From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Received for publication, December 12, 2002
, and in revised form, March 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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G subunits function indirectly to promote G protein activation,
because they do not contact G
subunits
(6,
7,
8). G
subunits can bind
receptors, as indicated by studies of rhodopsin and transducin, which show
that
binds activated rhodopsin and promotes activation of the
subunit (13,
14). Furthermore, rhodopsin
preferentially activates transducin containing its cognate
subunit
(
1; see Ref. 15); other
receptors likewise couple preferentially with G proteins containing certain
G
subunit isoforms in vitro
(16,
17,
18). Selective coupling
appears to be mediated by the diverged C termini of G
subunits, because
peptides corresponding to the last 14 residues of
1 stabilize the
active conformation of rhodopsin (metaII) and competitively inhibit
rhodopsin-mediated activation of transducin
(19). These and other
observations have suggested that receptors possess independent binding sites
for the C-terminal domains of G
and G
subunits
(13,
14,
19). Thus, one variation of
the "lever hypothesis" of G protein activation requires
simultaneous binding of the C termini of G
and G
to independent
sites on a receptor (5),
although other variations of this model do not
(2,
3).
In certain signaling pathways, receptor interaction with the C terminus of
G may have a non-essential role in G protein activation. Studies have
shown that
7, which interacts poorly with M2 or M4 muscarinic
acetylcholine receptors in vitro, supports 2-fold more efficient
coupling with these receptors than does a Go heterotrimer
containing
5, whose tail binds these receptors in vitro
(16). Furthermore, muscarinic
receptor coupling to Go in vitro is enhanced when the
heterotrimer contains
5 with a scrambled C-terminal sequence that does
not bind muscarinic receptors
(20). However, it remains to
be established whether receptor binding to the C termini of G
subunits
is an obligate or modulatory event for G protein activation in
vivo.
We established previously that the C-terminal domain of G promotes
receptor-G protein coupling by analyzing mutations affecting the G
subunit (STE18 gene product) of the yeast Saccharomyces
cerevisiae. In yeast, a single type of G protein heterotrimer transduces
signals from mating pheromone receptors
(21), providing a simple
system to evaluate G
function genetically and biochemically. We found
that G
truncated at position 94 (13 residues preceding the
CAAX box) associates with G
but strongly impairs receptor-G
protein coupling in vitro (agonist binding was low affinity and
insensitive to
GTP
S)1
(22). This receptor coupling
defect was not because of lack of C-terminal prenylation or palmitoylation,
because G
bearing a substitution (C107S) inactivating its CAAX
box associated with G
and G
and supported efficient receptor-G
protein coupling in vitro (agonist binding was high affinity and
sensitive to GTP
S). Therefore, peptide sequences between residue 94 and
the CAAX box of yeast G
, equivalent to the receptor binding
domains of mammalian G
subunits
(14,
15,
18,
19,
23,
24), were proposed to promote
receptor-G protein coupling.
Here we have investigated whether the C-terminal domain of yeast G
is required for receptor-mediated signaling in vivo.By analyzing the
function of a diverged family of Ste18 homologs from several species of
budding yeast and amino acid substitutions affecting the C terminus of Ste18
we provide evidence indicating that the C-terminal domain preceding the
CAAX box of G
is dispensable for receptor-mediated signaling
in vivo.
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EXPERIMENTAL PROCEDURES |
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Plasmids, Oligonucleotide-directed Mutagenesis, and Sequence AnalysisSTE18 was expressed from the PGK promoter, and terminator sequences were cloned into the yeast high copy plasmid pRS425 as follows. A fragment containing the PGK promoter and terminator separated by a multicloning site containing restriction sites for EcoRI, HindIII, and BamHI was excised from the pPGK vector (25) by digesting with XhoI and SalI and inserted into pRS425 (26) cut with XhoI and SalI. The resultant plasmid was digested with SalI and NotI and circularized to produce pRS425PGK, which has unique EcoRI, HindIII, and BamHI sites for expression cloning. Wild-type and mutant forms of the STE18 coding region were cloned as HindIII-BamHI fragments into pRS425PGK, which allowed expression of wild-type and mutant forms of HA-tagged Ste18. The N-terminal HA tag fully preserves Ste18 protein function (22). All point mutations made in the STE18 gene expressed from the ADH promoter were constructed in pVT-HA-Ste18 (22) using the QuikChangeTM site-directed mutagenesis method (Stratagene). The STE18 internal deletion mutant removing sequences encoding amino acids 94105 was constructed using the ExSiteTM mutagenesis method (Stratagene), which resulted in the additional substitutions N92T and A93R. All mutations were confirmed by DNA sequencing using appropriate primers.
Cloning and Expression of STE18 Homologs from Five Divergent Saccharomyces SpeciesThe genome sequences of five species of yeast of varying relatedness to S. cerevisiae (Saccharomyces bayanus (strain NRRL Y-11845), Saccharomyces castellii (NRRL Y-12630), Saccharomyces kluyveri (NRRL Y-12651), Saccharomyces kudriavzevii (IFO 1802), and Saccharomyces mikatae (IFO 1815); provided by M. Johnston, Washington University Genome Sequencing Center) were searched by BLAST for STE18 homologs. As is the case in S. cerevisiae, the genome of each of these species of yeast encoded a single STE18 homolog (data not shown). Contig sequences encoding each STE18 homolog were provided by P. Cliften and M. Johnston (Washington University Genome Sequencing Center) and will be deposited in Gen-BankTM by those investigators. Primers used to amplify only the coding region of each STE18 homolog were designed such that the resultant amplification products could be cloned as HindIII-BamHI fragments into pRS425PGK as follows. Genomic DNA from each species of yeast (obtained from M. Johnston, Washington University School of Medicine) and the following primers were used: forward S. bayanus, 5'-GCCCCCAAGCTTATGTCTGCAGTTCAGAACTCG-3' and reverse S. bayanus, 5'-CGCGGATCCTTACATAAGCGTACAACAAGC-3'; forward S. kudriavzevii, 5'-GCCCCCAAGCTTATGTCTGCTATTCAGAACTCG-3' and reverse S. kudriavzevii, 5'-CGCGGATCCTTACATAAGCGTACAACAAGC-3'; forward S. mikatae, 5'-GCCCCCAAGCTTATGTCTGTACTTCAAGATTCACC-3' and reverse S. mikatae, 5'-CGCGGATCCTTACATAATCGTACAGCAAAC-3'; forward S. kluyveri, 5'-GCCCCCAAGCTTATGTCTTCAGAAGAGCAGCAACC-3' and reverse S. kluyveri, 5'-CGCGGATCCTTACATAATTGCACAGCAGTTG-3'; forward S. castellii, 5'-GCCCCCAAGCTTATGTCACAACAGATAAAAACACC-3' and reverse S. castellii, 5'-CGCGGATCCTTACATTATAGCACAGCAACC-3'. The amplification conditions using Taq polymerase were as follows: 96 °C for 5 min; 30 cycles of 96 °C for 30 s, 43 °C for 2 min, and 72 °C for 2 min; 72 °C for 10 min. The resultant PCR fragments were sequentially digested with BamHI and HindIII and cloned into pRS425PGK to express the native, untagged forms of these Ste18 homologs in S. cerevisiae. The correct nucleotide sequence of each construct was confirmed by sequencing.
Random Mutagenesis of S. cerevisiae STE18 and Identification of STE18
Dominant-negative MutantsA plasmid suitable for performing random
mutagenesis of codons 79110 in the STE18 gene was constructed
by introducing a silent KspI site into sequences encoding amino acids
7779 and a KpnI site immediately downstream of the
STE18 stop codon in plasmid pVT-HA-STE18, resulting in
pVT-HA-STE18-KK. Oligonucleotides containing 1% of the three incorrect bases
at each position spanning the coding region for amino acids 79110 of
Ste18 were synthesized and inserted into pVT-HA-STE18-KK cleaved with
KspI and KpnI. Introduction of the library into
Escherichia coli produced a pool of 1500 bacterial transformants that
were used in the following ways. Sequencing of 16 random plasmids revealed
that one was wild-type, and the remaining contained mutations affecting
various codons throughout the targeted region, as expected. To select for
dominant-negative STE18 mutations, we introduced the pool of 1500
plasmids into the wild-type S. cerevisiae strain RK5116B.
Portions of the transformation mixture were plated on selective media that
lacked or contained a dose of -factor (1 µM) sufficient
to arrest growth of the parent strain expressing wild-type Ste18. Colonies
resistant to
-factor were recovered at a frequency of 3%. Plasmid
dependence of the
-factor-resistant phenotype was demonstrated by
recovery of
-factor sensitivity following plasmid loss on media
containing 5-fluoroorotic acid. Plasmids conferring
-factor resistance
were isolated from yeast cells and sequenced. The same pool of mutagenized DNA
was also subjected to a screen for STE18 dominant-negative mutations
in which wild-type cells (RK51116B) were transformed with the library,
and random colonies were picked and screened for impaired response to
-factor in growth arrest (halo) assays.
Pheromone Response AssaysLong term (48 h) pheromone-induced
growth arrest (halo) assays were used to determine the ability of cells to
respond to varying concentrations of agonist (-factor) as described
previously (27). Sterile paper
disks containing various amounts of synthetic agonist (
-factor; 15
pmol, 50 pmol, 150 pmol, 500 pmol, 1.5 nmol) were applied to lawns of cells
embedded in soft agar. After incubation at 30 °C for 2 days, zones of
growth inhibition were measured, and plates were scanned electronically to
record images. Unless indicated otherwise, halo assays were performed with
ste18
cells expressing various wild-type or mutant
STE18 alleles from the ADH or PGK promoter on high
copy plasmids. Although various STE18 homologs and S. cerevisiae
STE18 alleles were expressed from strong promoters on high copy plasmids,
this would not result in overexpression of G
complexes,
because G
and G
subunits were expressed from their normal
chromosomal loci and therefore were limiting.
Quantitative Mating AssaysCells were grown to mid-log phase
in synthetic media lacking leucine to select for plasmids. Approximately 1
x 107 cells of the tester strain of the appropriate mating
type and 2 x 106 cells of the experimental strain (a
ste18 mutant of either mating type that expressed various
STE18 alleles from plasmids) were mixed, collected on 0.45-µm
nitrocellulose filters, incubated for 3 h at 30 °C on YPD media,
suspended, diluted, and plated in triplicate on appropriate media to select
for diploids. The number of viable haploids of the experimental strains
present before mating was quantified by plating on appropriate selective
media. Mating efficiency (%) was calculated by dividing the number of diploids
produced by the number of haploids of the limiting strain and multiplying by
100.
ImmunoblottingYeast cells were grown to mid-log phase and harvested, and total cell protein was extracted by performing an alkaline lysis procedure (28). Proteins were resolved by SDS-PAGE (16% gel), transferred to nitrocellulose, and blocked in 5% non-fat milk in phosphate-buffered saline + 0.1% Tween-20 (PBST). Blots were incubated with the HA.11 monoclonal antibody (BAbCO) diluted 1:1000 in PBST, washed, and incubated with a goat anti-mouse horseradish peroxidase conjugate (Cappel) diluted 1:2000 in PBST, washed, and visualized by enhanced chemiluminescence (Amersham Biosciences) with x-ray film (Eastman Kodak Co.).
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RESULTS |
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Despite exhibiting varying degrees of sequence conservation in their
putative receptor coupling domains, all of the Ste18 homologs could correct
the signaling defect of S. cerevisiae cells carrying a disruption of
the chromosomal STE18 gene. As indicated by quantitative assays of
agonist (-factor)-induced growth arrest
(Fig. 2), expression of Ste18
homologs most similar to S. cerevisiae Ste18 (S. bayanus, S.
mikatae,or S. kudriavzevii) fully restored agonist
responsiveness to the S. cerevisiae ste18
mutant (zones of
-factor-induced growth inhibition were equivalent to those of
ste18
cells expressing the S. cerevisiae STE18 gene
from a plasmid). More strikingly, expression of either of the more highly
diverged Ste18 homologs (S. kluyveri and S. castellii)
nearly completely restored agonist responsiveness to the ste18
mutant (zones of growth inhibition were slightly smaller or turbid). Because
the C-terminal domains of these latter two Ste18 homologs are quite diverged
relative to S. cerevisiae Ste18, these results suggested that
specific amino acid sequences or motifs preceding the CAAX box of
Ste18 may not be critical for receptor-G protein coupling and signaling.
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Missense Mutations Affecting the C-terminal Domain of Ste18 Preserve
Receptor-dependent SignalingTo provide an independent means of
investigating whether C-terminal peptide sequences preceding the CAAX
box of S. cerevisiae Ste18 are important for receptor-mediated
signaling, we constructed and analyzed several types of missense mutations
affecting this domain. We first used alanine scanning mutagenesis targeted to
residues 99105, just upstream of the CCAAX box (residues
106110), which is prenylated and palmitoylated and required for
activation of the downstream mitogen-activated protein kinase cascade
(30,
31,
32). Pairs of residues were
substituted with an Ala-Ala dipeptide to create six mutants (M99A,S100A;
S100A,N101A; N101A,S102A; S102A,N103A; N103A,S104A; S104A,V105A) that were
analyzed for their ability to function when expressed from a plasmid in a
ste18 mutant. As indicated by quantitative assays of
agonist-induced growth arrest, expression of each of these alleles in a
ste18
mutant resulted in wild-type or nearly wild-type
response to agonist (Fig.
3A), suggesting that the side chains of residues
99105 are dispensable for receptor-mediated signaling.
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As an alternative to alanine scanning mutagenesis, we altered the charge distribution of selected residues within the C-terminal domain of Ste18. In one mutant, lysine 95 was changed to aspartic acid, and in another mutant three uncharged residues (Met-99, Ser-100, Asn-101) were all changed to aspartic acid. Expression of either of these mutants from a plasmid in cells carrying a deletion of the chromosomal STE18 locus fully restored agonist-dependent signaling (Fig. 3B), further suggesting that specific amino acid sequences in the C-terminal domain of Ste18 are not essential for receptor coupling and signaling in vivo.
Although suggestive, the preceding results could not exclude the
possibility that the C-terminal domain of Ste18 contains several sequences or
motifs that mediate receptor coupling in a functionally redundant manner. To
address this possibility, we introduced a string of four alanine residues at
positions 9497 and 102105 or eight alanine residues at positions
98105. Strikingly, expression of any of these STE18 alleles
from a plasmid fully rescued the ability of a ste18 mutant to
respond to agonist (Fig.
3C).
We also considered that Ste18 containing a string of eight alanine residues
(positions 98105) preceding the CAAX box could cause a
quantitative defect in receptor-mediated signaling that was difficult to
detect by in vivo signaling assays. Therefore, to increase the
sensitivity of these assays we expressed plasmid-borne STE18 alleles
in a ste18 mutant that expressed
-factor receptors
lacking their C-terminal domains. Receptors lacking their C-terminal tail were
used, because they are partially impaired for G protein coupling in
vitro (33), which is
manifested in vivo by zones of agonist-induced growth inhibition that
are slightly turbid and have less distinct margins
(Fig. 3D). Despite
this partial impairment in function, truncated
-factor receptors
signaled with similar efficiency whether wild-type Ste18 or mutant Ste18
containing eight alanine residues was expressed from a plasmid in
ste18
cells (Fig.
3D). Therefore, this mutant form of Ste18 appeared to be
highly functional even when receptor-G protein coupling was partially impaired
by other mechanisms.
Mutations throughout Sequences Encoding the C-terminal Domain Preserve
Ste18 FunctionTo address whether receptor coupling information is
present elsewhere within the C-terminal domain of Ste18, we performed random
oligonucleotide mutagenesis of sequences encoding residues 79110.
Sequences further upstream were spared, because they probably are required for
association with G, as suggested by structural studies of mammalian
G
complexes (34).
A pool of plasmids created by random oligonucleotide mutagenesis of this
region contained point and frameshift mutations as indicated by sequencing
random clones (data not shown). This plasmid pool was subjected to two types
of analyses to identify mutations that potentially impair receptor coupling.
In the first, we selected for plasmids that encode dominant-negative G
subunits. Dominant-negative G
mutants were selected by their ability to
allow wild-type cells to form colonies on medium containing a high dose of
agonist (
-factor; 1 µM) sufficient to arrest the growth
of control cells. This approach yielded only mutations that inactivated or
removed the CAAX box of Ste18 (truncation, frameshift, or missense
mutants; see Table II), which
is required for membrane targeting
(31).
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Because the previous selection method might have been biased for very
strong dominant-negative STE18 mutations that nearly completely block
signaling, our second approach used a screen that is capable of identifying
plasmids in the mutagenized library that encode weaker dominant-negative
G subunits. The screen was performed by introducing the library of
mutagenized plasmids into wild-type cells, picking colonies at random, and
assaying for impaired agonist-induced signaling, as indicated by formation of
smaller or turbid zones of agonist-induced growth inhibition. Despite the
ability of this assay to detect partial loss of function mutants
(35), the only mutations that
caused a detectable dominant-negative phenotype affected the CAAX box
(truncation, frameshift, missense, or point mutations; see
Fig. 4 and
Table II). Therefore, these
results suggested that a functionally critical receptor-coupling domain may
not be present within residues 79107 of Ste18.
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Ste18 Deleted for Residues Preceding Its CAAX Box Is
FunctionalThe preceding results suggested that sequences preceding
the CCAAX box of Ste18 are not required for receptor-mediated
signaling. As a further test of this hypothesis, we constructed an internal
deletion mutant (94105) of Ste18 that lacks the region of the
C-terminal tail implicated biochemically in receptor coupling
(22) but preserves the
CCAAX box. The CCAAX box was retained, because
palmitoylation and prenylation of Ste18 is required for downstream signaling
(30,
31,
32), although these
modifications are not required for receptor-G protein coupling in
vitro (22). Strikingly,
expression of this STE18 internal deletion allele
(
94105) from a plasmid fully restored the ability of a
ste18
mutant to respond to agonist (
-factor; see
Fig. 5). Expression of an
N-terminally HA-tagged version of this internal deletion mutant protein, as
well as two other mutants analyzed in this study (98105Ala; C107S, a
CAAX mutant) was confirmed by immunoblotting
(Fig. 6). Therefore, the
C-terminal region of Ste18 immediately preceding the CCAAX box is
dispensable for
-factor receptor-mediated G protein activation and
signaling.
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Signaling by Neither the a-Factor Nor the
-Factor Receptor Requires the Domain of Ste18 Preceding the
CCAAX BoxThe final possibility we considered was that the
C-terminal tail of Ste18 may be required for coupling to one type of mating
pheromone receptor but not the other. Indeed, previous studies have indicated
that the yeast G
subunit uses different regions of its C terminus to
couple with
-factor versus a-factor receptors
(36), the only two receptors
in yeast that couple with the G protein containing Ste18
(37). Accordingly, because the
preceding experiments examined the effects of Ste18 mutations on
-factor receptor signaling, we subsequently determined whether the tail
of Ste18 is required for a-factor receptor signaling. These experiments
could not use assays of a-factor-induced growth arrest, because fully
processed and prenylated a-factor is unavailable in sufficient
quantity. As an alternative, we compared the mating efficiencies, a sensitive
and quantitative assay of receptor-dependent signaling, of cells expressing
a-factor receptors and either wild-type Ste18 or mutant Ste18 lacking
residues 94105. As a control, we also quantified the mating
efficiencies of cells expressing
-factor receptors and either wild-type
Ste18 or mutant Ste18 lacking residues 94105. Remarkably, cells
expressing a-factor receptors and mutant Ste18 mated as efficiently as
controls expressing wild-type Ste18 (Table
III). Cells lacking a STE18 plasmid did not mate (data
not shown). Similarly, cells expressing
-factor receptors mated with
equivalent efficiency whether they expressed wild-type or mutant Ste18
(Table III), and the ability of
these cells to mate required the presence of a STE18 plasmid (data
not shown). Therefore, we conclude that signaling by neither the
-factor receptor nor the a-factor receptor in vivo
requires the C-terminal domain preceding the CAAX box of Ste18.
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DISCUSSION |
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The results presented here and our previous studies suggest that caution
must be exercised when assessing the potential importance of certain
receptor-G protein contacts solely by biochemical approaches. In the in
vitro system used to detect receptor-G protein coupling in yeast plasma
membrane fractions, agonist binding affinity of the -factor receptor is
sensitive to GTP
S (i.e. becomes low affinity) only under
conditions of high salt concentration and moderately elevated pH
(40). At physiological salt
concentration and pH, in contrast, agonist binding to
-factor receptors
in plasma membrane fractions is GTP
S-insensitive (i.e. remains
high affinity; see Ref. 40).
Therefore, only at high salt concentration and elevated pH is it possible to
show that
-factor receptor-G protein coupling in vitro
requires the G
tail
(22). However, these reaction
conditions may decrease the stability of receptor-G protein complexes such
that a functional interaction between the receptor and the G
tail
becomes evident. In contrast, the
-factor receptor-G protein interface
may be more stable in vivo and therefore tolerates loss of sequences
preceding the CAAX box of G
.
How do our results impact current models of receptor-mediated G protein
activation in which receptors are hypothesized to act as a lever to open the
nucleotide binding pocket of G
(3,
5)? One variation of this model
suggests that simultaneous contact between the receptor and the C-terminal
domains of G
and G
is required for receptors to open the
nucleotide binding site of G
and catalyze nucleotide exchange; however,
our results exclude this model, at least in yeast. Instead, they support
mechanisms in which the receptor contacts G
, G
, and/or other
regions of G
to activate the G protein. They are also consistent with a
model in which receptor interaction with G
is responsible for
perturbing the
5/
6 loop via movement of the
5 helix
(2,
3), which does not invoke an
essential role for receptor-G
interaction.
Are the C-terminal domains of G subunits dispensable for
receptor-mediated G protein activation in mammalian cells? Although this
question requires further investigation, several considerations suggest that
contacts between the tails of G
subunits and mammalian G
protein-coupled receptors are unlikely to be essential for G protein
activation in vivo. First, the mechanism of receptor-mediated G
protein activation is conserved from yeast to humans, because similar domains
of yeast and mammalian receptors and G proteins are required for receptor-G
protein coupling (22,
35,
36,
40,
41,
42,
43). Second, sequences
preceding the CAAX boxes of mammalian and yeast G
subunits are
not conserved, yet several types of mammalian receptors expressed in yeast can
activate a G protein consisting of a yeast/mammalian G
chimera, yeast
G
(Ste4), and yeast G
(Ste18)
(44,
45,
46). Third, G
5 carrying
a scrambled amino acid sequence preceding its CAAX box does not bind
M2 muscarinic acetylcholine receptors but can support efficient coupling of
Go
and G
1 with these receptors in
vitro (20). Fourth,
G
7, which does not appear to interact with M2 or M4
muscarinic acetylcholine receptors in vitro, supports more efficient
coupling with these receptors than does a G protein containing
G
5, whose C-terminal tail can bind these receptors in
vitro (16).
In apparent contrast, however, there is evidence suggesting that the
G tail is important for coupling between muscarinic receptors and G
proteins in vivo
(24). Cytoplasmic injection of
a geranylgeranylated peptide corresponding to the wild-type C terminus of
5 strongly impairs the ability of muscarinic receptors to inhibit
N-type Ca2+ channels in primary sympathetic neurons,
whereas a scrambled version of this peptide or wild-type peptides
corresponding to the C termini of
7 or
12 have no effect
(24). These observations are
consistent with the hypothesis that muscarinic receptors have a functionally
important binding site for the C terminus of
5. However, they are
equally likely to suggest that the
5 peptide blocks the ability of
G
subunits to inhibit N-type Ca2+ channels
by interfering with G
binding to the channel.
Because the C-terminal domains of G subunits clearly have the
ability to bind receptors (14,
15,
16,
17,
18,
19,
20,
22,
24), this interaction may have
novel functions in yeast or mammalian cells. For example, this interaction
might allow G
subunits to remain bound to receptors after
G
subunits are activated, which could promote downstream signaling via
G
effectors that cluster with receptors. Indeed, the blocking
effect of the
5 C-terminal peptide on muscarinic receptor-mediated
inhibition of N-type Ca2+ channels could be because of
release of G
subunits from receptors that potentially cluster
with N-type channels, decreasing the local concentration of G
required to maximally inhibit these channels. Tethering of G
subunits to receptors also has the potential to promote resetting of the
system to the inactive state by facilitating capture of G
subunits
after GTP hydrolysis. Therefore, further studies of receptor-G
interaction may reveal new insights into mechanisms that control the
efficiency, fidelity, and kinetic control of G protein signaling.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Cell Biology and
Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St.
Louis, MO 63110. Tel.: 314-362-1668; Fax: 314-362-7463; E-mail:
kblumer{at}cellbio.wustl.edu.
1 The abbreviations used are: GTPS, guanosine
5'-3-O-(thio)triphosphate; HA, hemagglutinin.
2 M. Johnston, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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