore filters, and crude membranes were recovered by centrifugation (10,000 rpm, 5 min, 4 °C, SS34 rotor). After resuspending the membranes to 4 × 107
cell equivalents/ml, binding of 2 nM cAMP was measured in
triplicate in the presence or absence of 100 µM GTPS
(5 min, on ice) by spinning the membranes through silicon oil (80%
light AR20: 20% heavy, P-AR-200) as described previously (51).
Growth
stage cells were washed once in H buffer (20 mM Hepes/KOH,
5 mM KCl, pH 7.0), resuspended to 2 × 107
cells/ml in H buffer, and shaken (15 min, 300 rpm, 22 °C). Aliquots (50 µl) were pipetted in duplicate into wells of a 96-well filtration plates, which were prewashed with 400 µl of H buffer. Using a multichannel pipetter, 100 µl of 45Ca2+
uptake mix (H buffer, 10 µM CaCl2, 500 µM CoCl2, ~5 µCi of
45CaCl2) was added to each of one set of wells.
To measure cAMP-stimulated Ca2+ uptake, the same mix
containing 150 µM cAMP was added to the duplicate wells.
The reaction was terminated by the addition of 100 µl of 775 mM nonradioactive CaCl2 at 40 s, a time at
which cAMP-stimulated Ca2+ entry into suspensions of cells
is complete. Samples were filtered using a filtration manifold, and
washed three times with 200 µl of ice-cold H buffer containing 10 mM CaCl2. Filters were air-dried and exposed to
autoradiographic film for 24 h. For quantitation, filters were
punched out, vortexed in 100 µl of 1% SDS, and assessed for
radioactivity using scintillation counting. Fig. 1
illustrates this rapid filtration assay. Nonstimulated
car1 cells accumulated low levels of
45Ca2+, and this level did not increase
detectably in the presence of cAMP. In contrast,
car1
cells expressing wild-type cAR1
accumulated significantly more 45Ca2+ in the
presence of 100 µM cAMP than did untreated controls.
Eight randomly selected clones expressing different mutant cARs also showed cAMP-stimulated 45Ca2+ entry, while two
clones showed no stimulated Ca2+ entry. Wells not receiving
cells did not bind appreciable amounts of
45Ca2+. Quantitation of the wild-type
cAR1-induced Ca2+ response indicated that it was ~4-fold
higher than Ca2+ entry into unstimulated cells (Table I).
For certain experiments, cAMP-dependent
45Ca2+ influx into suspensions of cells was
assessed as described elsewhere (36).
cAR1 Phosphorylation and Immunoblot Analysis
Growth stage amebae (1 × 106) were washed once in PB and resuspended in PB containing 5 mM caffeine, to restore cAR1 to the 40-kDa form, and 10 mM dithiothreitol, to inhibit phosphodiesterase activity. cAMP-induced cAR1 phosphorylation was measured as described previously (52). Cells were solubilized in Laemmli buffer, electrophoretically separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose, and immunoblotted with cAR1 antiserum as described elsewhere (53), except that an enhanced chemiluminescence kit was used for detection. Autoradiographs of immunoblots were digitized and analyzed as described previously (44).
We performed
mutagenesis of wild-type cAR1 using two degenerate oligonucleotides
designed to introduce 1-4 mutations into two regions of the third
intracellular loop designated B
(Ile190-Asn197) and C
(Lys198-Gln205). The mutant cAR1 libraries
were subcloned into an extrachromosomal expression vector (pMC34), and
transformed into car1 cells. The cells were
divided into 96-well plates, and after selection, growth-positive wells
were individually streak-plated on bacterial lawns to obtain clones.
After 7 days, 18 aggregation-negative clones as well as 53 aggregation-positive clones (one per plate) were reselected into
24-well plates containing selective media.
car1 cells do not show significant levels
of cAMP binding sites, but expression of exogenous cAR1 in these cells
increases the number of binding sites at least 20-fold (43). The
selected clones were assessed for cell surface [32P]cAMP
binding sites in the presence of ammonium sulfate using a filtration
assay. Fifty-seven clones showed high levels of cAMP binding, 10 clones
showed low levels of cAMP binding, and 4 clones did not bind cAMP (data
not shown). Clones exhibiting cAMP binding sites and two with no
detectable cAMP binding sites were selected for further
characterization.
Plasmids from 60 individual clones were rescued and sequenced through
the third intracellular loop. Of these, 22 mutant receptors (B and C
region clones) are shown in Table I. Of the rest, 5 receptors were partially characterized and are not shown, 14 receptors contained the wild-type cAR1 sequence, 12 receptors had a single Lys207 Asn which was observed in clone Ia-9, 2 receptors had frameshift mutations, and the other 5 possessed sequences
identical to certain of the mutant receptors presented in Table I.
Further characterization of the clones (discussed below) indicated the
presence of at least four classes of mutant receptors: those with
properties indistinguishable from wild-type (wtl), cAMP binding
affinity mutants with normal signal transduction (class III), general
activation mutants that are defective in all responses (class IV), and
signal transduction mutants that are defective in specific G
protein-dependent responses and/or G protein-independent
Ca2+ influx (class I). Each of the mutant cell lines was
reconstructed following initial biochemical characterization by
reintroducing isolated plasmid DNA into car1
cells to recapitulate the phenotype and to eliminate the possibility of
multiple plasmids in a single clone.
We assessed the cAMP-dependent
cAR1 phosphorylation of wild-type cAR1 and several representative
mutants (IIIa-1, I-7, I-14, and IV-8) by monitoring the parallel change
in the apparent molecular mass of the receptor from 40 to 43 kDa on
polyacrylamide gels. The reduction of electrophoretic mobility arises
from the phosphorylation of Ser303 and Ser304
present in the C-terminal domain of cAR1 (41). The EC50 of this response can be determined if the reaction is carried out to
steady-state at increasing doses of cAMP. Fig.
2A illustrates the profile of each mutant;
Fig. 2B shows data representative of that used to derive
this plot. Wild-type cAR1 began to respond at low nanomolar
concentrations of cAMP and responded maximally between 10 and 100 nM. The EC50 of the response was 23 nM. A similar profile was obtained for I-7. Higher
concentrations of cAMP were required to induce receptor phosphorylation
in mutant IIIa-1 (EC50 = 78 nM) and in I-14
(EC50 = 428 nM). IV-8 was the most severely impaired, showing no detectable response even in the presence of 100 µM cAMP.
To determine whether the high EC50 values of IIIa-1, I-14,
and IV-8 were due to a reduced ability to bind cAMP, Scatchard analysis
was performed under physiological conditions (Fig. 3 and
Table II). Wild-type cAR1 exhibited a minor cAMP binding
site with a Kd of 9 nM and and a major
site with a Kd of 450 nM, values similar
to earlier estimates (54). Mutant IIIa-1, showed a 2-fold reduction in
affinity with Kd values of 22 and 1000 nM. Mutant I-14 displayed an even lower affinity with
Kd values of 96 and 1600 nM. Despite
lacking any detectable phosphorylation response even at 100 µM cAMP, IV-8 bound cAMP comparatively well, with a
single affinity site of 220 nM. All of these receptors
displayed high levels of high affinity cAMP binding sites in the
presence of ammonium sulfate, which stabilizes the binding of cAMP to
cAR1 (data not shown).
|
A rapid filtration assay was used to assess cAMP-dependent
Ca2+ entry into car1 cells
expressing wild-type cAR1, IIIa-1, I-7, I-14, or IV-8 (Table I). The
relative ability of IIIa-1, I-14, and IV-8 to trigger the
Ca2+ response was the same as their relative ability to
elicit agonist-induced cAR1 phosphorylation. For example, IIIa-1 was as
effective as wild-type cAR1 in promoting cAMP-induced uptake of
extracellular Ca2+, I-14 responded weakly, and IV-8 did not
respond. Similar results were obtained when cAMP-mediated
Ca2+ entry was measured by the standard centrifugation
assay (36), rather than by the rapid 96-well filtration assay (data not
shown). However, despite having the same EC50 as wild-type
cAR1 for agonist-induced phosphorylation, I-7 showed an impaired
cAMP-dependent Ca2+ response. The amount of
cAMP-dependent Ca2+ entry into I-7 and I-14 was
standardized to the levels of cAR1 binding sites measured in the
presence of saturating concentrations of [3H]cAMP and
ammonium sulfate as described in Milne and Devreotes (36). Wild-type
cells accumulated 11 ± 2.3 Ca2+ ions/receptor, I-7
accumulated 2.1 ± 0.7 Ca2+ ions/receptor, and I-14
accumulated 0.6 ± 0.3 Ca2+/receptor (±S.E.,
n = 3).
The ability of the mutant receptors to mediate G
protein-dependent events was examined in several assays.
The ability of cells to aggregate when plated on starvation agar was
used as an initial test. Cells lacking cAR1, G2, or G
cannot
carry out chemotaxis or cell-cell signaling and remain as smooth
monolayers (43, 55-57). Expression of IIIa-1, I-7, or I-14 in
car1
cells restored aggregation and supported
later development, although not as efficiently as wild-type cAR1.
Expression of IV-8 did not restore aggregation (Fig. 4).
Next, GTP
S inhibition of the binding of 2 nM
[3H]cAMP to membranes was measured. This response, used
generally to assess G protein coupling to seven helix receptors (1), is
absent in cells lacking G
2 or G
(58, 59). The measurement was
carried out on membrane preparations from cells that were starved in
the presence of exogenous cAMP pulses to induce expression of G2. All
of the cell lines expressed similar levels of cAR1 and the G2
- and
-subunits (data not shown). 100 µM GTP
S effectively reduced the binding of 2 nM cAMP to wild-type cAR1 and
IIIa-1, but mutants I-7 and I-14 were noticeably impaired, and IV-8
showed no detectable response (Fig. 5). Similar results
were obtained when cAMP binding was measured in the presence of 10 nM [3H]cAMP (data not shown), which provided
greater sensitivity for the lower affinity mutants.
Characterization of Additional car1
Selected cAR1 mutants obtained from an earlier
mutagenesis of the N-terminal one-third of the third intracellular loop
(Thr182-Val189, A region clones) were also
expressed in car1 cells for analysis of G
protein-dependent and -independent responses. In our
previous work, most of these mutant cARs were analyzed in wild-type AX3
cells, which also contain endogenous cAR1 (44). The functional
properties of each mutant cAR1 are illustrated in Table I. Immunoblot
analysis of each of these mutant cell lines revealed that they
expressed receptor protein at levels ~0.5-2-fold of levels seen in
the wild-type cAR1/car1
control (data not
shown).
One striking result is apparent. Despite its small size, many mutations
can be introduced throughout the third loop of cAR1 with no loss of
function. For example, 11 mutants (wtl-1 through wtl-11) induced
wild-type patterns of electrophoretic mobility shift with maximal
responses occurring in the presence of 50 nM cAMP (Table
I). Similar results were obtained for M11.12, a mutant cAR1 in which
Ser183 and Ser195 present in the third
intracellular loop have been replaced with Gly residues by
site-directed mutagenesis.3 Each of these
mutants showed a cAMP-dependent Ca2+ uptake
that was at least 2.3-fold higher than the amount of Ca2+
accumulated by nonstimulated cells. This degree of stimulation is
comparable to that elicited by wild-type cAR1, which shows ~2-fold
stimulation when cells are assayed in suspension for
cAMP-dependent Ca2+ uptake (44). Each of the
mutants coupled to G proteins since they effectively rescued the
aggregation-deficient phenotype of car1 cells.
Moreover, binding of 2 nM [3H]cAMP to
membranes prepared from each of these cAR1-containing mutants (wtl-11
not examined) was reduced by at least 30% in the presence of 100 µM GTP
S.
Twenty of the remaining mutants showed a defective phosphorylation
response at 50 nM cAMP but responded fully at 10 µM cAMP. Only 2 receptors, IIIa-1 and IIIa-2, had defects
strictly related to cAMP binding. These latter receptors displayed a
decreased sensitivity for cAMP in the mobility shift assay, but at
saturating concentrations of cAMP behaved like wild-type cells in this
response and in cAMP-dependent Ca2+ entry. They
also effectively underwent GTPS inhibition of cAMP binding. Mutant
IIIa-1 had a single amino acid substitution Arg184
Gly
close to the N-terminal side of the loop. Mutant IIIa-2 altered the
same amino acid residue Arg184
Cys, although it had
several additional alterations. The remaining receptors appeared to
have defects in cAMP binding affinity, as assessed by the mobility
shift assay; however, these mutants also had additional defects in
signal transduction.
Examination of the class I mutants indicated that all of these
receptors showed essentially wild-type levels of cAR1 phosphorylation at 10 µM cAMP, but displayed specific defects in signal
transduction. Certain mutant receptors appeared to separate the
pathways leading to G protein-dependent responses and G
protein-independent Ca2+ entry. For example, I-1 and I-2
had wild-type levels of stimulated Ca2+ entry, but were
impaired in GTPS inhibition of cAMP binding. In contrast, I-3, I-4,
I-5, and I-6 displayed the opposite pattern of coupling: they all
showed good GTP
S inhibition of cAMP binding, but I-3 displayed no
stimulated Ca2+ entry, and I-4, I-5, and I-6 were markedly
defective. (These findings were confirmed in two independently
constructed clones of I-3 and I-4.) The other class I mutants, however,
were defective in both signaling pathways, showing less than 30%
inhibition of cAMP binding in the presence of GTP
S and reduced
levels of stimulated Ca2+ entry. Surprisingly, even the
most defective of the class I mutants still restored development of
car1
cells.
Certain amino acid substitutions gave rise to mutant receptors with
severely compromised function. These receptors, designated as class IV,
appeared to be general activation mutants, since they were uniformly
impaired in all G protein-dependent and -independent responses. These mutants typically showed less than 50% of the agonist-induced phosphorylation response at saturating concentrations of cAMP, displayed an absent or highly impaired ability to promote Ca2+ entry, displayed little GTPS inhibition of
[3H]cAMP binding, and did not rescue the
aggregation-minus phenotype of car1
cells
(Table I). Detailed data for a representative class IV mutant, IV-1, is
shown in Figs. 2 and 5. It shows impaired cAR1 phosphorylation
responses, even at saturating concentrations of cAMP (EC50 = 178 nM), binds cAMP with a Kd of 117 nM, and shows strongly impaired or absent G
protein-dependent signaling (44). We previously found
several other mutants in the N-terminal region of the third loop (IV-2,
IV-3, and IV-5) that were impaired in their ability to promote
cAMP-induced receptor phosphorylation, even at high concentrations of
cAMP. Other mutants (IV-4, IV-6, and IV-7) showed similar cAR1
phosphorylation profiles. All of these mutants were strongly impaired
or blocked in their ability to activate G protein-dependent
events and G protein-independent Ca2+ influx (Table I).
Several of these mutants (IV-1, IV-2 IV-3, IV-4, IV-5, and IV-6)
introduced or deleted charged amino acid residues in the N-terminal
side of the third intracellular loop. Mutant IV-7 also had a single
mutation in this area as well as several other in the central and
C-terminal side of the loop, including Tyr204
Asp. The
most defective signal transduction mutant, IV-8, bound cAMP (Fig. 3)
but failed to elicit any response (Figs. 2, 4, and 5 and Table I). This
mutant has two alterations in amino acids (Ser183
Pro,
Thr186
Ser) adjacent to the fifth transmembrane helix.
The proline substitution is likely the more important determinant of
the IV-8 phenotype since mutant I-9, although not wtl, contains the
same Thr186
Ser substitution and was able to elicit all
responses and rescue development. Introduction of a proline in the
adjacent residue, Arg184, also caused severe defects in
signal transduction; mutant IV-4 underwent cAMP-dependent
receptor phosphorylation, although it did not stimulate
Ca2+ entry or rescue aggregation.
Our analysis suggests that the third intracellular loop of cAR1
can tolerate many amino acid substitutions, with the retention of G
protein-dependent and -independent signaling responses.
Agonist-induced phosphorylation was the least influenced response,
whereas cAMP-dependent Ca2+ entry and coupling
to G proteins were influenced in a greater number of mutants. However,
despite the presence of biochemical deficiencies in certain of the
mutants, many functioned sufficiently well to rescue the development of
car1 cells.
Of the receptors with defective function, mutants IIIa-1 and IIIa-2 showed defects strictly in cAMP binding affinity, since they effectively elicited all responses but required high concentrations of cAMP to induce receptor phosphorylation (Table I and Figs. 2, 4, and 5). The EC50 of agonist-induced receptor phosphorylation of the other class IIIa and many class I mutants suggests that they also likely have defects in affinity. While impairment of cAMP-dependent cAR1 phosphorylation could be due to an inability of the receptor to interact with receptor kinases or undergo conformational changes to expose the phosphorylation domain, Scatchard analysis of IIIa-1 and I-14 supports the idea that at least certain of these mutants bind cAMP with reduced affinity relative to wild-type cAR1 (Fig. 3).
How might alterations in the amino acid sequence of the third
intracellular loop domain influence the ability of mutant IIIa-1 to
bind cAMP? Studies of rhodopsin and the -adrenergic receptor indicate that ligand binding occurs within the membrane bilayer in a
pocket arising from tight interactions between the seven transmembrane
helices (60). Since ligand-induced changes in the spatial arrangement
of helices induce conformational changes in the intracellular domains
required to couple to downstream proteins, it is plausible that at
least certain amino acid changes in the third intracellular loop of
cAR1 might trigger conformational changes, which alter the cAMP binding
pocket within the membrane. In addition, mutant I-14 replaces
Lys207 with Asn near the cytoplasmic border of the sixth
transmembrane helix and likely also disrupts the relative orientation
of the helices. This is probably not due to replacement of the
positively charged Lys207 residue or due to changes in the
size of the amino acid side chain since mutant wtl-9
(Lys207
Ile) did not influence cAMP binding affinity.
Rather, insertion of Asn may impair helix packing through its ability
to form an additional hydrogen bond through its amide group (61). It
remains to be determined if amino acid substitutions in the third
intracellular loop introduce conformational changes in the receptor
directly or whether they alter interactions with cAR1-binding proteins that modulate binding affinity. Regardless of the mechanism, this study, together with the mapping of mutations within the transmembrane and extracellular domains of cAR1 that modulate cAMP binding affinity (62, 77), suggests that ligand binding to cAR1 is complex, requiring
multiple intracellular and extracellular domains.
A number of receptors with defects in signal transduction were also identified. General activation mutants (class IV) were defective in all G protein-dependent and G protein-independent signal transduction (Table I). One of the most severely impaired receptors isolated in this or in a companion study (77), IV-8, failed to elicit any response (Figs. 2, 4, and 5), despite its ability to bind cAMP comparatively well under physiological conditions (Fig. 3). This mutation thus uncouples ligand binding from all subsequent downstream signaling events. In contrast, class I receptors showed more selective defects (Table I). For example, receptors I-1 and I-2 were specifically impaired in coupling to G proteins, possibly due to a reduced ability to bind the G protein or to activate it once it is bound. Similarly, receptors unable to activate cAMP-dependent Ca2+ influx (I-3, I-4, I-5, and I-6) may be impaired in their ability to bind or activate the yet unidentified downstream effector(s) which trigger Ca2+ entry. It is less likely that cAR1 itself mediates Ca2+ entry since there do not appear to be sufficient numbers of acidic amino acid residues in the transmembrane region to form an effective Ca2+ binding domain. Although very few of these mutants were isolated, they provide important biochemical evidence complementary to earlier genetic analysis (36, 37) that G protein-dependent signaling through cAR1 can be dissociated from G protein-independent Ca2+ signal transduction. It remains to be determined if these mutants influence other G protein-independent events triggered by cAR1, namely, the activation of a MAP kinase (38) and regulation of gene expression events occurring during development (39, 40).
Surprisingly, even though the loop was heavily mutagenized, the specific defect in G protein-dependent signaling seen in I-1 and I-2 was rare and incomplete. In general, functional cARs could mediate all responses, whereas mutants lacking one response were defective in all others. Moreover, many of the mutant cARs retained function despite extensive changes in amino acid sequence, which in many instances, introduced or removed acidic or basic amino acid residues (mutants wtl-1, wtl-3, wtl-5, wtl-6, wtl-7, wtl-8, IIIa-1, I-3, I-4, I-5, I-6, I-8, I-9, I-10, I-12, I-13, and I-15). Interestingly, wtl-8 disrupts a motif containing basic amino acids that is conserved in a large number of seven-helix receptors (2) and thought to be important for interactions between the receptor and G proteins (24). These results are consistent with several models. One possibility is that the loop is required for G protein coupling but the alteration of single or several amino acids was not severe enough to alter the binding affinity of the receptor for G proteins. A second possibility is that the loop may not be needed for specific interactions and that other domains of cAR1 couple to G proteins, as has been suggested for the N-formyl peptide receptor (23, 63). If this were so, why do certain mutations in the third loop block all functions? We propose that domains within the third intracellular loop may act as a hinge. Agonist binding may remove a constraint on the wild-type receptor that holds it in a resting conformation, permitting the generation of intermediate states that interact with G proteins, the components involved in G protein-independent signaling and the receptor kinases required for desensitization. The third intracellular loop will likely be essential for the general activation of many, if not all, seven-helix receptors, since activation mutants and constitutively active mutants have been mapped to this region (22, 30, 64-66).
In light of these findings, the interpretations from earlier mutagenesis studies of other seven-helix receptors implicating the involvement of the N-terminal and C-terminal domains of the loop for the activation of G proteins need to be reassessed. All of those studies analyzed only G protein-dependent signal transduction and do not preclude the possibility that the mutations impair the ability of the receptor to undergo a general activation step. This would, of course, block subsequent steps in the recognition and activation of G proteins. Recent biochemical evidence suggests that certain mammalian G protein-coupled receptors may also activate G protein-independent signals (67-69) other than receptor desensitization (70-72); these may provide useful systems to address this issue.
The results of the third loop mutagenesis and the random mutagenesis of
cAR1 (77) suggest that binding of agonist causes a series of
conformational changes in the receptor during the activation process. A
model depicting these steps is illustrated in Fig. 6. We
propose that cAMP binding to cAR1 leads to an activated state of the
receptor, L-cAR1*, which is able to interact with a receptor kinase.
Additional conformational change(s) lead to the formation of L-cAR1**
enabling the receptor to interact with G proteins or with components
required for Ca2+ entry. Mutants of class II most likely
limit access of cAMP (77), while class III mutants may influence
interactions of the agonist with the binding site. The reduction in
affinity for cAMP does not prevent the formation of active receptor
intermediates, since saturating concentrations of cAMP restores
downstream signaling events in most of these mutants. In contrast, the
general activation mutants of class IV effectively bind cAMP, but show
markedly reduced or absent responses. These signaling defects were not
overcome by high concentrations of cAMP, suggesting that the receptors are unable to undergo conformational changes required for the generation or stabilization of any active cAR1 intermediates. In class
I mutants, cAMP elicits essentially wild-type phosphorylation responses
yet activates poorly G protein-dependent events or G protein-independent influx of Ca2+. These data suggest that
there may be a hierarchy among signaling functions; generation of the
L-cAR1* intermediate is sufficient for receptor phosphorylation, while
L-cAR1** likely is required for Ca2+ influx and coupling to
G proteins. A few class I mutants, such as I-1 and I-3, were
specifically defective either in coupling to G proteins or in
cAMP-dependent Ca2+ influx. These receptors may
attain the L-cAR1** conformation but fail to interact or activate the G
proteins or the factor(s) required for ion fluxes, respectively.
Alternatively, these mutations potentially could block the formation of
yet additional cAR1 intermediates essential for one or both of the
responses.
Our model implies that the receptor must go through an intermediate, L-cAR1*, which is able to be phosphorylated before it forms L-cAR1**, which then mediates other signal transduction events. All of our data are consistent with this model. However, it is conceivable that there are mutants which attain the conformation required for coupling to G proteins and G protein-independent events, but which do not attain the conformation required to interact with receptor kinase. Our experimental designs may have precluded the identification of this particular type of mutant. The third loop mutagenesis targeted a very small region of the receptor, which may not be involved in receptor phosphorylation, whereas the general mutagenesis focused on the characterization of aggregation-deficient clones. In fact, deletion of all of the sites within cAR1 which undergo agonist-induced phosphorylation does not impair aggregation4 (41).
Our identification of these mutant classes, together with recently emerging information of rhodopsin (73, 74), suggests that the activation of G protein-coupled receptors may be more complex than previously envisioned and may involve multiple intermediates. Given the advances in determining the structure of other seven-helix receptors (75, 76) and progress in the purification of cAR1,5 these mutants will provide an important tool for structural determination of cAR1 intermediates during the activation process.
We thank Jane Borleis and Dr. Dale Hereld for providing mutant Mll.12.