(Received for publication, November 9, 1995; and in revised form, December 6, 1995)
From the
Amino acids in the third intracellular loops of receptors play
pivotal roles in G-protein coupling. To define their structural
requirements, we have subjected the N- and C-terminal regions of this
loop (Ni3 and Ci3, respectively) of the m5 muscarinic receptor to
random saturation mutagenesis. (see Burstein, E. S., Spalding, T. A.,
Hill-Eubanks, D., and Brann, M. R. (1995) J. Biol. Chem. 270,
3141-3146 and Hill-Eubanks, D., Burstein, E. S., Spalding, T. A.,
Bräuner-Osborne, H., and Brann, M. R.(1996) J.
Biol. Chem. 271, 3058-3065). In the present study, we have
extended our analysis of Ni3 by constructing libraries of receptors
with all possible amino acid substitutions at the residues we
previously identified as functionally important and characterizing
their functional phenotypes. Numerous hydrophobic substitutions were
well tolerated at Ile and Thr
and caused
constitutive activation in two cases, establishing that hydrophobicity
is structurally favored at these positions and that many amino acid
side chains are compatible with this structural role. Similarly,
hydrophobic and polar, but not charged, substitutions were observed at
Tyr
, but in contrast to results for Thr
,
most substitutions at Tyr
substantially decreased maximum
response and increased the EC
for carbachol, demonstrating
that the specific side chain of residue 217 participates in G-protein
coupling. Arg
allowed the widest range of substitutions
of the residues tested, but only basic residues were well tolerated.
All other substitutions significantly increased (up to 100-fold) the
EC
for carbachol without significantly affecting maximal
response. There were no significant changes in the ligand binding
properties of these mutant receptors. We conclude that Ile
and Thr
fulfill a structural role, forming the
foundation of the G-protein-coupling pocket, whereas Tyr
and Arg
contact G-proteins through specific side
chain interactions. We propose that G-proteins are recruited to
receptors by ionic interactions and that hydrophobic residues
participate in activation.
Muscarinic receptors consist of five genetically defined
subtypes (m1-m5), which belong to a superfamily of seven
transmembrane receptors that couple to
G-proteins(1, 2, 3, 4, 5, 6, 7) .
Although the crystal structures have been solved for two G-protein
-subunits in both the active and inactive
conformations(8) , to date, the structural basis for
receptor/G-protein coupling is not well understood and awaits the
availability of high resolution structures of receptors. Most of our
current knowledge of the structural basis of receptor/G-protein
coupling was determined from molecular experiments in which receptor
domains have been systematically deleted, exchanged, and/or
mutagenized. 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 are required for
function(13, 14, 15, 16) .
To
define the structural requirements of Ni3 and Ci3, we have subjected
these regions of the m5 muscarinic receptor to random saturation
mutagenesis(17, 18) . In these studies we constructed
libraries of receptors containing mutations randomly distributed
throughout Ni3 and Ci3. We then used a functional assay we have
developed called R-SAT (receptor selection and amplification technology ((17, 18, 19, 20) ), patent pending)
to screen these libraries and identify the mutant receptors that
retained the ability to transduce proliferative signals. We grouped
phenotypically similar receptors and compiled lists of their associated
substitutions, and based on these mutation patterns, we were able to
ascertain structure/function relationships. Both regions were predicted
to form -helical extensions by our analysis and generally showed
conservation of hydrophobic residues and little conservation of charged
residues, with the exception of an invariant arginine in Ni3 (18) and an invariant lysine in Ci3(17) . In the
present study, we have extended our analysis of Ni3 by constructing
point mutant libraries of residues we previously identified as
functionally important and characterizing their functional phenotypes
allowing us to distinguish their specific roles.
We used a PCR-based protocol (see (17) ) to construct point mutation libraries at each of four residues within Ni3 previously shown to be functionally important ((18) ; see Fig. 1). We identified functional receptors based on their ability to amplify NIH 3T3 cells in a ligand-dependent manner using an assay called R-SAT ((17, 18, 19, 20) , patent pending). We screened each library in the presence or absence of a single dose (100 µM) of carbachol to identify functional receptors. The positive clones were then sequenced and subjected to a detailed dose-response analysis. (see Table 1and Fig. 2).
Figure 1: Schematic depicting the m5 muscarinic receptor. The Ni3 region is expanded to show the individual amino acid residues. The residues shown previously to be functionally important (18) are numbered. In this study, point mutant libraries containing all possible substitutions were constructed for each of these residues and their phenotypes characterized in functional and binding studies.
Figure 2: Dose/response curves of several Ni3 point mutants analyzed by R-SAT (see ``Experimental Procedures''). Responses were measured as absorbance at 420 nm and were normalized to the maximum response of wild-type m5, which was typically 0.5 absorbance units. Points are the means ± S.E. of duplicate determinations. Curves are representative of two to three experiments. A, open circles: wild-type m5; filled squares: I216L; open triangles: Y217T; filled inverted triangles: R223M. B, filled triangles: T220V assayed at the indicated concentrations of carbachol; filled squares: T220V assayed at the indicated concentrations of atropine; open circles: wild-type m5 assayed at the indicated concentrations of carbachol; open squares: wild-type m5 assayed at the indicated concentrations of atropine.
At
position 220 the observed amino acid substitutions were predominantly
hydrophobic, and all were well tolerated (Table 1). In fact,
substitution by valine and cysteine each resulted in a constitutively
active receptor with a decreased the EC for carbachol (Table 1, Fig. 2). The constitutive activity could be
completely reversed by the negative antagonist atropine (Fig. 2). Substitutions with large hydrophobic residues (leucine
and phenylalanine) or with glycine were also well tolerated, increasing
EC
values only 6-fold or less, although the maximum
response was only 70% for phenylalanine. We observed no charged
substitutions, suggesting these amino acids are not permitted at
position 220.
We observed a similar pattern at position 216 where
hydrophobic and medium sized polar substitutions were best tolerated,
causing little or no phenotypic changes from wild-type m5 (Table 1). Again, most of the allowed substitutions were well
tolerated. However the large aromatic residues phenylalanine and
tyrosine, and the small residue glycine increased the EC values 11-, 26-, and 84-fold, respectively, and had maximum
responses of only 23, 64, and 67% of wild type, suggesting that amino
acid size was also an important structural constraint.
At position
217, again both polar and hydrophobic, but not charged, substitutions
were observed, but in contrast to the results described above, almost
every observed mutation at 217 increased EC 10-fold or
greater and decreased maximum response 20% or more (Table 1). Only the phenylalanine mutant retained a wild-type
phenotype. Surprisingly, glycine was well tolerated. Possibly other
residues compensate/fill the void created by glycine. These data
indicate there are more stringent constraints on the particular amino
acid side chain at position 217 than at positions 216 or 220.
Arg permitted the widest range of mutations of the
residues tested, including polar, nonpolar, basic, and acidic
substitutions, but only basic residues were well tolerated (Table 1). All other mutations increased EC
values
25- to over 100-fold. However the maximum responses were almost all
80% of wild type, in contrast to the results for Tyr
where maximum responses of less than 60% were typical. These data
indicate that loss of positive charge at position 223 reduces the
efficiency with which receptors couple to G-proteins, although it does
not prevent full activation from occurring.
We evaluated the
[H]NMS and carbachol binding properties of a
number of the Ni3 mutants described above to determine if the observed
phenotypic changes could be accounted for by changes in expression
levels or ligand affinities. As shown in Table 2, there were
little or no differences in the binding properties of these receptors.
Therefore, the effects of the introduced mutations were primarily upon
the abilities of these mutant receptors to couple to G-proteins.
Our results strongly support our initial findings (18) that Ni3 forms an -helix with a conserved hydrophobic
face, terminating with an invariant arginine. That study identified
Ile
, Tyr
, Thr
, and
Arg
as the functionally important residues in Ni3. In the
present study we have extended our analysis by analyzing libraries of
receptors with all possible amino acid substitutions at these residues.
Significantly, charged substitutions were not permitted at
Ile
, Tyr
, or Thr
, but basic
residues were strongly favored at Arg
. Furthermore,
proline substitutions, which can disrupt
-helices, were never
observed. These results are entirely consistent with our earlier work.
Comparison of the proportion of well tolerated to poorly tolerated
substitutions at each residue allows one to make inferences about their
functional roles (see Table 3). Many different hydrophobic and
medium sized polar amino acids were well tolerated at positions 216 and
220, but few other substitutions were observed. If the function of
residues 216 and 220 is to form the backbone of the G-protein-coupling
pocket, then substitutions that do not disrupt the architecture of this
domain should be fully compatible, while amino acids which distort this
domain would not be functionally permitted. This would explain why a
large proportion of the observed amino acid substitutions at these
positions are well tolerated. Similar mutations were allowed at
position 217; however, most had deleterious effects on receptor
function. This suggests that Tyr engages in more specific
amino acid side chain interactions with G-proteins than either
Ile
or Thr
(discussed below). Finally, only
basic residues were well tolerated at position 223, but many
radical substitutions were allowed, indicating that positive charge at
this position is critical for G-protein coupling. The tolerance of
Arg
to radical substitutions suggests that these
mutations do not destroy the overall structure of the
G-protein-coupling site.
We tabulated the observed substitution
patterns at each tested position in m5 with the residues found at the
analogous positions in the biogenic amine receptor family (Table 3). In general, the well olerated substitutions we
identified in m5 are represented at these positions in other receptors,
indicating that there is an evolutionary as well as functional
conservation of these residues. In particular, this relationship holds
for Ile and Thr
, where all but one residue
(Ser in the G
-coupled m2 and m4 receptors) is represented
as a well tolerated residue in m5. It is surprising that there is so
little conservation of basic residues at the position analogous to
Arg
in the other monoamine receptors (Table 3)
considering how strongly favored this property was in our analysis.
This apparent discrepancy can be resolved when one considers that
alignments of receptor sequences are currently based on homology and
undoubtedly become less reliable further from the
transmembrane/cytoplasmic border. Thus, assumptions about what
constitutes an analogous residue to Arg
in other
receptors may be wrong in some cases. It is noteworthy that in all of
the monoamine receptors that do not have a basic residue at the
position analogous to Arg
, there is a basic residue
located either one position before, and/or three positions after
it(2, 26, 27, 28) , which might
perform the same function. Therefore it is reasonable to speculate that
there is a general requirement for positive charge in this domain in
all monoamine receptors, although this remains to be proven for other
receptors(31) . We propose that Arg
functions to
recruit G-proteins to the receptor by an ionic mechanism. Based on our
earlier results(18) , Arg
is situated at the end
of an
-helical extension into the cytoplasm, appropriately located
to fulfill this function.
The analogous (to Tyr)
tyrosine in the m3 muscarinic receptor has been proposed to be a key
determinant of G-protein-coupling specificity(25) . This
residue is conserved in all the muscarinic subtypes that couple to
phosphoinositide turnover, but is replaced by a serine in the subtypes
that couple to inhibition of adenylyl cyclase (see (7) ).
Significantly, we found that substitution of serine caused the greatest
decrease in receptor function of all mutations observed at
Tyr
. Thus, within the muscarinic receptor family,
Tyr
may well be a major determinant of G-protein-coupling
specificity. However examination of all the residues found at that
position in the monoamine receptors indicates that this model is too
simplistic to explain how receptor/G-protein specificity. For example,
tyrosine is found in many G
- and G
-coupled
receptors, and we saw that phenylalanine, which is also found in
numerous G
-coupled receptors, was well tolerated at
Tyr
(see Table 3). Furthermore other epitopes,
notably the i2 loop(29, 30) , contribute to coupling
specificity, so the exact requirements remain obscure. Possibly,
specificity is determined by cooperation between multiple epitopes.
We isolated two constitutively activated receptors, T220V and T220C
(see Fig. 2; Table 1). We found that the m5 receptor can
also be constitutively activated by mutations at position 465 in TM6. ()In that study we observed that every observed substitution
(11 total) activated the receptor, which led us to speculate that
residue 465 stabilizes the inactive state of the receptor. A similar
interpretation was used to explain activation of the
1B-adrenergic
receptor(22) . In contrast to those results, most substitutions
at position 220 were not activating. This may indicate that Thr
forms the G-protein-coupling pocket and is involved in the
structural transition between active and inactive states of the
receptor, explaining why mutations cause either gain or loss of
function.
The potential for Ni3 to form an amphipathic -helix
has been appreciated for several years(32) , based on the
observation that mastaporan, a peptide toxin from wasp venom, directly
activates G-proteins and adopts an amphipathic
-helical
structure(33) . Despite intensive study of this working
hypothesis, the critical determinants for G-protein coupling in Ni3 had
been unresolved. Subsequent mutagenesis studies on this domain have
alternatively implicated the hydrophobic
residues(15, 21, 31, 34) , the
hydrophilic residues(16) , or the amphipathic nature of
Ni3(35) . These apparent contradictions can now be resolved
with our data which elucidate for the first time the precise identities
and functional roles of the residues in Ni3 critical for G-protein
coupling. Together our results for Ni3 ( (18) and this paper)
and Ci3 (17) indicate both domains form
-helices
terminating with functionally conserved basic residues. Thus we propose
that G-proteins are recruited to receptors by an ionic mechanism and
that protein/protein interactions occur through hydrophobic
interactions.