(Received for publication, January 29, 1997, and in revised form, April 29, 1997)
From the Division of Biology, California Institute of Technology, Pasadena, California 91125
Ca2+ ion concentration changes
are critical events in signal transduction. The
Ca2+-dependent interactions of calmodulin (CaM)
with its target proteins play an essential role in a variety of
cellular functions. In this study, we investigated the interactions of
G protein subunits with CaM. We found that CaM binds to known
subunits and these interactions are
Ca2+-dependent. The CaM-binding domain in
G
subunits is identified as G
residues 40-63. Peptides
derived from the G
protein not only produce a
Ca2+-dependent gel mobility shifting of CaM but
also inhibit the CaM-mediated activation of CaM kinase II. Specific
amino acid residues critical for the binding of G
to CaM were
also identified. We then investigated the potential function of these
interactions and showed that binding of CaM to G
inhibits the
pertussis toxin-catalyzed ADP-ribosylation of G
o subunits,
presumably by inhibiting heterotrimer formation. Furthermore, we
demonstrated that interaction with CaM has little effect on the
activation of phospholipase C-
2 by G
subunits, supporting the
notion that different domains of G
are responsible for the
interactions of different effectors. These findings shed light on the
molecular basis for the interactions of G
with Ca2+-CaM and point to the potential physiological
significance of these interactions in cellular functions.
In response to external stimuli or changes in ligand
concentration, the activated seven-transmembrane receptors of the cell interact with heterotrimeric G proteins. These in turn activate or
inhibit a variety of intracellular proteins through either the G
subunits with GTP bound or the free G
subunits or both, leading
to the generation of second-messenger molecules and changes in the
patterns of cellular metabolic activity and growth. Whereas direct
interaction of G protein
subunits with effectors has been known for
a long time, recently it has been demonstrated that G
subunits
also play critical roles in effector activation, in modulating the
interaction among various G protein pathways, and in regulating
cellular functions. For example, the G
dimers have been shown to
participate in interactions with adenylyl cyclases (1), phospholipases
(2, 3), phosducin (4),
-adrenergic receptor kinases (5-7), Bruton
tyrosine kinase (8), inositol trisphosphate kinases (9, 10),
mitogen-activated protein kinase (11), and a number of ion channels
(12-16). The activation of phospholipase C-
2 and
3 by G
has been suggested to account for the
PTX1-sensitive increase in intracellular
Ca2+ in response to a number of chemoattractants (17, 18)
and other ligands, thus regulating intracellular Ca2+
concentration.
Calmodulin (CaM) has been known to act as an intracellular calcium
sensor protein. When the intracellular Ca2+ concentration
increases, CaM can bind up to four Ca2+ ions, changing its
conformation and regulating cellular functions such as activation or
inhibition of a large number of enzymes (19, 20), ion channels (21),
and receptors (22). These Ca2+-dependent
interactions of CaM with its target proteins have played an important
role in intracellular Ca2+ signaling and in various
cellular functions including cell growth and differentiation. There are
reports demonstrating that G protein complex can bind to CaM
when passed through the CaM-agarose column (23, 24); however, the
precise nature of the interaction is not clear.
In this report, we investigated the interaction of Ca2+-CaM
with G subunits and the potential physiological significance of this interaction. We found that CaM not only binds to G
subunits purified from brain but also to the most diverse G
5L
complex from retina. We then identified and characterized the
CaM-binding domain of the G
subunit by using synthetic peptides and
site-specific mutation. Furthermore, we showed that binding of CaM to
inhibits the
-dependent PTX-catalyzed
ADP-ribosylation of G
subunits. Using both brain G
subunits
and the CaM-binding peptide derived from the
subunit, we
demonstrated that the Ca2+-CaM-dependent
activation of CaM kinase II could be inhibited at a molar ratio of 1 (peptide/CaM). These studies provide insight into the molecular basis
for the interactions of
with Ca2+-CaM and the
potential functions of these interactions in modulating cellular
signaling and other functions.
Materials
Synthetic peptides were obtained from the Peptide Synthesis Facility (Beckman Institute, California Institute of Technology). Peptides were purified by high performance liquid chromatography and verified by mass spectrometry. Bovine brain CaM was purchased from Calbiochem. Phosphatidylethanolamine and phosphatidylinositol-4,5-diphosphate (PtdInsP2) were purchased from Avanti Polar Lipids (Alabaster, AL) and Boehringer Manheim, respectively. [3H]PtdInsP2, myo-[2-3H]inositol, and [32P]NAD were obtained from DuPont NEN. Pertussis toxin was purchased from List Biological Laboratories Inc.
CaM Binding Assay of G Protein 5L
Subunits
Bleached bovine rod outer segment membranes were prepared from
bovine retina as described (25). Transducin was removed from the
membranes by 6 × hypotonic elution with 100 µM
GTPS in the presence of 3 mM MgCl2 (26). The
remaining membranes were solubilized for 12 h at 4 °C in 20 ml
of a buffer containing 50 mM Hepes, pH 8.0, 100 mM NaCl, 3 mM MgCl2, 10 mM 2-mercaptoethanol, and 1% (by mass) sodium cholate.
Unsolubilized material was pelleted for 40 min at 100,000 × g, and the supernatant was diluted with 30 ml of a buffer
containing 50 mM Hepes, pH 8.0, 100 mM NaCl, 3 mM MgCl2, 10 mM 2-mercaptoethanol,
and 1.67 mM CaCl2 (final concentration: 1 mM CaCl2, 0.4% sodium cholate). The diluted
supernatant was then applied at a flow rate of 0.3 ml/min to a
CaM-Sepharose column (10-ml bed volume, Pharmacia Biotech Inc.)
equilibrated with the above described buffer. The column was washed
with 50 ml of the above buffer. Proteins bound to the CaM-Sepharose
column were then isocratically eluted with a buffer containing 50 mM Hepes, pH 8.0, 100 mM NaCl, 3 mM
MgCl2, 10 mM 2-mercaptoethanol, 10 mM EDTA, and 0.4% sodium cholate. Fractions of 600 µl
were collected. 20 µl of the indicated fractions were separated by 10% SDS-PAGE and immunoblotted using the G
5-specific
antiserum CT215 as described (27).
Gel Mobility Shifting and Gel Overlay Assays
High affinity binding of G protein peptides to CaM was
demonstrated by the gel mobility shifts of CaM in 12.5% nondenaturing polyacrylamide gels in the presence or absence of 4 M urea.
Different concentrations of G
peptide were incubated with 1 µM CaM at room temperature for 30 min. To determine the
Ca2+ dependence and binding stoichiometry, gels were run in
the presence of either 0.5 mM CaCl2 or 2 mM EGTA. Proteins were visualized by Coomassie Brilliant
Blue staining.
Fluorescence Measurements and Determination of Dissociation Constants
Fluorescence emission spectra were obtained using SLM 4800 spectrofluorimeter. Excitation was at 295 nm. Excitation and emission band-passes were both 10 nm. Total fluorescence was determined by integration of emission spectra. The fluorescence titration data was used to determine the dissociation constant as described previously (28). By plotting the fraction of bound peptide as a function of free CaM concentrations, we obtained the peptide-CaM titration curve. The dissociation constant for the peptide-CaM binding was obtained from the curve fitting.
Site-specific Mutagenesis
Mutations in the putative CaM-binding site of G1
were generated by polymerase chain reaction with the high fidelity DNA
polymerase, Pfu (Stratagene). The mutations were confirmed
by DNA sequencing done in the DNA sequencing facility at Caltech. The
constructs were subcloned into pCDNA3.1 vector (Invitrogen, San
Diego, CA).
In Vitro Translation and Binding Assays
Transcription and translation were carried out using a rabbit
reticulocyte lysate system from Promega at 30 °C for 90 min with
amino acid mixtures without methionine. 5 µCi of
[35S]methionine (>1000 Ci/mmol, DuPont NEN) was added to
the reaction mixture to monitor the synthesis of new proteins. 1-2
µg of cDNAs were used in each translation. 2.5 µl aliquots of
the translation mixture were separated on 12% SDS-PAGE. The newly
synthesized proteins were identified by autoradiography. After in
vitro translation, the protein mixture was incubated with
CaM-agarose beads in the presence of Ca2+ for 30 min at
room temperature. Then the CaM-agarose beads were washed extensively to
get rid of nonspecific binding. Proteins bound to the beads were eluted
by a buffer containing 10 mM EGTA. The eluted G proteins
were separated by SDS-PAGE and visualized by autoradiography.
ADP-ribosylation of Go Subunits by PTX
Pertussis toxin-catalyzed ADP-ribosylation of Go was
performed as described previously (29). Briefly, 0.1 µg of
recombinant G
o was mixed with 0.1 µg of purified rat brain
subunits in the absence or presence of Ca2+-CaM and
incubated for 10 min at room temperature before the addition of the
reaction mixture (20 mM Tris-HCl, pH 8.0, 1 mM
EDTA, 2 mM MgCl2, 2 mM
dithiothreitol, 0.5 µM 32P-labeled NAD
(20,000 cpm/pmol), and 10 µg/ml pertussis toxin). Reactions were
incubated at room temperature for 30 min and terminated by adding
5 × SDS-PAGE sample buffer. Samples were resolved on SDS-polyacrylamide gels and stained with Coomassie Blue. Labeling of
proteins was detected by autoradiography.
Enzymatic Activity Assays of CaM Kinase II
The Ca2+-CaM-dependent activity of CaM
kinase II was assayed essentially as described previously (30).
Briefly, different concentrations of G peptide were preincubated
with Ca2+-CaM for 30 min at room temperature. The
phosphorylation reactions contained 50 mM Hepes buffer, pH
7.5, 10 mM magnesium acetate, 1 mM
CaCl2, 0.1 mM [
-32P]ATP, 1 µM CaM, 5-10 µg of CaM kinase II peptide substrate
(Chemicon International, Inc., Temecula, CA), and purified rat brain
CaM kinase II (Calbiochem). All assays were initiated by the addition of kinase.
COS-7 Cell Transfection and Phosphoinositol Phospholipase C Activity Assays
In Vivo Transfection AssaycDNAs encoding PLC-2,
G
, and CaM were cotransfected into COS-7 cells with LipofectAMINE
(Life Technologies, Inc.). Then the cells were labeled with 10 µCi/ml
myo-[2-3H]inositol the following day. 48 h after transfection, the activity of PLC-
2 was assayed by
determining the levels of inositol phosphates as described previously
(31, 37).
Phospholipid vesicles containing 50 µM
[3H]PtdInsP2 and 500 µM
phophatidylethanolamine were prepared by mixing with chloroform solution, drying under a stream of N2, then sonicating with
88 mM Hepes buffer, pH 7.5, and 18 mM LiCl.
Assays were performed in a 70-µl reaction mixture containing 20 ng of
PLC-2, 1.7 µM G
from bovine retina, 50 µM CaCl2, 10 mM LiCl, and
phospholipid vesicles. The reaction mixtures were incubated at 30 °C
for 10 min in the presence of different concentrations of CaM. The
reactions were stopped by adding 0.35 ml of chloroform/methanol/HCl
(500:500:3). The released Ins-1,4,5-P3 was extracted by
adding 0.1 ml of 1 M HCl with vigorous vortexing. The
aqueous phase separated after centrifugation was subjected to
scintillation counting.
To
demonstrate the direct interaction of G subunits with CaM,
purified bovine brain
subunits were incubated with CaM-agarose gel in the presence of 1 mM CaCl2. After
extensively washing with Ca2+-containing buffer, the bound
subunits were eluted by buffer containing EGTA. As shown in Fig.
1A, brain
binds to CaM, and this
binding is Ca2+-dependent. Direct binding was
also observed in a gel overlay assay with 125I-labeled CaM
(data not shown). There are five different G protein
subunits that
have been characterized in mammalian systems. Among them,
G
5 is the most diverse form and is expressed
predominately in neuronal cells (27). A splice variant of
G
5,
5L, is found only in rod outer
segment membrane. To examine whether CaM binding is a common feature in
different
subunits, G
5L
extracts were obtained
from bovine retina and passed through a CaM-agarose fast protein liquid
chromatography column in the presence of Ca2+. Proteins
bound to the CaM-agarose column were eluted with buffer containing 10 mM EDTA and detected by specific antibodies against the
G
5 subunit. Like other
subunits,
5L
also binds to Ca2+-CaM (Fig.
1B), suggesting that the CaM-binding domain is conserved in
all G
subunits.
Identification of a CaM-binding Domain of G
To
identify the CaM-binding domain on G subunits, the amino acid
sequences of
were compared with those of known high affinity
CaM-binding proteins. We found that the N terminus of all G
subunits
contains a putative CaM-binding domain that exhibits the
characteristics typical of CaM-binding peptides (32). Fig. 2 shows the alignment of the N-terminal domain sequences
of G
subunits with well known CaM-binding domains of rat olfactory cyclic nucleotide-gated channel (RAT OCNC), skeletal muscle
myosin light chain kinase (SK-MLCK), Ca2+ pump,
calcineurin, Ras-like GTPase Kir/Gem, murine inducible nitric oxide
synthase (iNOS), and CaM kinase II. To demonstrate that the
small domain from amino acid residues Val-40-Trp-63 is indeed the
CaM-binding domain of G
subunits, synthetic peptides representing
the putative CaM-binding domain were prepared and tested for their
abilities to bind CaM in the presence of Ca2+ using both
gel mobility shifting assay and tryptophan fluorescence assays.
Binding of peptides to CaM was first assayed by nondenaturing
polyacrylamide gel shifting assay. Depending on the charges and
hydrophobicity of the peptide, high affinity binding of the peptide to
CaM was detected as a complex band with increased or decreased mobility
compared with the unbound CaM band. In the presence of
Ca2+, the mobility of CaM was decreased by the peptide
corresponding to G residues 40-63 (Fig.
3A). Several ratios of the peptide to CaM
were used. In the absence of peptide, there is a single, fast-moving
band reflecting pure Ca2+-CaM (Fig. 3A, CaM
alone). At ratios of 0.25 and 0.75 of peptide to CaM, two bands were
visible on the gel, the fast-moving CaM band and the lower mobility
peptide-Ca2+-CaM complex. At a 1:1 molar ratio of peptide
to CaM, all of the CaM was gel-shifted, and the intensity of the
peptide-Ca2+-CaM band was increased as the result of
complex formation (Fig. 3A). At still higher molar ratios,
no new band was detected on the gel nor did the peptide-CaM complex
band change in intensity, indicating a 1:1 binding stoichiometry of
peptide to CaM and the absence of multivalent peptide-CaM complexes
(Fig. 3A). Similar results were obtained when gel shift
assays were performed in the presence of 4 M urea. However,
in the presence of Ca2+ chelator, EGTA, only the pure CaM
bands were detected on the gel and no peptide-CaM complex band was
visible (Fig. 3B), indicating the complex formation is
Ca2+-dependent. Peptides from other regions of
the G
subunit had no effect on the mobility of CaM (data not
shown).
Since the synthetic peptide contains a tryptophan residue, whereas CaM
contains none, binding of G peptide to CaM was directly studied by
monitoring the changes in tryptophan fluorescence (Fig. 4). When the peptide was excited at 295 nm, it exhibited
an intrinsic tryptophan fluorescence emission peak at 353 nm. Addition
of CaM in the presence of Ca2+ not only caused a blue shift
of the fluorescence peak but also increased the maximal fluorescence
intensity (Fig. 4A). Since Ca2+ alone has no
effect on the emission spectrum, the observed change must have resulted
from the binding of Ca2+-CaM to the peptide. The blue shift
in fluorescence emission and the increase in total intensity indicate
that the environment of the G
peptide became hydrophobic, presumably
due to interactions with the hydrophobic domains of CaM upon formation
of complex, further confirming the CaM binding property of the peptide.
The changes in fluorescence property upon formation of the peptide-CaM complex were fully reversed by the addition of EGTA (Fig.
4A), indicating that the binding of CaM to the peptide is
Ca2+-dependent. Titration of fixed amounts of
peptide with different concentrations of CaM showed a saturation
pattern. As shown in Fig. 4B, when a 0.1 µM
concentration of peptide was used, the fluorescence intensity increased
almost linearly with increasing CaM concentrations and reached a
plateau at 0.1 µM CaM, confirming a 1:1 tight binding
between the peptide and CaM. When the fraction of bound peptide was
plotted as a function of free CaM concentration, the dissociation
constant (Kd) obtained was ~40 nM
(Fig. 4C), indicating high affinity binding to
Ca2+-CaM. The actual binding affinity could be higher than
40 nM; however, the limitation of this fluorescence
measurement does not allow us to test low peptide concentration.
Direct interaction of calmodulin with G
peptide assayed by measuring the changes in tryptophan fluorescence
spectrum. A, Ca2+-dependent
interactions of the G
peptide with CaM, measured by the emission
spectrum of the peptide and peptide-CaM complex. When the peptide (0.1 µM) was excited at 295 nm, the peptide exhibited a
fluorescence emission spectrum with a maximal emission peak at 354 nm
(peptide w/Ca2+). In the presence of
Ca2+, addition of CaM not only caused a blue shift of the
fluorescence peak but also increased the total fluorescence intensity
(peptide + Ca2+-CaM). Addition of EGTA
completely reversed these fluorescence changes (peptide + Ca2+-CaM + EGTA), indicating that the
interactions between the G
peptide and CaM is
Ca2+-dependent. B, the G
peptide
was titrated with different concentrations of CaM in the presence of
0.5 mM CaCl2. C, determination of
the affinity constant for the interaction between CaM and G
peptide. The fraction of bound peptide was calculated and plotted against free
CaM concentration. The curve was fitted by a ligand binding equation
with a dissociation constant of ~40 nm.
Mutation Analysis of the Putative CaM-binding Domain of G
To identify the key amino acids essential for CaM
binding, two types of mutations were carried out in the putative
CaM-binding domain by site-directed mutagenesis. One set contains a
number of basic amino acid residues (Arg-42, Arg-48, Arg-49), and the other group involves hydrophobic residues (Ile-43, Leu-51, Leu-55). These specific amino acids were individually replaced by alanine residues. Mutant proteins were then compared with wild type protein for
their ability to bind Ca2+-CaM. Both wild type and mutant
cDNA were translated in vitro together with 2 subunit
in rabbit reticulocyte lysates in the presence of
35S-labeled methionine. Fig. 5A
shows the in vitro translated wild type and mutant G
proteins separated by SDS-gel and detected by autoradiography. The
amount of wild type protein and mutant proteins synthesized are similar
under these assay conditions. The in vitro translated
protein mixtures were incubated with CaM-agarose beads to assess the
CaM binding ability. After extensive washing with
Ca2+-containing buffer (100-200 bead volume), the bound
protein was eluted with EGTA buffer. As shown in Fig. 5B,
like the wild type G
protein, one of the mutants (R42A) retained its
ability to bind CaM. However, the binding affinities of other mutants
(I43A, L55A, R48A/R49A, and L51A) for CaM were reduced by varying
degrees under the same assay conditions. For instance, mutations at
residues Arg-48, Arg-49, and Leu-51 dramatically reduced the CaM
binding ability of the protein (Fig. 5B), suggesting that
these residues are critical in the interactions. To further probe the
conformation and activity of mutant G
subunits, we analyzed the
formation of
dimers using in vitro translated
proteins and coimmunoprecipitation with specific anti-G
2
antibodies. The wild type G
and its mutant proteins can be
precipitated by
2 antibodies, and there is little difference in their ability to interact with
subunits (data not
shown), indicating that mutant
subunits could still fold into the
native conformation and retain affinity for
subunits.
Effects of CaM Binding on ADP-ribosylation of G
To explore the possible physiological functions of CaM
binding to G subunits, we first examined the effects of CaM
binding on
-dependent pertussis toxin-catalyzed
ADP-ribosylation of G
o subunit. PTX is a bacterial toxin that
catalyzes the ribosylation of the C-terminal cysteine residues of
G
o, G
i, and G
t subunits. PTX modification blocks the
interactions of G
subunits with receptors and thus blocks the
ligand-mediated signal transduction. The labeling of G
o by PTX
requires the G
o
heterotrimer rather than the free G
o
subunit (33). Thus, ADP-ribosylation of
o can be used as a sensitive
indicator of formation of
o
heterotrimers. Incubation of CaM
with bovine brain G
subunits in the presence of 0.1 mM Ca2+ inhibited the ribosylation reaction, as
shown in Fig. 6. The reduction of labeling is
concentration-dependent. At equal molar concentrations of
CaM and
subunits, the PTX-catalyzed ribosylation of G
o was
almost completely blocked by Ca2+-CaM. Since
Ca2+ alone had no effect on the reaction, the simplest
interpretation of this observation is that Ca2+-CaM formed
a complex with G
subunits and this complex lost its ability to
interact with G
o subunit; therefore, CaM inhibited the
-dependent ADP-ribosylation of G
o.
Binding of CaM to G
To further investigate the nature of CaM
binding on G subunits, we examined its effects on
-stimulated PLC-
2 activity. As shown in Fig.
7A, CaM has little effect on PLC-
2
activity stimulated by
subunits in an in vitro
reconstituted system. To confirm the observations obtained in the
reconstitution experiments, cDNAs encoding PLC-
2, G
, and
CaM were transfected into COS-7 cells. Coexpression of PLC-
2 and
subunits increased inositol 1,4,5-trisphosphate release 3-5
fold (Fig. 7B, column 3). However, cotransfection
of CaM expression plasmids had little effect on the
-stimulated
PLC-
2 activity (Fig. 7B, column 4). These
results indicated that in the G
subunit, the domain responsible for
PLC-
2 activation is distinct from the domain binding to CaM,
suggesting that different domains of the
subunit are involved in
interactions with different effector proteins.
Inhibition of Ca2+-CaM-dependent Enzyme, CaM Kinase II, by G
The putative
CaM-binding domain of G subunits was further characterized by using
the synthetic peptide derived from G
to inhibit
Ca2+-CaM-dependent activation of CaM kinase II.
Concentration-dependent effects of the peptide on CaM
kinase II activity were examined. As shown in Fig.
8A, the peptide was a potent inhibitor of CaM kinase II activity. At a 1:1 binding ratio of G
peptide to CaM, the
peptide totally inhibited the
Ca2+-CaM-dependent activation of CaM kinase II,
suggesting that the binding affinity of G
peptide is comparable to
the affinity of CaM kinase II toward Ca2+-CaM and should be
in the nM range. Other peptides from the N-terminal regions
had no effect on the Ca2+-CaM-stimulated enzymatic activity
(data not shown). To examine whether G
subunits can also inhibit
the Ca2+-CaM-dependent CaM kinase II activity,
Ca2+-CaM was incubated with brain G
subunits for 30 min at room temperature. Then the Ca2+-CaM-stimulated CaM
kinase II activity was assayed. Fig. 8B shows that brain
G
subunits inhibited 70-80% of Ca2+-CaM-stimulated
CaM kinase II activity, indicating that
can competitively bind
to Ca2+-CaM.
Previous studies have demonstrated that G protein subunits can act
as potent inhibitors of the Ca2+-CaM-stimulated
phosphodiesterase activity (23, 24), probably through interactions of
G with CaM. In this report, we show direct binding of G
subunits with CaM. This CaM-binding property of G
is
Ca2+-dependent and conserved in known G
subunits, including the most diverse
5 subunit. We also identified
and characterized the CaM-binding domain in
subunits using three
different methods. In the gel mobility shifting assays and tryptophan
fluorescence assay, a conserved 25-amino acid peptide in the N-terminal
region of G
subunits was found to interact with CaM. By modifying
specific amino acids in this region, we identified some key residues
(Arg-48, Arg-49, Leu-51, Ile-43, Leu-55) that play an important role in the binding of
to CaM.
The interaction between G subunits and CaM is
Ca2+-dependent. Ca2+ is an
important intracellular messenger in many cellular functions including
cell growth and development (34, 35). Many seven-transmembrane receptors activate the G protein subunits, which in turn activate PLC-
, resulting in the production of inositol 1,4,5-trisphosphate and diacyglycerol from PtdInsP2. Inositol
1,4,5-trisphosphate acts as an intracellular second messenger by
binding to the inositol 1,4,5-trisphosphate receptors in the
endoplasmic reticular membrane, triggering the release of
Ca2+ from the endoplasmic reticulum and therefore
increasing the intracellular Ca2+ concentration.
Ca2+ is also believed to organize and stabilize CaM domain
structure in a conformational state that can bind target proteins (32, 36). The calmodulin concentration in some cells, e.g.
neuronal cells, is in the order of micromolar. Calcium release can thus convert 10-50% of these molecules to the Ca2+-bound form.
The affinity of G
for Ca2+-CaM is sufficiently high
so that under these conditions much of the free
in the cells
should be in the CaM-bound form. Therefore, an increase in
intracellular Ca2+ concentration could selectively regulate
the interactions of G
with a number of other proteins through
binding with CaM.
The interaction of G with Ca2+-CaM could play an
important role in the cross-talk mechanism between different G protein
pathways (37). Ca2+-CaM has been shown to regulate the
formation and hydrolysis of cAMP. In the G
s-coupled pathway, both
adenylyl cyclases and phosphodiesterases are
Ca2+-CaM-dependent, which could serve as the
convergence point for Ca2+-dependent and
G
s-dependent stimuli.
The primary structure of the identified CaM-binding domain of G
shows features similar to some other CaM-binding proteins and
inhibitors (21, 28, 38-45). For instance, the identified
CaM-binding domain contains a high percentage of hydrophobic residues
and an excess of positively charged residues, a property found in all
CaM-binding peptides. It is believed that the basic residues contribute
to CaM binding via electrostatic interactions with acidic residues in
CaM, whereas the hydrophobic amino acids seem to play a more important
role in CaM binding through interactions with the hydrophobic patches
of the globular domains of CaM (38, 39). From x-ray structural
analysis, 80% of all contacts are van der Waals interactions, and the
interaction appears to involve two key hydrophobic amino acids
separated by eight residues, although flexibility does exist in the
interaction because of the flexible central helix of CaM (36). CaM
contains two globular domains, and these two domains are connected by a
long alpha helical segment (32, 36). When different CaM binding targets
are recognized, this long central segment allows different relative
positioning of the two lobes of CaM. Therefore, although the secondary
structure of the G
CaM-binding domain is not a typical amphipathic
helix (46), CaM could still position itself to bind to the G
protein, possibly by stabilizing a change in the conformation of the
N-terminal domain of G
protein. On the other hand, a number of
CaM-binding proteins have been identified and shown to contain sites
that are not typical amphipathic helices but consist of basic amino acids interspersed with nonpolar amino acids (52-54).
The binding of CaM to G interfered with the formation of
G
trimers as assayed by the inhibition of PTX-catalyzed
ADP-ribosylation of G
o. Based on the secondary structure of the
subunit, the N-terminal region of the
subunit is in close proximity
to the N-terminal portion of G
subunit (47-49). It has been
reported that effector activation by the
subunits is blocked
upon the addition of the G
subunit presumably by heterotrimerization
(1, 13). Thus, by interacting with the N-terminal domain of
subunit, the Ca2+-CaM complex could affect the heterotrimer
formation of G
.
Interaction of CaM with has little effect on the G protein
subunit-activated PLC-
2 activity. These results support the
notion that interaction of different
-responsive effectors is
mediated by distinct domains of G
(50, 51). By using a series of
chimeras between Dictyostelium and mammalian
subunits, a
small C-terminal segment of G
was identified as responsible for the
activation of PLC-
2 (50). The CaM-binding domain of G
identified
in this report is located in the N-terminal region. This region of G
was suggested to play a role in the interaction of adenylyl cyclase
type 2, the muscarinic receptor-gated atrial inwardly rectifying
potassium channel (GIRK1), and in the activation of mitogen-activated
protein kinase pathways (50, 51). It will be of great interest to
determine whether binding of CaM to G
can affect the activation
of the potassium channels (GIRK) and mitogen-activated protein kinase
pathways.
We thanks Dr. Silvia Cavagnero (Division of Chemistry, Caltech) for her help and discussion with the fluorescence measurements of the peptide-protein interactions and Dr. A.R. Means (Duke University) for cDNA clones of calmodulin.