(Received for publication, July 21, 1995; and in revised form, August 11, 1995)
From the
We used random mutagenesis and phenotypic rescue of adenylyl cyclase-null Dictyostelium cells to isolate loss-of-function mutations in the enzyme. Mutants were (i) catalytically inactive or (ii) resistant to chemoattractant receptor and guanosine 5`-3-O-(thio)triphosphate stimulation. Both classes of mutants harbored substitutions within the cytoplasmic C1a domain. Mutations that inactivated the enzyme were often at highly conserved positions. Those that blocked activation were grouped in two distinct regions: one close to the plane of the plasma membrane and another halfway within the C1 loop. Missense mutations or deletions within the transmembrane domains resulted in missorting of the protein. Our screen provides a simple and efficient method to separately define the sites of catalysis and regulation of this important class of enzymes.
Adenylyl cyclases catalyze the conversion of ATP into the second
messenger cAMP. Modulation of adenylyl cyclase activity by hormone and
neurotransmitter receptors, through heterotrimeric guanine
nucleotide-binding regulatory proteins (G proteins), underlies a wide
variety of physiological events(1) . The genes for eight types
of mammalian, one Drosophila, and one Dictyostelium adenylyl cyclase all encode proteins that are predicted to span
the membrane 12 times and contain two large cytoplasmic domains (about
40 kDa each)(2, 3, 4, 5) . Each
cytoplasmic domain contains a region of homology (designated C1a and
C2a) with the catalytic domains of several adenylyl and guanylyl
cyclases(2, 3) . Deletion analysis and site-directed
mutagenesis have shown that the interaction between the two halves of
the enzyme is necessary for activity and suggested that the two
homologous cytoplasmic domains are not
equivalent(6, 7) . ()
The receptor and G
protein regulation of the adenylyl cyclase (ACA) ()in Dictyostelium is analogous to that in mammalian
cells(8, 9) . The chemoattractant receptor, cAR1,
mediates, in addition to the events involved in chemotaxis, the
activation of ACA and synthesis of cAMP. The regulation of ACA is
similar to mammalian type II and IV adenylyl cyclases, which are
synergistically stimulated by G protein
-subunits and
activated by G
(10) . Indeed, genetic and
biochemical analyses have established that receptor and GTP
S
activation of ACA requires two components: the
-subunits of
the heterotrimeric G protein G2 and a novel cytosolic protein named
CRAC for cytosolic regulator of adenylyl cyclase(11, 12) .
Proper regulation of ACA
is essential for the early stages of development during which 10 cells spontaneously aggregate and differentiate into fruiting
bodies. Secretion of cAMP serves to relay chemotactic signals to distal
cells which, in turn, migrate toward the center to form aggregates.
Consequently, aca
, g
, and crac
cells cannot carry out
aggregation and remain as smooth monolayers when plated on non-nutrient
agar(5, 11, 12) . In this study we used
complementation of the aca
phenotype as a
convenient and efficient readout to identify loss-of-function mutations
in ACA.
Random mutagenesis of the ACA gene was achieved by
error-prone PCR. We mutagenized a 1.7-kilobase pair region of the ACA
cDNA corresponding to five predicted transmembrane helices as well as
the C1 domain (Fig. 1). The mutagenized cDNAs were subcloned
into an extrachromosomal expression vector. We used a Dictyostelium-specific shuttle vector (pCP33), which was shown
to give high transformation efficiencies (>10)
and 100% segregation (i.e. after transformation, each
transformant contains a unique mutated plasmid). pCP33 was derived from
the p155d1 plasmid constructed by Hughes et al.(24) .
It contains sequences needed for extrachromosomal replication derived
from Dictyostelium nuclear plasmid Ddp1, the neomycin gene,
and a bacterial backbone for replication in Escherichia coli.
The ACA gene was cloned downstream of the actin-15
promoter(25) , which causes the ACA gene to be overexpressed
compared with wild type (data not shown). The library was
electroporated into aca
cells, and the
resulting transformants were screened by clonally spreading them on K. aerogenes lawns (see ``Experimental
Procedures''). Wild-type ACA transformants aggregate to form
``rough'' plaques with 100% efficiency; loss-of-function
mutants that fail to aggregate are readily detected as
``smooth'' plaques (Fig. 2A).
Figure 1: Random mutagenesis of the ACA gene. Model of adenylyl cyclase depicting the mutagenized region (shadedarea).
Figure 2:
Phenotypic screen and protein expression
of ACA mutants. A, representative phenotypic screen; the cells
were mixed with K. aerogenes and plated on SM agar plates.
This picture, taken 5 days after plating, shows one mutant and five
wild-type plaques. Bar represents 0.5 cm. B, Western
analysis of selected aggregation-deficient mutants using a peptide
antibody directed against the C terminus of ACA. Detection was
performed using enhanced chemiluminescence. WT refers to aca cells in which the wild-type ACA gene is
overexpressed.
Strong and
weak PCR mutagenesis conditions were used, and the results of the
screens are presented in Table 1. A total of 120
aggregation-deficient transformants were analyzed for ACA expression
using a C-terminal peptide antibody that specifically labels a
160-kDa band in cells overexpressing a WT ACA gene (Fig. 2B). Forty mutants showed protein expression. The
level of expression varied between mutants and was always greater (up
to 10-fold) or equal to that observed in wild-type parental cells
(AX3). Upon rescreening for development on non-nutrient agar, the 40
protein-expressing mutants all remained aggregation-deficient. The
non-regulated adenylyl cyclase activity of these clones was measured in
the presence of MnSO
and revealed that 17 of the 40 mutants
had activity (Table 1). Eight mutants (4 active, 4 inactive) were
selected at random and their mutated plasmid was recovered and
retransformed into aca
cells. Each plasmid
recapitulated the phenotype. Seven of the eight recapitulated mutants
showed high protein expression and were either catalytically inactive
(I1, I2, I3, I4) or active (U1, U2, U3) (Fig. 3, A and B). One mutant (U4) showed no protein expression upon
retransformation and was discarded. Comparison of the enzymatic
activities in lysates prepared from AX3 cells and from aca
cells overexpressing wild-type ACA (Fig. 3B) shows that aggregation can occur over a
10-fold range of adenylyl cyclase activity. Clearly, U1, U2, and U3
fall within this limit. Presumably, these active mutants are
aggregation-deficient because they are ``uncoupled'' from
surface receptor or G protein stimulation.
Figure 3:
Biochemical analysis of selected ACA
mutants. The plasmids from mutants I1-I4 and U1-U3 were
transformed into aca cells. The resulting
transformants were compared with the aca
cell line overexpressing the wild-type plasmid. A, ACA
protein expression detected as in Fig. 2. B, basal and
MnSO
-stimulated adenylyl cyclase activity. The enzymatic
activity of the AX3 cells (parental cell line of aca
cells) is shown as a control for normal
expressing levels of ACA. Gray and whitebars represent basal and unregulated (MnSO
) conditions
respectively. C, adenylyl cyclase activity following cAMP
stimulation of catalytically active mutants. Filledsquares, WT; squares, U1; filleddiamonds, U2; diamonds, U3. D,
GTP
S-stimulated adenylyl cyclase activity of U1-U3. Gray and singlehatchedbars represent basal
and GTP
S conditions. Black and doublehatchedbars represent basal and GTP
S
stimulation in the presence of CRAC. Whitebars represent unregulated activity (MnSO
). The lower
levels of ACA activity reported here, compared with previously
published results, are due to the lower substrate concentration used.
The results presented are representative of at least two independent
experiments.
To characterize the
biochemical defect in U1, U2, and U3, we dissected the receptor-G
protein-adenylyl cyclase cascade. To determine if receptor-mediated
activation of adenylyl cyclase was normal, we measured ACA activity, in
the presence of CRAC, following the addition of exogenous cAMP to
intact cells (see below) (Fig. 3C). Under such
conditions, cells overexpressing wild-type ACA show a rapid increase in
enzyme activity, peaking 1-2 min after addition of cAMP, followed
by a return to basal levels within 7-10 min. Neither U1, U2, nor
U3 showed chemoattractant-mediated activation of adenylyl cyclase,
explaining why they fail to aggregate. To assess the integrity of the
interaction between cAR1 and G2, we performed an in vitro GTP-induced inhibition of binding assay. In this assay, the
presence of GTP, which promotes the uncoupling of the G protein
-subunit form both the receptor and its
- and
-subunits,
considerably reduces the affinity of the agonist-receptor interaction.
All three mutants showed normal high affinity binding to cAMP. In
addition, in the presence of GTP, binding was reduced by 68, 65, 67,
and 69% for WT, U1, U2, and U3, respectively (data not shown). Since
these results showed that the defects in these mutants are downstream
of G protein activation, we performed an in vitro GTP
S
stimulation assay. As shown in Fig. 3D, U1, U2, and U3
exhibited no G protein-mediated activation of adenylyl cyclase.
Moreover, these defects were not reversed by the addition of excess
exogenous CRAC (Fig. 3D). Due to the limited
availability of CRAC in lysates of cells overexpressing wild-type ACA,
this addition typically potentiates GTP
S stimulation.
We sequenced the mutagenized inserts, and the results are summarized in Fig. 4. The overall frequency of mutagenesis was very low, even under strong PCR mutagenesis conditions, and ranged from 1 to 8 base pair changes/mutant sequenced. Of a total of 23 point mutations identified, 6 were transversions. Twenty of the 23 mutations resulted in a change of amino acid and 3 were silent. Interestingly, we also isolated a mutant bearing an in-frame 213-nucleotide (71-amino acid) deletion within the second and third transmembrane domain (U1). Out of the 20 missense point mutations characterized, only two occurred within the predicted transmembrane domains (I2, I3).
Figure 4: Sequence analysis of ACA mutants. A, topological scheme depicting point mutations of catalytically inactive and G protein-insensitive mutants. The grayline depicts the conserved C1a domain. Circle, I1 (G629C); filledcircle, I2 (N333D, G633S); square, I3 (N330T, L460S, K510R, E595D, I640N, W681R); filledtriangle, I4 (L409I, V445A, D488G, N690D, N744S, T747A); boxedarea, U1 (deletion 237-308); filledstar, U2 (V427I, S642F); invertedtriangle, U3 (L405S, F421S, C713R). B, amino acid sequence of ACA within the C1a loop (positions 626-672). The mutated G629 (I1), G633 (I2), and I640 (I3) residues are marked with an arrow. Alignment of the presumed catalytic domains of adenylyl and guanylyl cyclases from bacteria (adenylyl cyclase from Brevibacterium liquefaciens), sea urchin (Strongylocentrotus purpuratus membrane-bound guanylyl cyclase), yeast (adenylyl cyclase from Saccharomyces cerevisiae and Schizosaccharomyces pompe), Dictyostelium (aggregation (C1a domain) and germination specific adenylyl cyclases), trypanosome (Trypanosoma brucei expression site-associated gene), Drosophila (adenylyl cyclase, C1a domain), and mammalian (Type I-V adenylyl cyclases, C1a domain, soluble and membrane-bound guanylyl cyclases) species was obtained from W. J. Tang and A. G. Gilman. Residues that are identical in at least 75% of these sequences were included in the design of the consensus sequence shown. x refers to hydrophobic residues.
For the catalytically
inactive mutants, we discovered that point mutations clustered within a
highly conserved stretch of glycine residues located between positions
626 and 672 for three of the four mutants analyzed (Fig. 4B). Indeed, I1 displayed a single point mutation
at a highly conserved glycine residue (G629C) (Fig. 4A). For I2 the introduction of a hydroxyl group
on a neighboring conserved glycine residue (G633S) as well as an
asparagine to aspartate change at position 333 also rendered the
protein inactive. In addition, among its six substitutions, mutant I3
carried an isoleucine to asparagine (position 640) change at a
conserved hydrophobic residue flanking one of the highly conserved
glycine residues (Fig. 4B). These results suggest that
this stretch of conserved residues within the C1a domain of ACA is
important for catalytic activity. Mutant I4 exhibited six missense
mutations located throughout the C1 domain (Fig. 4A).
Interestingly, one of them (D488G) corresponded to a site-directed
mutant of a highly conserved aspartate residue (D354A) shown to render
the mammalian type I adenylyl cyclase catalytically inactive while
still capable of binding to G.
Two of
the three GTPS-insensitive mutants, U2 and U3, displayed two and
three point mutations, respectively, within the C1a domain (Fig. 4A). Tang and Gilman (26) showed that a
soluble type I/type II chimera lacking transmembrane domains retains
sensitivity to G
stimulation. Our results also suggest
that regulatory sites lie within the cytoplasmic domains, along with
the catalytic sites. Analysis of their location revealed that the
substitutions grouped in two distinct regions: one close to the plane
of the plasma membrane and another halfway within the C1 loop. The
cluster close to the membrane is intriguing in that regions of
cytoplasmic loops abutting the fifth, sixth, and seventh transmembrane
domains of surface receptors are critical for G protein
interaction(27) . Our data suggest that similar
membrane-apposed regions are involved in G protein regulation of
adenylyl cyclases. We are currently separating the two groups of
mutations in U2 and U3 and isolating additional mutants to more
precisely define this regulatory domain. Our results are different but
not mutually exclusive from those of Chen et al.(28) ,
who, using mammalian type II adenylyl cyclase peptides, recently
demonstrated that G
-subunits may interact with a region of
C2a.
Although U1 had similar biochemical properties as U2 and U3, it
displayed a 71-amino acid deletion within the second and third
transmembrane domain (Fig. 4A). Thus it would be
predicted to contain only four transmembrane domains in its N-terminal
half. Since this mutant showed normal catalytic activity, we were
confident that it acquired an appropriate conformation. However, we
reasoned that U1 might not be targeted to the correct subcellular
location; missorting could preclude its appropriate receptor and G
protein regulation. Xiao and Devreotes ()recently
demonstrated that cAR1 and ACA co-localize within specific plasma
membrane subdomains. We carried out their subcellular fractionation
procedure as an additional assessment of all the mutants. While both U2
and U3 exhibited a normal ACA distribution, U1 showed a drastically
lower amount of ACA associated with cAR1 (data not shown). We propose
that U1 is resistant to receptor and GTP
S stimulation mainly
because it cannot access activated G
-subunits. Indeed,
following cAMP activation of wild-type cells, CRAC is translocated to
membranes. In g
cells this
CRAC membrane association does not occur and GTP
S-stimulated
adenylyl cyclase activation cannot be measured(21) .
The catalytically inactive mutants I2 and I3 also exhibited abnormal subcellular distributions (data not shown). However, U1 illustrates that while co-localization with cAR1 may be required for appropriate regulation, it is not essential for catalytic activity. We conclude that the defects in I2 and I3 are most likely due to the mutations in C1a as described above. Interestingly, these two missorted mutants also harbored mutations within the predicted transmembrane domains (as did U1; Fig. 4A). These results suggest that correct sorting may be dependent on features within the transmembrane domains.
Using our screen, both catalytically inactive and receptor/G
protein-resistant adenylyl cyclase mutants can be efficiently and
simply isolated from a randomly mutagenized population of molecules.
Indeed, this is the first report of independent mutations that clearly
separate these defects. Moreover, we have devised a way to isolate
gain-of-function adenylyl cyclase proteins by transforming the
mutagenized ACA libraries into crac cells
and isolating aggregation-competent clones. We can use suppression of
the crac
phenotype to screen for ACA mutants
with high, unregulated activity or for those that retain regulated
activity in the absence of CRAC, bringing more insight into the
G
-subunit regulation of adenylyl cyclases.