(Received for publication, March 27, 1995; and in revised form, December 4, 1995)
From the
The third intracellular loop (i3) plays a critical role in the
coupling of many receptors to G-proteins. In muscarinic receptor
subtypes, the N- and C-terminal regions (Ni3 and Ci3) of this loop are
sufficient to direct appropriate G-protein coupling. The relative
functional contributions of all amino acids within Ni3 was evaluated by
constructing libraries of m5 muscarinic receptors containing random
mutations in Ni3 and screening them using high throughput assays based
on ligand-dependent transformation of NIH 3T3 cells. In receptors that
retained a wild type phenotype, the pattern of functionally tolerated
substitutions is consistent with the presence of three turns of an
helix extending from the transmembrane domain. All of the amino
acid positions that tolerate radical substitutions face away from a
conserved hydrophobic face that ends with an arginine, and
helix-disrupting proline substitutions were not observed. All of the
mutant receptors with significantly compromised phenotypes had amino
acid substitutions in residues predicted to form the hydrophobic face.
Similar data from the Ci3 region (Burstein, E. S., Spalding, T. A.,
Hill-Eubanks, D., and Brann, M. R.(1995) J. Biol. Chem. 270,
3141-3146) are consistent with the presence of a single helical
turn extending from the transmembrane domain, with an alanine that
defines G-protein affinity. Functionally critical residues of Ni3 and
Ci3 are predicted to be in close proximity where they form the
G-protein-coupling domain.
Muscarinic acetylcholine receptors are members of a 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, TM1-TM7(1, 2, 3, 4) . ()This family consists of five genetically defined subtypes,
m1-m5(5, 6, 7, 8, 9, 10) ,
that can be divided into two functional classes. m1, m3, and m5 couple
to pertussis toxin-insensitive G-proteins to potently stimulate
phospholipase C (G
), 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 (G
). m2 and m4 do
not stimulate mitogenesis, nor do they transform NIH 3T3
cells(9, 10, 11) .
Studies using chimeric receptors prepared from subtypes representing the two functional classes (e.g. chimeric m1/m2 and m3/m2 receptors) have shown that the third intracellular loop (i3) is the region that defines subtype specificity for distinct G-proteins (12, 13, 14, 15) . With the exception of the N- and C-terminal regions (Ni3 and Ci3, adjacent to TM5 and TM6, respectively), the majority of the i3 loop can be deleted without impairing G-protein coupling in muscarinic (16, 17) and adrenergic (18, 19) receptors, and exchange of the Ni3 region is sufficient to redirect G-protein coupling in these chimeric receptors(13, 14, 15, 20) .
Computational analysis of the primary sequences of the Ni3 regions
of several receptors has predicted that this region may form an
amphipathic helix(21, 22, 23) . A role
for amphipathic
helices in G-protein activation has also been
indicated by analysis of the toxin mastoparan, which forms an
amphipathic
helix and directly activates G-proteins(23) .
Comparison of the sequences of the intracellular domains of various
G-protein-coupled receptors with that of mastoporan led to the
suggestion that the distribution of charged residues within these
structures may be the critical determinant of G-protein coupling (21, 22, 23) . Using site-directed
mutagenesis, these charged residues have now been shown to have little
or no contribution to G-protein coupling in muscarinic (24) and
-adrenergic (25) receptors. These data suggest that the
charged residues were not conserved because of their functional role
and thus highlight a major limitation inherent in predictions of
function based on sequence comparisons within gene families, namely
that sequence homology is as often a reflection of evolutionary
relatedness as it is on function.
Random saturation mutagenesis overcomes the constraining need to infer structural significance from sequence comparisons, since all amino acids in a given region are subjected to mutations(26, 27, 28) . Furthermore, since screens are based on retention of function rather than disruption of function, the likelihood of misinterpretation inherent in evaluation of ``negative'' data is significantly reduced. A broad range of allowed amino acid substitutions can be identified from which patterns can be inferred and critical mutation-intolerant residues can be singled out for further study. The ability to establish structure-function relationships using random saturation mutagenesis is limited by the capacity to functionally screen the extremely large numbers of mutated receptors that must be generated.
We have developed two efficient methods for applying
random saturation mutagenesis to the study of muscarinic receptors.
Both approaches stem from the observation that muscarinic receptors
induce ligand-dependent transformation of NIH-3T3 cells, with a
dose-response relationship that is similar to that of phospholipase C
stimulation(29) . In the first approach, functional receptors
are cloned from a library of mutant receptors by virtue of their
ability to induce foci in response to agonist treatment. This approach
allows large numbers of mutant receptors to be screened simultaneously
for functional clones and is especially suited to the isolation of
clones with rare phenotypes. We have also found that this
ligand-dependent transformation of NIH 3T3 cells can be monitored using
a reporter gene. In our assay, ligands select and amplify cells that
express functional receptors (receptor selection and amplification
technology, R-SAT, patents pending(30, 32) . ()This assay allows graded responses to be measured, thus
permitting a quantitative evaluation of mutant receptor phenotypes. We
have shown that this response can by mediated by G
(32) and have used this approach to study the Ci3 region
of the m5 receptor(30) . In the present study, we used these
two complementary strategies to identify residues within the Ni3 domain
of the m5 muscarinic receptor that are required for signal transduction
and to establish structural motifs that define receptor-G-protein
interactions.
Figure 1: Preparation of m5 muscarinic receptors with random mutations in the N-terminal region of the third intracellular loop (Ni3). A, diagram of the location of Ni3 with respect to the five TM domains of the m5 receptor. B, strategies for the preparing and screening mutant m5 receptors. Two PCR products were prepared such that the reverse primer (p2) for the first product comprised the TM5 domain and the forward primer (p3) for the second product comprised the Ni3 region. Mutations were introduced into the Ni3 region by introduction of random bases during synthesis of the p3 primer. The two PCR products were blunt-end-ligated to join the wild type TM5 domain with the mutated Ni3 region and cloned into the Hm5pCD plasmid at restriction sites introduced in the outer primers (p1 and p4). Mutant receptors were transiently expressed in NIH 3T3 cells and screened for functional responses to carbachol. Pools of mutant receptor clones were screened for the ability to induce foci, and individual mutant receptor clones were screened using high throughput R-SAT assays.
to obtain the dissociation constant K and
the number of binding sites B
, where A = [
H]NMS specifically bound and X = free concentration of [
H]NMS.
to obtain minimum response (A), maximum response (D), and EC (where R is absorbance (A
) at concentrations X of carbachol).
Curves were generated by nonlinear least squares regression using the
graphics/analysis program KaleidaGraph(TM) (Abelbech).
Our strategy for preparing a library of m5 muscarinic
receptors, saturated with mutations in the N-terminal 20 amino acids of
the third intracellular loop (Ni3 domain), is illustrated in Fig. 1and described under ``Experimental Procedures.''
Incorporation of mutations directly into PCR primers used in the
construction of recombinant receptors allows precise control of the
rate of base misincorporation and insures a random distribution of
substitutions. The protocol used in the synthesis of PCR primers in
these studies was expected to yield a nucleotide substitution rate of
11%. In 21 recombinants selected at random from an E. coli library of 680 clones, the observed nucleotide substitution
rate was 10%, with an average of 4.9 amino acid substitutions per
recombinant receptor (Table 1A). Nine of the sequenced clones
(43%) contained stop codons and thus would be unlikely to express
functional receptors. With the exception of the extreme 3` end,
mutations were randomly distributed throughout the sequenced fragment.
The lower rate of mutations in the extreme 3` end was not unexpected as
oligonucleotides with these substitutions would be inefficient PCR
primers and thus would be selected against during construction of the
library of mutants. Also as expected from the relatively low rate of
base misincorporation that was used, the vast majority of amino acid
changes were caused by single base changes. In fact, only 15% of the
amino acid changes were due to two simultaneous base changes in a
codon.
The same pool of 680 mutant receptors were expressed in AV-12
cells and tested for their ability to bind the muscarinic antagonist
[H]NMS. No detectable
[
H]NMS binding is observed in AV-12 cells prior
to transfection. Using equal amounts of DNA for transfection, the pool
of mutant receptor DNAs expressed fewer binding sites (54 ± 16
fmol/mg) than did wild type m5 receptor DNA (220 ± 28 fmol/mg).
The affinity of the pool of mutant receptors (K
of
104 ± 8 pM) was similar to that of wild type (140
± 11 pM). These data suggest that
25% of the
mutant receptors bind [
H]NMS. The presence of
stop codons explains the inactivity of the majority of mutant receptors
(see above). Assuming the
170 receptors that are active within the
pool have an average of 4.9 amino acid substitutions per receptor, it
can be estimated that more than 800 individual amino acid substitutions
(averaging
40 substitutions at each position) were functionally
screened in our study.
To identify receptors that couple to
G-proteins and induce functional responses, we screened mutant
receptors using two high throughput assays that are based on the
ability of G-coupled receptors to transform NIH 3T3 cells
in response to the agonist treatment. Fig. 2illustrates the
dose-response relationships of carbachol in the two assays. Carbachol
induces focus formation with an EC
of 1,960 ± 280
nM and R-SAT amplification responses with an EC
of 123 ± 22 nM. The greater potency of carbachol
in the R-SAT assay may be related to the shorter time course of this
assay (4 days) versus the focus assay (14 days). In the
shorter assay it is likely that high levels of transiently expressed
receptors are present, while in the focus assays lower levels of stably
expressed receptors are likely to predominate. Using focus assays large
numbers of mutant receptors can be screened for function (several
hundred recombinants per plate) and thus are practical for
identification of rare phenotypes.
Figure 2:
Carbachol dose-response relationships for
receptors transiently expressed in NIH 3T3 cells. Functional responses
were measured using focus formation (open triangles) and R-SAT (filled squares). Focus data represents the number of
macroscopic foci per 10-cm dish (mean of data from two
plates) after 14 days of treatment with carbachol. R-SAT data represent
the absorbance at 405 nm (mean of duplicate determinations) after 4
days of treatment with carbachol. Lines are fits of the data to the
mass action relationship described under ``Experimental
Procedures''.
We screened the pool of 680
mutants for receptors that induce formation of foci. No foci were
observed in the absence of the muscarinic agonist carbachol. In the
presence of carbachol, a total of 23 independent mutant receptor
sequences were recovered (3.4% of the total library), suggesting that
14% of the receptors that bind radioligand also couple to
G-proteins and transduce the functional response that yields foci.
Sequences isolated from these foci had an average of 2.5 amino acid
substitutions per receptor, far fewer than observed in the randomly
selected clones. In contrast to the clones selected at random, amino
acid substitutions in the active muscarinic receptors expressed in foci
were not randomly distributed. At some positions, multiple nonconserved
substitutions were observed, while at other positions, either no
substitutions or only highly conserved amino acids were allowed (Table 1B).
The major limitation of these screens is that
formation of a foci is an all or none event that is difficult to
quantitate. Because R-SAT assays are much less labor intensive than
focus assays, it is practical to screen individual clones. Detailed
phenotypic studies are simplified by the ready availability of the
cloned plasmid DNA, as no labor-intensive rescue or subcloning steps
are required. The pharmacologies of many reference muscarinic ligands
at cloned receptors as assayed using R-SAT have been described in
detail. Overall, the pharmacologies of both agonist and
antagonist ligands are similar when assayed using R-SAT and more
traditional functional assays, and R-SAT assays allow a precise
discrimination of full and partial agonists(30) .
Using the
R-SAT assay, an additional 380 clones were selected at random and
individually assayed in the presence and absence of carbachol (100
µM). As in the focus assay, no receptors with
agonist-independent activity were identified. Receptors that responded
to carbachol were then tested for their carbachol dose-response
relationships. These functional receptors were ranked according to the
robustness of their functional responses and divided into two groups.
It should be emphasized that the receptors represented a continuum of
phenotypes, and thus the placement of a few clones into one group or
the other may not be statistically significant. These clones with only
slightly compromised function are marked with an asterisk. Group 1
receptors (n = 14 or 4%) displayed essentially wild
type responses to carbachol, whereas Group 2 receptors (n = 13) are compromised with respect to EC,
percent maximum response, or both (Table 2). The carbachol
dose-response relationships for representative Group 1 and Group 2
receptors are shown in Fig. 3. Group 1 receptors averaged
significantly fewer substitutions (2.9) than Group 2 (3.9). The pattern
of amino acid substitutions in Group 1 receptors were not random, with
many residues that were either not mutated or which only tolerated
highly conservative substitutions. The rates and patterns of
substitutions observed in receptors identified in foci and Group 1
receptors identified by R-SAT are nearly identical (Table 1B and Table 2). These data suggest that only receptors with essentially
wild type responses to carbachol were able to form foci and that the
two screening procedures sampled relatively comprehensive sets of
mutations that are tolerated for the wild type phenotype.
Figure 3: Carbachol dose-response relationships of randomly mutated m5 receptors. A, group 1 receptors with essentially wild type functional phenotypes, and B, group 2 receptors with functionally compromised phenotypes. R-SAT responses were normalized to a wild type receptor evaluated in the same experiment. Lines are fits of the data to the mass action relationship described under ``Experimental Procedures.''
The
sequences of receptors that form foci and robust responses in R-SAT
both predict the same amino acids as being required for function. The
positions of these critical residues are presented in Table 3.
The first two amino acids only tolerate a few hydrophobic
substitutions; consistent with these residues forming the last turn of
the TM5 helix. The next residue (Cys
) tolerates
radical substitutions to all amino acid classes, including several
charged substitutions. Arginine 215 tolerates a hydrophobic
substitution and change of charge. Isoleucine 216 only tolerates
substitution with hydrophobic residues of similar size, and Tyr
was not mutated. The next two positions tolerated many radical
substitutions, and only the highly conserved alanine was substituted
for Thr
. The next two positions tolerated a diversity of
radical substitutions, and Arg
was not mutated. In the
last 8 amino acid positions of the sequence, all of the positions
tolerated multiple nonconserved substitutions. The only exceptions were
the 3` end, where lack of substitutions may have been due to a low
mutation rate that was an artifact of the library construction method
(see above).
Proline substitutions were not observed in the 12 amino
acids preceding Arg. In seven of these positions mutation
to proline would have required two base changes, making these
substitutions rare. On the other hand, at the other five positions
substitution with proline should have been a common event. Proline
substitutions were tolerated in the more cytoplasmic end of the
sequence.
Examination of the sequences of Group 2 receptors
indicates that all but one of these receptors have amino acid
substitutions in residues that were conserved in the Group 1 receptors
(see Table 2), thus confirming the functional importance of these
residues. The one exception was Arg which had a wild type
maximum response and only a modest increase in EC
.
Receptors R262, R284, R361, R255, and R373 all had essentially wild
type EC
values, but these receptors had decreased maximum
responses. All of these receptors had substitutions in
Tyr
, a residue which was not mutated in the Group 1
receptors. All of the remaining receptors had significant increases in
EC
, and all of these receptors with the exception of
Arg
had substitutions in the highly conserved Ile
and/or Tyr
. Arg
and Arg
had radical substitutions in the highly conserved
Thr
. Arg
was not mutated in any of the
receptors that responded to carbachol.
During the course of these
studies, no receptors with substantially decreased EC values for carbachol or much greater then wild type maximal
response were isolated, nor were receptors that responded in the
absence of added agonist. It is possible that such
``activating'' mutations would have been masked by the fact
that most of our mutant receptors had multiple amino acid
substitutions. This possibility seems unlikely as we have isolated many
receptors with multiple amino acid substitutions that are not
compromised, and we have isolated ``activated'' m5 receptors
from a library of receptors with random mutations in TM6(37) .
A growing body of evidence suggests that the Ni3 regions of
G-protein-coupled receptors exist as amphipathic
helices(21, 22, 23, 38, 39) .
Our data provides compelling evidence that the Ni3 region of the m5
receptor forms an
helical structure and identifies all of the
functionally critical residues within this structure (Fig. 4).
First, no charged substitutions were tolerated in receptors with wild
type phenotypes until Cys
, consistent with this residue
marking the beginning of the more hydrophilic Ni3 region. Second in a
helical representation, all of the conserved, largely hydrophobic,
amino acids are clustered on one face of the helix which extends
approximately 10 residues (
3 turns of a helix) into the
intracellular space to the completely conserved arginine
(Arg
). The opposing face of this helix, where most of the
charged residues are located, tolerates multiple, nonconservative
substitutions. And third, the lack of helix-disrupting proline
substitutions between the TM domain and Arg
provides
additional evidence that this region may form a helical structure with
functional importance. Proline substitutions are tolerated after
Arg
(20) . Finally, a comparison of this Ni3
domain with the corresponding domains of the three
1 receptor
subtypes, which also couple with the G-protein G
and form
foci, reveals a similar pattern of hydrophobic residue conservation,
suggesting that this may be a general feature among these functionally
related receptors.
Figure 4:
Proposed locations of functionally
critical residues in Ni3 and Ci3. A, viewed from the
intracellular surface toward the interior of the cell, with lighter and wider lines nearer the viewer. Transmembrane (TM) domains are arranged sequentially (TM1-TM7) in the preferred counterclockwise orientation,
as proposed based on comparison with the structure of rhodopsin (as
discussed in recent reviews; Refs. 40 and 41). Ni3 and Ci3 are drawn as
a continuation of TM5 and TM6, respectively. The beginning of the
intracellular domain is indicated by the first residues (C and Q, respectively) that tolerate mutation to charges. The circled residues do not tolerate radical substitutions, and
the darker shading indicates residues that are also predictive
of the Gversus G
coupling selectivity
of muscarinic receptor subtypes. The extent of the helical structure is
predicted by the periodicity in tolerated substitutions and the lack of
tolerance of helix disrupting proline substitutions. B, linear
view of the Ni3 and Ci3 regions. Upper box indicates proposed
TM domains where charged substitutions are not tolerated. Shaded (also in bold) residues do not tolerate radical
substitutions. Darker shading is as in A.
The Ni3 region of muscarinic receptors has been
one of the most intensively studied regions using site-directed
mutagenesis, and many of the functionally critical residues that we
have now identified have been tested in the earlier studies of the
closely related m1 and m3 receptors. In the m1 receptor, a large
portion of the i3 loop after the arginine analogous to Arg can be deleted without altering receptor activation of
phosphatidylinositol metabolism (24) , and in the m3 receptor,
the analogous arginine has been implicated in
coupling(38, 39, 40) . Also in the m1
receptor, three of the charged residues that were predicted to be
involved in G-protein coupling, based on sequence comparisons, can be
mutated without altering function(24) . In the present study,
the analogous residues tolerate multiple nonconserved substitutions.
Finally, in m3 receptors the residue analogous to Tyr
has
been shown to be a major determinant of G-protein
selectivity(41) .
Our data identifies two additional
residues, Ile and Thr
, that together with
Tyr
form a functionally critical hydrophobic face of a
helical extension of TM5. Based on these predictions, all of the mutant
receptors with significantly compromised phenotypes had substitutions
in residues that can be assigned within a structural context. The
receptors that had wild type EC
values for carbachol and
substantially decreased maximal responses had substitutions in
Tyr
, a residue predicted to be within the TM domain.
Receptors with substantially increased EC
values for
carbachol all had substitutions in residues predicted to form the
hydrophobic face of Ni3. Because most of the receptors have multiple
amino acid substitutions, the assignment of individual residues to
specific mutant phenotypes is preliminary. In an extension of the
present study, we have now defined the precise functional roles of all
of these residues by site-directed mutagenesis(43) .
In a
parallel study of the Ci3 region, we have shown that this region also
has helical structure that is a critical determinant of G-protein
coupling(30) . However, that region was far more tolerant of
mutation then the Ni3, due to only a single turn of an helix
being essential for function. Alanine 441, which is positioned in the
last turn of that helical extension of TM6, was found to be a major
determinant of receptor affinity for G
. If one assumes that
G-protein-coupled receptors consist of seven TM domains that are
arranged counterclockwise (when viewed from the extracellular space)
sequentially from N- to C-terminal in a bundle around a ligand binding
site, then the location of the Ni3 region with respect to the rest of
the receptor can be approximated(3, 4, 42) .
In this orientation, highly conserved residues in the Ni3 and Ci3
regions (Tyr
, Thr
, Ala
,
Ala
) that we have now implicated in G-protein coupling
are in close proximity to one another (Fig. 4). It should be
noted that these are the only functionally required amino acids within
the i3 loop that are predictive of G-protein-coupling selectivity
(G
versus G
) of muscarinic receptor
subtypes (shaded residues of Fig. 4). Taken together,
these data suggest that G-protein coupling involves a domain formed by
Ni3 and Ci3. Additional evidence for the cooperation between the N- and
C-terminal regions of the i3 loop is provided by recent studies in
which co-expression of isolated N- and C-terminal regions of the i3
loops of various receptors inhibit signal transduction(31) .