(Received for publication, October 25, 1994)
From the
To derive structure-function relationships for receptor-G
protein coupling, libraries were created of human m5 muscarinic
acetylcholine receptors (m5) randomly mutated in the C-terminal region
of the third intracellular loop. Functional receptors were identified
based on their ability to amplify NIH 3T3 cells in a ligand-dependent
manner. These receptors either had wild-type phenotypes (Group 1) or
were functionally impaired (Group 2). No ``activated
receptors'' were identified. Tolerated substitutions in Group 2
receptors were randomly distributed and frequently included prolines
and glycines. In contrast, tolerated substitutions in Group 1 receptors
exhibited a periodicity proximal to transmembrane domain 6 where
proline and glycine substitutions were not observed. These observations
are consistent with a short -helical extension of the C-terminal
region of the third intracellular loop from transmembrane domain 6.
Mutations at Ala-441 were most commonly associated with impaired
function of Group 2 receptors. Twelve point mutations at Ala-441 were
tested, and all caused marked increases in EC
values with
little effect on maximal response or agonist binding affinity. These
results indicate that Ala-441 is a key determinant of m5 receptor
affinity for G proteins and exists within the structural context of a
short
-helix.
Muscarinic acetylcholine receptors are members of a large family
of receptors that mediate signal transduction by coupling with G
proteins. Most members of this family display significant sequence
homology, particularly within regions predicted to form seven
transmembrane domains (TM
1-7)()(1, 2) . Muscarinic receptors
consist of five genetically defined subtypes (m1-m5) (3, 4, 5, 6) that can be divided
into at least two functional classes. m1, m3, and m5 couple to
pertussis toxin-insensitive G proteins to potently stimulate
phospholipase C, promote calcium release from cytosolic stores,
modulate ion channels, stimulate mitogenesis, and transform NIH 3T3
cells. m2 and m4 couple to pertussis toxin-sensitive G proteins to
weakly stimulate phospholipase C, open inwardly rectifying potassium
channels, and inhibit adenylyl cyclase. The m2 and m4 subtypes do not
stimulate mitogenesis nor do they transform NIH 3T3
cells(7, 8) . By preparing chimeras between subtypes
from the two functional classes (e.g. chimeric m1/m2 and m3/m2
receptors), the third cytoplasmic loop (i3) has been shown to be the
region that defines subtype specificity for distinct G proteins (9, 10, 11, 12) . The central
portion of the i3 loop can be deleted without impairing coupling to G
proteins, indicating that only the N- and C-terminal regions of the i3
loop (Ni3 and Ci3, adjacent to TM5 and TM6, respectively) are required
for function(13, 14, 15, 16) .
However, the precise structural requirements of these domains for G
protein coupling remain largely unknown.
In order to comprehensively examine the structure-function relationships of receptor-G protein coupling, we have developed an efficient method for using random mutagenesis in mammalian systems. A major advantage of random mutagenesis is the ability to create thousands of amino acid substitutions with no bias regarding the type and location of substitutions made. By using a screen to identify mutants that retain function, one can identify which amino acid substitutions are functionally allowed. Activated phenotypes or ``gain of function mutations'' are particularly easy to identify in a screen based upon positive selection. When a domain is saturated with mutations, one can make inferences about amino acid substitutions that are never recovered as functional proteins(18, 19) .
A
reliable and facile screen is a prerequisite for any random mutagenesis
protocol. Our methods are based on the observation that
phosphatidylinositol-coupled muscarinic receptors induce
ligand-dependent focus formation in NIH 3T3 cells. In fact, the
dose-response relationships of transformation and phospholipase C
stimulation are identical(28) . Thus, functional muscarinic
receptors can be cloned from a library of mutants by virtue of their
ability to induce foci in response to agonist treatment. Furthermore,
we have found that this receptor-mediated stimulatory effect can be
monitored using a reporter gene assay, ()eliminating the
need to culture cells until foci develop and allowing us to measure
graded responses. We have used these methodologies to obtain the first
comprehensive look at the structurally important elements of the i3
loop of a G protein-coupled receptor.
Based on protocols that we have previously used to evaluate
induction of foci by ligand-occupied muscarinic receptors(28) ,
we developed a method for monitoring proliferative responses using a
reporter gene. In our assay technology, ligands select and amplify
cells that express functional receptors. These same cells also express
a reporter gene; thus receptor activity is assayed as a change in
enzyme levels (R-SAT, patent pending). As shown in Fig. 1A, when -galactosidase and m5 muscarinic
acetylcholine receptor expression vectors were co-transfected into NIH
3T3 cells, there was a time-dependent amplification of cells cultured
in the presence of carbachol compared with cells cultured in media
alone (Fig. 1). This amplification was rapid, becoming
significant by the second day of culture and plateauing by 5 days in
culture. Thereafter all assays were performed for 5 days. We then
evaluated the dose-response relationship of carbachol-induced
amplification and compared it with a focus-forming assay. As shown in Fig. 1B, the EC
for carbachol was
approximately 10-fold lower in R-SAT than focus assays. This difference
is probably due to higher expression levels of receptor in the shorter
assay (5 days compared with 14 days). The pharmacology of carbachol and
several other muscarinic agonists and antagonists as determined with
R-SAT is very similar to that determined with traditional functional
assays. A detailed pharmacological analysis will be presented
elsewhere.
Figure 1:
R-SAT assay. A, time course. m5 and -galactosidase cDNAs were
co-transfected into NIH 3T3 cells, and induced
-galactosidase
activity was assayed at the indicated times as described under
``Experimental Procedures.'' The responses were calculated
from the base line, and maximum responses were derived from least
squares fits of full dose-responses at each time point (filled
triangles, maximal response; open circles, basal
response). B, carbachol dose-response of the
-galactosidase amplification on day 5 and focus formation on day
14. Plotted are absorbance at 405 nm (filled triangles, R-SAT)
and number of macroscopic foci/10-cm
dish (open
squares, focus assay) versus carbachol concentration.
Assays and curve fits were performed as described under
``Experimental Procedures.''
We employed R-SAT in combination with random saturation mutagenesis to investigate the structure-function relationships of m5 receptor-G protein coupling. We used randomly doped oligonucleotides in a PCR-based construction method (Fig. 2) to generate a library of mutant m5 muscarinic acetylcholine receptors saturated with mutations throughout Ci3. Sequence analysis of unscreened libraries verified that there was a 10% nucleotide substitution rate with an average of greater than 5 amino acid substitutions per clone (not shown). A pool of recombinants representing 500 clones was evaluated for the ability to bind the antagonist NMS and to transform NIH 3T3 cells. We observed that transient expression of pCDm5-Ci3* in COS7 cells yielded 35% of the binding sites compared with cells transfected with an equivalent amount of wild-type pCDm5 (1.1 ± 0.2 versus 3.1 ± 0.3 pmol/mg protein) with essentially identical affinity for NMS (153 ± 26 pMversus 126 ± 19 pM), indicating that a third of the constructs expressed binding sites. The Ci3* pool of mutants was able to transform 3T3 cells 5-10% as efficiently as wild-type m5 (not shown), consistent with the notion that there was a strong functional selection against mutations within Ci3. Interestingly, although mutations within the analogous regions of other receptors are activating(20, 21, 22, 23, 24, 25, 26) , we detected no foci in the absence of carbachol.
Figure 2:
Mutagenesis of the Ci3 of m5. A,
region to be mutagenized. Wild-type sequence is indicated. B,
library construction strategy. PCR was performed using primers p1-p4. The p2 primer comprised residues
424-445. The outer primers (p1 and p4) contain ApaI and XbaI restriction sites for subsequent
cloning. The two PCR products were treated with T4 DNA polymerase to
create blunt ends, ligated to yield concatamers, and restricted with ApaI and XbaI to release the randomly mutated (*)
Ci3*ApaI-XbaI inserts. Inserts were ligated into an ApaI/XbaI fragment of the pcD-m5 yielding a
population of mutant m5 receptor DNA (pCDm5-Ci3*). Competent DH5
cells were transformed with pCDm5-Ci3*, amplified either as pools or as
individual transformants, and plasmid DNA was
isolated.
The results in the focus experiments with the pooled library indicated that there was a sufficiently high percentage of functional mutants to screen clones derived from this library individually, eliminating the need to retrospectively clone and identify positive receptors. Assuming that mutant receptors capable of producing foci in 3T3 cells would also be able to mount a response in the R-SAT assays, we expected a 5-10% positive clone rate. We screened 300 individual constructs in the presence or absence of a single dose (100 µM) of carbachol in triplicate and were able to identify 36 clones that responded. Three of these clones failed to respond a second time, and two clones had wild-type sequences.
The positive clones identified in the primary
screen were subjected to a detailed dose-response analysis, using doses
of carbachol ranging from 1 nM to 100 µM or
100-fold below to 1000-fold above the EC of carbachol for
wild-type m5. As shown in Fig. 3and Table 1, we isolated
receptors with EC
values ranging to 100-fold above the
wild-type EC
and maximal responses ranging from 25 to 125%
of wild type. Thus we effectively isolated the full range of receptor
phenotypes with rightward shifts in EC
or with decreased
maximum responses that we could have reliably identified under these
conditions (the EC
cannot be greater than the K
). However, we did not find any receptors with
greater than 3-fold leftward shifts in EC
or significantly
elevated activity. We would have expected, a priori, that
activated receptors would have been readily detected since R-SAT is
based upon positive selection and since mutations in Ci3 activate many
other
receptors(20, 21, 22, 23, 24, 25, 26) .
Figure 3: Dose-response curves in the R-SAT functional assay for Group 1 (fully functional receptors) (A) and Group 2 (functionally impaired receptors) (B) compared with m5.
We divided the functional mutant receptors into two groups based upon their pharmacological phenotypes ( Table 1and Fig. 3). Group 1 includes receptors with essentially wild-type characteristics, whereas Group 2 receptors are impaired but still functionally competent. To assess the molecular basis for the range of phenotypes observed, we sequenced each of these receptors. Examination of the receptors in Group 1 reveals that although mutations were randomly introduced, functionally tolerated mutations were clustered in residues 424-438 and rare in residues 439-445, suggesting there was functional selection against mutations at discrete residues. Indeed, there was an average of 3.7 substitutions per Group 1 receptor but 4.9 substitutions per Group 2 receptor supporting this notion.
To facilitate comparison of the molecular differences between Group
1 and Group 2 receptors, we compiled all the mutations into lists (Table 2). A consensus sequence of functionally conserved
residues that were never mutated can be derived for the Group 1
receptors consisting of Ala-440, Ala-441, and Leu-444 (Table 2).
In addition, Lys-439 was only conservatively mutated to arginine. Group
2 receptors had a much more random distribution of mutations, and 9 out
of 16 had mutations in the consensus sequence derived for Group 1,
confirming the functional importance of these residues. Merely
examining sequence conservation among muscarinic subtypes (Table 2) was not reliably predictive of the functional
importance of any specific residue. For example, the KERK motif (amino
acids 436-439) is well conserved, but only the second lysine
appeared to have functional importance based on our analysis (Table 2), which is consistent with earlier
observations(15, 16) . We assumed residues observed to
be frequently or radically mutated in receptors that retained full
function or were dispensable for function. Presumably, mutations
observed in both groups also did not account for the observed
phenotypic changes in Group 2 nor could differences in expression
levels account for the relative impairment of Group 2 receptors
compared with Group 1 receptors (see [H]NMS
binding in the legend to Table 1). Thus the impaired function of
Group 2 receptors is most likely related to the mutations within the
consensus sequence.
Another striking difference between Group 1 and
Group 2 receptors is the proline and glycine substitution patterns (Table 2, see helix breaks). Proline and glycine substitutions
are excluded from residues 439-445 in Group 1 receptors but are
distributed throughout Group 2 receptors, including residues
439-445. This is strong evidence for the existence of a short
-helical extension from TM6, which when disrupted impairs receptor
function. It is noteworthy that the mutation pattern in Group 1
receptors appears to become periodic beginning at Arg-438 with radical
mutations clustered 4 residues apart (Arg-438-Gln-442), also
suggesting the existence of a helical structure. The junction of the i3
loop and TM6 is predicted to be within the vicinity of
Gln-442(8) . Together, these data suggest that the TM6 helix
may extend into Ci3 for a single turn before adopting some other
conformation.
Inspection of the Group 2 mutants revealed that
mutations at Ala-441 were associated with large increases in EC (see clones 32, 135, 139, 166, and 202 in Table 1),
implying that Ala-441 is a critical determinant of receptor potency.
This hypothesis is complicated since the mutations at Ala-441 were
analyzed in the context of other mutations. Therefore, we constructed a
point mutant library of receptors randomly mutated to all of the
possible amino acid substitutions at Ala-441, rescued viable clones by
R-SAT, and analyzed each receptor as described above. We identified 12
unique amino acid substitutions at Ala-441, spanning all of the major
amino acid classes, and each caused significant increases in EC
without affecting maximal response (Table 3). Changes in
agonist binding affinity could not account for the increases in
EC
nor could differences in expression levels (Table 3, legend). Significantly, substitution of threonine,
which is found in the analogous position to Ala-441 in m2 and m4
(G
-coupled) receptors (see Table 2and (8) )
caused the largest shift in EC
suggesting Ala-441 also
influences G protein coupling selectivity. Proline, which disrupts
-helices, was also effective at reducing receptor potency.
Cysteine had little effect, perhaps because it is of similar size to
alanine. However basic, acidic, aromatic, and large hydrophobic
residues all had similar effects on function.
We have developed an efficient method for analysis of the structure-function relationships of G protein-coupled receptors by random mutagenesis. This approach was possible because of the development of a high throughput assay based upon cellular proliferation, which offers significant technical advantages over focus assays or second messenger assays for evaluating receptor function. Second messenger assays are too labor intensive to allow one to screen the large numbers of receptors necessary for studying structure-function relationships by random mutagenesis. Although focus assays are amenable to screening large numbers of clones, foci are not observed until at least 10 days of carbachol treatment(28) , while R-SAT gives measurable responses in 3-5 days. More significantly, one can easily measure graded responses using R-SAT, allowing one to characterize subtle effects of mutations upon receptor function, whereas focus assays only discriminate all or none responses.
Using R-SAT in combination with random saturation mutagenesis we
have discovered that the section of Ci3 proximal to TM6 regulates m5
receptor affinity for G proteins and that this function is most
severely impaired by proline substitutions or by substitutions at
Ala-441 (Fig. 4). Therefore we conclude that Ala-441 is a key
determinant of m5 affinity for G proteins and that Ala-441 is likely to
reside within the structural context of a short -helical extension
from TM6. We have found that overexpression of G
shifts the
EC
values of ``low potency'' receptors
(EC
> 1 µM) back to wild-type values, (
)further supporting the idea that the main effect of
mutations at Ala-441 is to reduce receptor affinity for G
,
although it is impossible to say whether or not Ci3 directly contacts
G
.
Figure 4:
Ratio of EC values of each
clone to the EC
of wild-type m5. Inset, plot for
the Ala-441 point mutants. A value below 1 represents a clone with
greater potency than wild-type m5. EC
values were
determined from R-SAT experiments performed as described under
``Experimental Procedures.'' P and D denote
proline substitutions or deletions observed within the predicted
helical segment of Ci3, respectively. Three-letter amino acids denote
observed substitutions for Ala-440. Group 1 and Group 2 clones are
defined as in Fig. 2and Table 1.
We could detect no evidence of elevated constitutive
activity of any of the Ci3 mutants in contrast to results for many
other receptors (20, 21, 22, 23, 24, 25, 26) .
Similarly, none of the Ala-441 point mutants were activated, in
contrast to studies on the -adrenergic receptor in
which all amino acid substitutions at the analogous position activated
that receptor(22) . These negative results are not due to
technical limitations since the activated phenotypes of mutationally
activated adrenergic receptors are readily observed using R-SAT. (
)Although we found no activating mutations within Ci3, we
have discovered that m5 is activated by mutations in TM6. (
)TM6 is also a site of activation for the leutinizing
hormone receptor(32) . Mutations in TM6 increase the basal
activity of m5 and also raise the affinity of m5 for agonists, very
similar to the effects of mutations within the Ci3 region of the
adrenergic receptors(20, 21) . Considering that TM6 is
immediately adjacent to Ci3 and is predicted to form an
-helix
extending into Ci3 (this paper) one might envision TM6-Ci3 as a single
functional unit extending down to Lys-439. In this context, the
function of the TM6-Ci3 domain of m5 may be quite similar to the
adrenergic receptors, but the details of how it operates to convert
agonist binding into G protein coupling may be reversed in the two
receptor families.