(Received for publication, September 5, 1996, and in revised form, December 16, 1996)
From the Department of Pharmacology, University of Tennessee,
Memphis, Memphis, Tennessee 38163 and Department of
Biomolecular Chemistry, University of Wisconsin,
Madison, Wisconsin 53706-1532
Previously, we have demonstrated that
epidermal growth factor (EGF) can stimulate adenylyl cyclase activity
via activation of Gs in the heart. Moreover, we have
recently shown that Gs is phosphorylated by the EGF
receptor protein tyrosine kinase and that the juxtamembrane region of
the EGF receptor can stimulate Gs directly. Therefore,
employing isolated cardiac membranes, the two-hybrid assay, and
in vitro association studies with purified EGF receptor and
Gs
we have investigated Gs
complex formation with the EGF receptor and elucidated the region in the receptor involved in this interaction. In isolated cardiac membranes, immunoprecipitation of EGF receptor was accompanied by
co-immunoprecipitation of Gs
. In the yeast two-hybrid
assay, the cytosolic domain of the EGF receptor and the N-terminal 64 amino acids of this region (Met644-Trp707)
associated with Gs
. However, interactions of these
regions of the EGF receptor with constitutively active
Gs
were diminished in the two-hybrid assay. Employing
purified proteins, our studies demonstrate that the EGF receptor,
directly and stoichiometrically, associates with Gs
(1 mol of Gs
/mol of EGF receptor). This association was not
altered in the presence or absence of ATP and therefore, was
independent of tyrosine phosphorylation of either of the proteins.
Peptides corresponding to the juxtamembrane region of the receptor
decreased association of the EGF receptor with Gs
.
However, neither the C-terminally truncated EGF receptor
(
1022-1186) nor a peptide corresponding to residues 985-996 of the
receptor altered association with Gs
, thus indicating
the selectivity of the G protein interaction with the juxtamembrane
region. Interestingly, peptides corresponding to N and C termini of
Gs
did not alter the association of Gs
with the EGF receptor. Consistent with the findings from the two-hybrid assay where constitutively active Gs
poorly associated
with the EGF receptor, in vitro experiments with purified
proteins also demonstrated that activation of Gs
by
guanosine 5
-3-O-(thio)triphosphate decreased the
association of G protein with the EGF receptor. Thus we conclude that
the juxtamembrane region of the EGF receptor, directly and
stoichiometrically, associates with Gs
and that upon
activation of Gs
this association is decreased.
The pleiotropic actions of epidermal growth factor
(EGF)1 are elicited by stimulation of a
number of second messenger systems by the ligand-activated EGF receptor
(1, 2). In addition to its well documented effects on the
mitogen-activated protein kinase cascade (3) and phospholipase C
(4), EGF has also been demonstrated to regulate the cAMP second
messenger system (5). We have previously demonstrated that in cardiac
myocytes EGF elevates cAMP accumulation (6) by augmenting the activity of adenylyl cyclase (7, 8) and that this increase in cAMP accumulation
also augments the beating rate and contractility in intact hearts (9).
EGF stimulates cardiac adenylyl cyclase by activation of the
-subunit of the stimulatory GTP-binding protein, Gs
(8), and the protein tyrosine kinase activity of the EGF receptor is
important in this modulation (10). More recently, we have demonstrated
that a 13-amino acid sequence in the cytosolic, juxtamembrane, region
of the EGF receptor is important for activation of Gs and
stimulation of adenylyl cyclase (11). In addition to the activation of
Gs by the juxtamembrane region of the receptor (11), we
have also shown that the EGF receptor protein tyrosine kinase can
phosphorylate Gs
on tyrosine residues and that this
phosphorylation of Gs
increases its ability to stimulate
adenylyl cyclase (12). Thus, the combined actions of the juxtamembrane
region of the activated EGF receptor on Gs (11) and
phosphorylation of Gs
by the EGF receptor protein
tyrosine kinase (12), may, in a mutually reinforcing manner, amplify
the signaling events leading to adenylyl cyclase stimulation.
The juxtamembrane region of the EGF receptor has also been shown to be
important for determining specificity of mitogenic signaling as well as
determining substrate specificity. Thus, the deletion of 8 amino acids
in the juxtamembrane region of the EGF receptor (amino acids 660-667)
alters the mitogenic activity of EGF (13). In addition, the mutation of
one amino acid residue in this region (Arg662) of the EGF
receptor alters the mitogenic signaling and pattern of protein
phosphorylations within the cells without altering the protein tyrosine
kinase activity of the receptor (14). Likewise, the juxtamembrane
region (Met644-Gly666) of the EGF receptor is
also important for the association of phosphatidylinositol-4 kinase and
phosphatidylinositol 4-phosphate 5-kinase (15). This latter region
encompasses the 13-amino acid region that activates Gs
(11). Because the juxtamembrane region of the EGF receptor has been
demonstrated to be important for activation of Gs (11), as
well as binding of proteins, and determining substrate specificity for
the EGF receptor protein tyrosine kinase (13-15), the studies
described herein were performed to characterize the association of EGF
receptor with Gs, and to elucidate the region(s) in the
receptor that are important for such an association. Our studies
demonstrate that in cardiac membranes, the activated EGF receptor
associates with Gs
. Moreover, our data from yeast
two-hybrid assays, as well as from in vitro association studies with purified proteins (EGF receptor and Gs
),
demonstrate that the juxtamembrane region of the EGF receptor is
important for association with Gs
and that the
GDP-liganded Gs
, not GTP·Gs
, is the
preferred form of the G protein which physically interacts with the EGF
receptor. Interestingly, the association between EGF receptor and
Gs
is stoichiometric, direct (not involving adapter
protein(s)), and does not involve the major phosphorylation sites on
the EGF receptor.
Hearts excised from male rats of the Harlan Sprague Dawley strain (180-200-g body weight) were homogenized in medium containing 5 mM Tris-HCl, pH 7.4, 250 mM sucrose, and 1 mM EGTA. Cardiac membranes were isolated from the homogenate by the methods previously described (7, 8). The cardiac membranes (300 µg of protein) were then incubated in adenylyl cyclase assay mixture described previously (7, 8) in the presence and absence of EGF (100 nM). Following incubation for 30 min at room temperature, the reactions were terminated by addition of lysis buffer described below and immunoprecipitations performed (see below).
Construction of Plasmids Encoding Chimeric ProteinsEssentially, this assay was performed using the plasmids
and yeast strains provided in the MatchmakerTM kit
(Clontech Laboratories Inc.). Employing the full-length human EGF
receptor cDNA as template (gift from Dr. Gordon Gill, University of
California at San Diego), and primers corresponding to nucleotides 1932-1953 (sense strand; primer sequence: 5-
GGG
ATGCGAAGGCGCCACATCGTTCGG-3
) and 3538-3561
(complementary strand; primer sequence: 5
-
AG
CTCATGCTCCAATAAATTCACTGCT-3
), the complete
cytosolic region of the EGF receptor (amino acids 644-1186;
nucleotides 2188-3816) was generated by PCR. The 5
primer introduced
an EcoRI site (underlined above) and the 3
primer was
tagged with BamHI and SalI sites downstream
(underlined above). The addition of the unique EcoRI site at
the 5
end facilitated the in-frame cloning of the cDNA
corresponding to the cytosolic region of EGF receptor into the plasmids
pGAD424 and pGBT9; these constructs are referred to as
pGAD424-EGFRC and pGBT9-EGFRC,
respectively.
The plasmids pGAD424 and pGBT9 contain the activating domain and
binding domain, respectively, of the GAL4 gene and expression is under
the control of the yeast alcohol dehydrogenase promoter. The
BamHI site at the 3 end (introduced by PCR) along with an internal BamHI site at nucleotide 2121 in the EGF receptor
cDNA facilitated the truncation of the chimeric constructs in
plasmids pGAD424-EGFRC and pGBT9-EGFRC so that
only N-terminal 64 amino acids (Met644-Trp707) in the
juxtamembrane region of the cytosolic domain of the EGF receptor were
expressed as fusion proteins with the activating and binding domains of
GAL4, respectively. These chimeric constructs in the plasmids
expressing the short form of the EGF receptor cytosolic domain are
referred to as pGAD424-EGFRCJM and
pGBT9-EGFRCJM. In addition, using a 5
primer corresponding
to amino acids Gly695-Phe699 (sequence:
5
-CAAAGT
CTCCGGTGCGTTC-3
) tagged with a
SmaI site (underlined) and 3
complementary primer
corresponding to nucleotides 3796-3816 described above, constructs
pGAD424-EGFRC
JM and pGBT9-EGFRC
JM were
also generated. These latter constructs encoded all of the cytoplasmic
region (amino acids 694-1186) of the EGF receptor devoid of the
juxtamembrane region (amino acids 645-694).
Employing the full-length Gs cDNA as template
(obtained from Dr. Alfred Gilman, University of Texas Southwestern
Medical Center) and primers corresponding to nucleotides 1-27 (sense
strand; sequence:
5
-ATTCTAGACC
CCATGGGCTGTCTCGGAAACAGC-3
)
and 1122-1143 (complementary strand; sequence:
GAGGTT
TTAGAGCAGCTCATACTGACG 3
), SalI
restriction endonuclease sites (underlined in sequences above) were
added on the 5
and 3
ends, respectively. The SalI site at
the 5
end facilitated the in-frame cloning of the Gs
cDNA into the plasmids pGAD424 and pGBT9 to generate plasmids pGAD424-Gs
and pGBT9-Gs
, respectively;
numbering system for nucleotides and amino acids used here are those
for the short form of Gs
(16). The constitutively active
form of Gs
(Q213L) (17) was obtained by a two-step PCR.
First, using Gs
cDNA as template along with 5
sense
primer mentioned above and a 3
complementary primer corresponding to
nucleotides 628-648 of Gs
with a T
A substitution
at nucleotide 638 (to substitute glutamine for leucine; sequence:
TTCATCGCGC
GGCCGCCCAC), a PCR product was generated.
Similarly, using a sense primer corresponding to nucleotides 628-650
of Gs
with an A
T substitution at nucleotide 638 and
the 3
complementary primer to Gs
mentioned above,
another PCR product was generated. Using the PCR products of
Gs
with the A
T and T
A substitutions in the
coding and noncoding strands, respectively, and using the 5
-most sense and 3
-most primers mentioned above, the full length Gs
cDNA coding for the constitutively active form of the protein was
generated by a second round of PCR. This latter cDNA encoding the
constitutively active, Q213L, form of Gs
(Gs
*) was then cloned into the SalI site of
pGAD424 and pGBT9 vectors to generate plasmids pGAD424-Gs
* and pGBT9-Gs
*, respectively.
The mutation of Gs
at nucleotide 638 was confirmed by
di-deoxynucleotide sequencing method (18). Likewise, all of the plasmid
constructs were sequenced to confirm that the cloning of the
appropriate cDNAs was in-frame for transcription and devoid of any
mistakes resulting from PCR.
The two-hybrid assay was performed
employing the HF7c strain of yeast (Clontech Laboratories Inc.)
transformed with the various constructs in plasmids pGAD424 and pGBT9.
Transformation of cells was performed as described by Clontech
Laboratories Inc. in the MatchmakerTM kit. The transformed
yeast cells were grown on plates containing either medium devoid of
L-leucine (Leu) and L-tryptophan
(Trp
) or medium in which L-histidine as well
as L-leucine and L-tryptophan (Leu
/Trp
/His
) had been
omitted. The plates were incubated at 30 °C for 3 days. Several of
the colonies from transformants were then individually streaked out
onto new plates containing the corresponding media.
To monitor -galactosidase activity in transformants growing on
Leu
/Trp
/His
medium, colonies
were individually grown in liquid medium lacking the three amino acids.
After overnight growth in this medium, when the cells had reached an
A600 nm of approximately 0.3, the cells were
harvested by centrifugation, resuspended in complete medium and grown
for an additional 3 h (A600 = ~0.5). At
this point, the cells were harvested by centrifugation, washed with
buffer containing 60 mM Na2HPO4, 40 mM NaH2PO4, 1 mM KCl, 1 mM MgCl2, pH 7.0, and then lysed in the same
buffer supplemented with Triton X-100 (final concentration, 0.2%). The
cell lysates were subjected to two freeze-thaw cycles with liquid
nitrogen and assayed for
-galactosidase activity employing the
chemiluminescence kit obtained from Clontech Laboratories Inc.
The
BL21(DE-3) strain of Escherichia coli transformed with the
plasmid pQE-60, containing cDNA encoding the 45-kDa form of bovine
Gs, was obtained from Alfred Gilman (University of Texas
Southwestern Medical Center, Dallas, TX). Expression of Gs
was induced with isopropyl
-D-thiogalactopyranoside, and the protein was purified
essentially as described by Graziano et al. (19). EGFR was
purified from A431 cells as described previously (20).
Purified EGF receptor (33 ng) and recombinant
Gs (25 ng) were incubated in a medium (final volume = 10 µl) containing 5 mM HEPES-NaOH, pH 7.4, 5 mM MgCl2, 2 mM MnCl2,
20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM
dithiothreitol, and 1 µM EGF with or without 10 µM ATP at room temperature for 60 min. The mixture was
supplemented with 500 µl of immunoprecipitation buffer containing the
following (final concentration): 25 mM Tris, pH 7.4, 0.5% Nonidet P-40, 1% Triton X-100, 1 mM EDTA, 150 mM NaCl, 20 µg/ml aprotinin, and 20 µg/ml leupeptin. To
immunoprecipitate the EGF receptor, monoclonal anti-EGF receptor
antibody (EGFR1, Amersham Corp.) was added to the above mixture to
reach a final concentration as 2-3 µg/ml. To immunoprecipitate
Gs
, 5 µl of CS1 antiserum raised against the C
terminus decapeptide (8) were added. As a control for EGFR1, an
irrelevant mAb BBC-4 against adenylyl cyclase (provided by Dr. Thomas
Pfeuffer, University of Würzburg, Germany) was used. Likewise,
nonimmune rabbit serum was employed as a control for CS1 antiserum. The
mixture was then incubated at 4 °C overnight with constant rolling.
Pansorbin suspension (20 µl of 10% suspension, Calbiochem), which
had been prewashed with immunoprecipitation buffer, was then added, and
the mixture was further incubated at 4 °C for 1 h with constant
rolling. The samples were centrifuged at 14,000 × g
for 1 min, and the resulting pellets were washed once each in 500 µl
of buffer containing high salt (25 mM Tris, pH 7.4, 500 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), medium
salt (25 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and no salt (25 mM Tris, pH 7.4, 0.1% Nonidet P-40). The final pellet was
resuspended in 30 µl of 2 × Laemmli sample buffer, heated, and
subjected to SDS-polyacrylamide gel electrophoresis. The association
between Gs
and EGF receptor was detected by employing
two different approaches. First, since we have shown that the EGF
receptor can phosphorylate Gs
(12), the incubation was
performed in the presence of 10 µM [
-32P]ATP, 2 mM MnCl2, and 5 mM MgCl2. Following incubation for 1 h at
room temperature, the respective proteins were immunoprecipitated with
either the EGFR1 mAb or CS1 antiserum, separated by SDS-PAGE, and the
phosphorylated Gs
and EGF receptors were detected by
autoradiography on Kodak X-Omat film. In the second approach, following
separation of proteins in the immunoprecipitate by SDS-PAGE and after
transfer onto nitrocellulose, the proteins were detected by Western
analyses with either EGFR1 mAb (CS1 immunoprecipitations) or CS1
antiserum (EGFR1 immunoprecipitates) employing the Amersham ECL system.
Whenever peptides were employed, these were added in the initial
incubation.
The monoclonal anti-EGF receptor antibody was
purchased from Amersham Corp. Antiserum against the C terminus
decapeptide of Gs was the generous gift of Dr. Graeme
Milligan, University of Glasgow. Peptides EGFR-13, EGFR-14, and
Gs
371-380 were synthesized as described previously
(11). Threonine phosphorylated EGFR-13 (P-EGFR-13) was custom
synthesized by Genosys Biotechnologies Inc. (Woodlands, TX). Peptide
EGFR 985-996 was purchased from Bacham Laboratories (Torrance, CA).
Peptides corresponding to amino acids 15-29 (Gs
15-29)
and 354-372 (Gs
354-372) were gifts from Dr. Heidi
Hamm (University of Illinois College of Medicine, Chicago). All of the
other chemicals employed were of the highest grade commercially
available.
Our initial studies to characterize the association between EGF
receptor and Gs were performed in isolated membranes derived from rat hearts. This system was selected because we have previously demonstrated that, in membranes isolated from rat hearts, EGF stimulates adenylyl cyclase activity via activation of
Gs
(7, 8). Essentially, rat heart membranes were
incubated under the conditions of adenylyl cyclase activity assay in
the presence or absence of ATP, with and without EGF. Reactions were terminated by addition of lysis buffer and the EGF receptor was immunoprecipitated. Following separation of proteins by SDS-PAGE, Western analysis with anti-Gs
antiserum, CS1,
demonstrated the presence of Gs
in the EGF receptor
immunoprecipitates of incubations performed in the presence of EGF and
ATP, but not in EGF receptor immunoprecipitates of incubations
conducted in the absence of EGF (Fig. 1). Moreover, ATP
was also required to observe association of the EGF receptor with
Gs
(Fig. 1). The presence or absence of EGF and/or ATP
in the incubation did not alter immunoprecipitation of the EGF receptor
(Fig. 1, bottom panel). We have previously demonstrated that
the EGF receptor protein tyrosine kinase activity is important for EGF
to stimulate adenylyl cyclase activity in cardiac membranes (10). These
earlier findings, along with the requirement for ATP and EGF to observe EGF receptor association with Gs
(Fig. 1), suggest that
the activation of the EGF receptor and perhaps autophosphorylation of
the receptor alter its conformation to make sites accessible for
association with Gs
. Indeed, Cadena et al.
(21) have shown that, upon activation and autophosphorylation of the
EGF receptor, the receptor undergoes a conformational change from a
compact to a more extended form.
To further characterize the association between EGF receptor and
Gs and to define the regions of the receptor which
interact with the G protein, additional experiments were performed
employing the yeast two-hybrid system (22). Yeast cells (HF7c strain) were transformed with plasmids pGAD424-EGFRC or
pGBT9-EGFRC and pGBT9-Gs
or
pGAD424-Gs
, respectively; these EGF receptor constructs
expressed only the cytosolic region of the receptor (Met644-Ala1186) as chimeric proteins with
either the activating (pGAD424 plasmid) or binding (pGBT9 plasmid)
domains of GAL4 gene product. Controls were performed with either the
two plasmids alone (not shown), or with the chimeric construct in one
plasmid and the other plasmid devoid of either the EGF receptor or
Gs
cDNA (Fig. 2A). Since the plasmids pGAD424 and pGBT9 encode for the LEU2 and TRP1 cDNAs, respectively, growth of cells on Leu
/Trp
medium indicated the successful transformation of cells with both
plasmids (right-hand panels in Fig. 2, A-C).
Likewise, since the GAL4 activating and binding domains have to come in
proximity of each other to initiate transcription of the HIS3 and lacZ
genes, growth of transformants on
Leu
/Trp
/His
medium and
expression of
-galactosidase activity indicated a productive
interaction between the proteins being tested. As shown in Fig. 2
(panels A-C), transformation of yeast HF7c cells with the
various constructs in plasmids pGAD424 and pGBT9 allowed comparable growth of cells on Leu
/Trp
medium. However,
when these same transformants were grown on Leu
/Trp
/His
medium, growth to
different extents was observed in only some of the transformants. Thus,
in the latter medium, transformation with plasmids expressing the
entire cytoplasmic region (EGFRC) (Fig. 2A) or
the N-terminal 64 amino acids (Met644-Trp707;
EGFRCJM) of the cytosolic region of the EGF receptor in
plasmid pGBT9 (Fig. 2B), and the Gs
in
plasmid pGAD424, robust cell growth was observed. Although growth of
cells on Leu
/Trp
/His
medium
was observed when the EGF receptor cytoplasmic domain (EGFRC) or the N-terminal 64 amino acids of this region
(EGFRCJM) and Gs
were expressed in the
reciprocating plasmids, the yeast did not grow as well as in the former
combination (Fig. 2, A and B). The better
interaction between cytosolic regions of the EGF receptor as chimeras
with the binding domain of GAL4 and Gs
or constitutively
active form of this G protein (Gs
*) as a chimera with
GAL4 activating domain as compared to the reciprocal chimeras of these
proteins (Fig. 2C) is not unusual and similar observations
have previously been reported for other proteins (reviewed in Fields
and Sternglanz (23)).
In the presence of Gs*, as indicated by growth of yeast
cells on Leu
/Trp
/His
medium,
the interactions with EGF receptor cytosolic regions, EGFRC
and EGFRCJM, was diminished (Fig. 2, A and
B). Notably, when interactions between Gs
and
the cytosolic region of EGF receptor in which the N-terminal amino
acids 645-694 had been deleted (constructs
pGAD424-EGFRC
JM and pGBT9-EGFRC
JM) were
tested (Fig. 2C), no growth on
Leu
/Trp
/His
medium was
observed in any of the combinations of the plasmids, except the
positive control with pGBT9-EGFRC and
pGAD424-Gs
. That these cells were successfully
transformed with the various constructs is demonstrated by growth of
all transformants on Leu
/Trp
medium (Fig.
2C, right panel).
The differences in activity of -galactosidase in the various
transformants were also consistent with the findings described above
with growth in medium lacking histidine (Fig. 2, panel D). Hence, both the entire cytosolic domain of the EGF receptor
(EGFRC) and the N-terminal 64 amino acids of this region
(EGFRCJM) demonstrated greater expression of
-galactosidase activity in the presence of Gs
as
compared to Gs
* (Fig. 2D). Moreover, as
observed for cell growth, transformation of cells with
Gs
(wild type and constitutively active) in plasmid
pGAD424 and EGF receptor constructs in pGBT9 yielded higher
-galactosidase activities than expression of these proteins in the
reciprocal vectors (Fig. 2D). Since controls performed with
either EGFR cDNA constructs (EGFRC and
EGFRCJM) or Gs
constructs paired with the
empty plasmids did not grow on
Leu
/Trp
/His
medium,
-galactosidase activity in these cells could not be monitored.
Likewise, since transformants with plasmids constructs encoding the
cytosolic region of EGF receptor without the N terminus 50 amino acids
(deletion of residues 645-694; EGFRC
JM) also did not
grow in the absence of histidine,
-galactosidase activity could not
be monitored. These findings in the two-hybrid system therefore suggest
that the EGF receptor and Gs
interact with each other
and at least a portion of the 64 amino acids (residues 644-707) in the
juxtamembrane region of the receptor are required for this interaction.
Additionally, the data from the yeast two-hybrid experiments (Fig. 2,
B and D) demonstrate that the juxtamembrane region of the EGF receptor interacts better with the wild type Gs
as compared to the constitutively active form of this protein, Gs
*.
In order to determine whether the association between the EGF receptor
and Gs is direct (i.e. not involving other
proteins) and to further delineate the region(s) on the EGF receptor
which associate with Gs
, in vitro association
experiments were performed employing the purified EGF receptor and
Gs
. Since Gs
is phosphorylated by the EGF
receptor (12), experiments were performed wherein, after
phosphorylation of Gs
, either the EGF receptor or the
Gs
were immunoprecipitated, and 32P-labeled
proteins in the immunoprecipitate were detected by autoradiography following separation by SDS-PAGE. As demonstrated by the data in Fig.
3, immunoprecipitation of the EGF receptor resulted in co-immunoprecipitation of Gs
. Likewise
immunoprecipitation of Gs
was accompanied by the
presence of EGF receptor. In controls performed with either an
irrelevant monoclonal antibody (BBC-4) or nonimmune serum, neither EGF
receptor nor the Gs
was immunoprecipitated (Fig.
3A). Similarly, in additional control experiments with EGF
receptor or Gs
alone in the incubation mixture, the
anti-EGF receptor antibody (EGFR1) did not immunoprecipitate Gs
, and the anti-Gs
antiserum (CS1) did
not immunoprecipitate EGF receptor (see e.g. Fig.
3B). The requirement for ATP to observe association between
the EGF receptor and Gs
in experiments with cardiac
membranes (Fig. 1) would suggest that autophosphorylation of the EGF
receptor is important for interaction with the G protein. Therefore, to
further elucidate the role of phosphorylation of EGF receptor and/or
Gs
, if any, in association of the two proteins,
incubations were performed in the presence or absence of unlabeled ATP.
Following immunoprecipitation of EGF receptor, the Gs
in
the immunoprecipitate was detected by Western analysis with CS1
antiserum. The data in Fig. 3B demonstrate that the amount of Gs
co-immunoprecipitated with EGF receptor was the
same whether or not ATP was present; the phosphorylation states of Gs
and EGF receptor in these experiments were confirmed
by probing the Western blot with anti-phosphotyrosine antibodies (not
shown). Thus, the data in Fig. 3B demonstrate that the
association of the EGF receptor with Gs
is independent
of ATP and the phosphorylation state of the EGF receptor and
Gs
. Notably, the purified EGF receptor preparation
contains EGF (~5-8 µM), and therefore, experiments in
the absence of the growth factor are not possible. However, since the
purified EGF receptor which is not tyrosine phosphorylated does not
require ATP to associate with Gs
, it would appear that
the ligand bound, nonphosphorylated, pure EGF receptor is already in a
conformation in which the site(s) on the receptor are accessible for
binding the G protein. Conversely, in intact cardiac membranes, the
presence of ATP is required to observe EGF- dependent association of
the G protein with the receptor (Fig. 1). These latter data suggest
that in its native membrane environment, as demonstrated by Cadena
et al. (21), the EGF receptor changes its conformation upon
autophosphorylation and assumes a structure in which the
Gs
binding sites are accessible.
The experimental strategy described in Fig. 3A also allowed
the determination of stoichiometry of the EGF receptor and
Gs association. In these studies, the EGF receptor (33 ng) and Gs
(500 ng) were incubated in the presence of
[
-32P]ATP, and phosphorylation of both proteins was
allowed to reach stoichiometry as described previously (12). An aliquot
of the incubations was directly applied to SDS-PAGE, and following
separation of known amounts of proteins, the stoichiometry of
phosphorylation of each protein was calculated. This also allowed the
determination of specific radioactivity of each protein. Consistent
with our previous report (12) the stoichiometry of phosphorylation of Gs
was 2 mol of Pi/mol of Gs
,
and that of the EGF receptor was 4.5 mol of Pi/mol of
receptor. The remainder of the incubation was immunoprecipitated with
the anti-EGF receptor antibody, and following separation of proteins by
SDS-PAGE, the radioactivity associated with EGF receptor and
Gs
was determined. By dividing the radioactivity
associated with EGF receptor and Gs
in the
immunoprecipitate by the specific radioactivity of each protein (obtained from the stoichiometry of phosphorylation), the amount of
each protein in the immunoprecipitate was calculated. By this method,
the stoichiometry of association between the two proteins was
determined to be 0.9 mol of Gs
associated with 1 mol of
EGF receptor, i.e. the association between the receptor and Gs
is stoichiometric. Because the phosphorylation states of the EGF receptor and Gs
were not important for
association of these proteins (Fig. 3B), all subsequent
studies were performed in the absence of ATP.
The juxtamembrane region of the EGF receptor activates Gs
(11) and this region of the receptor has also been reported to bind
proteins and determine substrate specificity with respect to the
proteins that are phosphorylated (13-15). Moreover, our data from the
two-hybrid assay suggested that the juxtamembrane region of the EGF
receptor is important for association with Gs (Fig. 2).
Therefore, additional in vitro association studies were aimed at elucidating whether or not the juxtamembrane region of the EGF
receptor is important for physical interactions with Gs
. In our approach we employed peptides EGFR-13 and EGFR-14, which correspond to amino acids 645-657 and 679-692 in the juxtamembrane domain of the EGF receptor (11). We have previously demonstrated that
EGFR-13 is a potent activator of Gs (11). As demonstrated by the data in Fig. 4A, in the presence of
EGFR-13 and EGFR-14 the amount of Gs
which
co-immunoprecipitated with the EGF receptor was markedly diminished.
Densitometric analyses demonstrated a 95% decrease in association in
the presence of EGFR-13 and EGFR-14. This decrease in EGF
receptor-Gs
association was not the result of decreased
EGF receptor immunoprecipitation, since the latter remained constant as
determined by Western analysis of the immunoprecipitate with the
anti-EGF receptor antibody (Fig. 4B). Previously we have
shown that phosphorylation of EGFR-13 on threonine residue
corresponding to Thr654 in the EGF receptor diminishes its
ability to activate Gs and stimulate adenylyl cyclase (11).
Likewise, phosphorylation of EGFR-13 (P-EGFR-13) also abolished the
ability of the peptide to compete for association of Gs
with the EGF receptor (Fig. 4D). These findings demonstrate
that the effects of EGFR-13 are specific. Since both EGFR-13 and
EGFR-14 are basic peptides, additional controls were performed with
polylysine and polyarginine (Fig. 4D). Neither polyarginine
nor polylysine competed for the association between EGF receptor and
Gs
(Fig. 4D). These findings, along with the
observation that neither a peptide corresponding to amino acids
985-996 of the EGF receptor (Fig. 4C) nor three other
peptides corresponding to sequences in Gs
(discussed
later) decreased the association of EGF receptor with
Gs
, demonstrate that the effects of EGFR-13 and EGFR-14
are specific. Thus the data with EGFR-13 and EGFR-14 demonstrate that
the juxtamembrane region of the EGF receptor (amino acids 645-692) is
important for association with Gs
. Previously we
demonstrated that micromolar concentrations of EGFR-13 are required to
activate Gs (11). However, in the present study millimolar
concentrations of the peptide are required to compete for the
association of EGF receptor with Gs
(Fig. 4,
A and C). This difference in concentration
probably relates to the fact that in the Gs activation
studies (11) only the peptide and Gs were present. On the
other hand, in the present study, the peptide is being utilized to
compete for association between the receptor and Gs
.
Thus it would appear that the affinity of the full-length EGF receptor
for Gs
is high, and therefore, high concentrations of a
peptide corresponding to a sequence within the receptor are required
for competition. Previously we have also shown that the 13-amino acid
region (EGFR-13; amino acids 645-657), but not the 14-amino acid
region (EGFR-14; amino acids 679-692), is important for activation of
Gs by the EGF receptor (11). This coupled with the
competition studies with the peptides EGFR-13 and EGFR-14 (Fig. 4,
A and C) suggest that within the juxtamembrane
domain of the EGF receptor, regions which activate Gs
(e.g. amino acids 645-657) (11) as well as other regions
not involved in activation of Gs (e.g. amino
acids 679-692) (11) participate in the association.
Although the studies with peptides corresponding to the juxtamembrane
region of the EGF receptor suggest that this region of the receptor is
important for association with Gs (Fig. 4, A
and C) these experiments do not completely rule out the
participation, if any, of the C terminus region of the receptor that
harbors the autophosphorylation sites (24-26) to which SH2
domain-containing proteins such as Shc and phospholipase C
bind (3,
4). Therefore, to determine whether or not the C terminus of the EGF
receptor is involved in Gs
association, experiments were
performed with the purified full-length and truncated EGF receptor
(
1022-1186). In the truncated receptor all amino acids after
threonine 1022 are deleted. In these studies equal amounts of the
full-length receptor and truncated receptors were employed. As shown in
Fig. 4C, the amount of Gs
co-immunoprecipitated with the truncated receptor was the same as that
associated with the full-length EGF receptor. Moreover, a peptide
corresponding to amino acids 985-996 of the EGF receptor did not
decrease association between EGF receptor and Gs
(Fig.
4C). Therefore, the data in Fig. 4 demonstrate that the
juxtamembrane region, but not the C terminus region, of the EGF
receptor is involved in its association with Gs
.
The importance of the juxtamembrane region of the EGF receptor in
association with Gs (Fig. 4) and activation of
Gs by this region (11) would suggest that upon activation
of Gs, the
-subunit does not associate with the
receptor. Although some support for this contention is provided by the
data with yeast two-hybrid assay (Fig. 2, A, B,
and D), additional experiments with purified proteins were
performed to determine whether or not the active, GTP
S-bound, form
of Gs
associates with the EGF receptor. As demonstrated
by the data in Fig. 5, in immunoprecipitates of the EGF
receptor, in the presence of GTP
S (1 µM), the
co-immunoprecipitation of Gs
was reduced by an average
of 85%. GTP
S did not alter the immunoprecipitation of the EGF
receptor (not shown). Notably, the effects of GTP
S are specific
since neither GDP (1 µM, Fig. 5) nor another nucleotide
such as ATP (Fig. 3) affected the co-immunoprecipitation of
Gs
with the EGF receptor. The data in Fig. 5 are also consistent with the findings from the two-hybrid assay which compared the association of the wild type and constitutively active
Gs
with EGF receptor (Fig. 2) and demonstrate that upon
activation, Gs
is no longer associated with the EGF
receptor.
Employing chimeric proteins, antibodies, and peptides, several studies
have shown that the C terminus region of Gs is important
for activation of the G protein by receptors (see e.g. Refs.
8, 24, and 25). Indeed, employing CS1 antiserum, which is directed
against the C terminus decapeptide of Gs
, we have
previously shown that this region of Gs
is important for
its activation by the EGF receptor and
-adrenergic receptors (8).
More recently, employing peptides, studies from Hamm's laboratory have
shown that the activation of Gs
by
-adrenergic receptors can be obliterated by peptides corresponding to the C
terminus of Gs
. Therefore, to determine whether or not the C terminus regions of Gs
is also important for
association with EGF receptor, in experiments similar to those
described in Fig. 4, the ability of peptides corresponding to amino
acid residues 15-29, 354-372, and 371-380 of Gs
to
compete for the association of the EGF receptor with Gs
was investigated. Although the C terminus of Gs
is
important for its activation by EGF receptor in terms of stimulation of
adenylyl cyclase (8), none of the peptides tested altered the
association of Gs
with the EGF receptor (data not
shown). These data (not shown) indicate that the region of the
Gs
molecule which is involved in association is not
necessarily the same region that is important for activation of this G
protein by receptors in terms of mediating a signal. Moreover, our
findings that the three peptides corresponding to sequences in
Gs
at concentrations as high as 1 mM did not alter association of the EGF receptor with Gs
(not
shown) indicate that the effects of EGFR-13 and EGFR-14 as observed in Fig. 4 are specific. Three-dimensional structure studies of the transducin and Gi
proteins have demonstrated that the N
terminus of the
-subunits of heterotrimeric G proteins interacts
with the
-subunits (26, 27). Additionally, the N terminus region of
-subunits of G proteins has been implicated to interact with receptors (26). However, the inability of a peptide corresponding to
amino acids 15-29 of Gs
to compete for association
between the EGF receptor and Gs
(not shown) suggests
that the region of Gs
, which is important in
interactions with
-subunits (26, 27) and which may interact with
receptors, is not the region important in association with the EGF
receptor. Given our observation that GTP
S decreases the association
of Gs
with the EGF receptor and the findings from the
three-dimensional structures of G protein
-subunits (28, 29) that
GTP binding most markedly changes the conformation in the three switch
regions (28, 29), it is tempting to speculate that perhaps one of the
switch regions and/or a domain proximal to this switch is involved in
association with the EGF receptor. Whatever the case, presently, the
precise region of Gs
which associates with EGF receptor
remains unknown and forms the subject of further investigations.
The association of single transmembrane protein tyrosine kinase
receptors with adapter proteins that participate in the signaling to
serine/threonine kinases has been well documented (see Refs. 30 and 31
for reviews). However, the interactions of this family of receptors
with G protein-mediated processes has been less well defined. Likewise,
although a large number of studies have demonstrated that regions in G
protein -subunits or domains in heptahelical receptors which
activate these G proteins are important for transducing signals (8,
24-29, 32, 33), to date the association of G protein
-subunits with
heptahelical receptors has only been demonstrated for the angiotensin
AT2 receptor (34). However, even in the case of the angiotensin AT2
receptor, the region of the receptor that associates with
Gi
2 and Gi
3 remains unknown (34).
Moreover, whether or not the association of AT2 receptor with
Gi
2 and Gi
3 is direct or involves another
protein also remains to be investigated. In this respect, the data
presented in this study are the first to show the direct, and
stoichiometric, association of a G protein
-subunit with a receptor
which can activate this G protein (8, 11, 12). Although the association
of Gi
with EGF receptor has been suggested (35), in that
study, phospholipase C
was shown to be associated with EGF receptor
as well as Gi
. Therefore, whether the association of
Gi
with the EGF receptor is direct or indirect via
phospholipase C
remains unknown. Similarly, although Nishimoto
et al. (36) have demonstrated the association of
Go with amyloid precursor protein (APP), heterotrimeric
Go was demonstrated to be associated with APP, and
therefore, whether or not the Go
-subunit associates
directly with APP is also not known. Since the regions within the EGF
receptor and the
-adrenergic receptors that activate Gs
are similar (11, 32) and because the region (EGFR-13) in the EGF
receptor that activates Gs also modulates association of
the receptor with Gs
, it is tempting to speculate that the similar motif in the third cytosolic loop of the
-adrenergic receptor would also be involved in association of that receptor with
Gs
.
Interestingly, both EGFR-13 and EGFR-14 compete for the association
between EGF receptor and Gs. Since the association of
the two proteins is stoichiometric (1 mol of Gs
:1 mol of
EGF receptor), it is possible that there are at least two contact sites
for Gs
on the EGF receptor; one site at amino acids 645-657 (EGFR-13) and the other at amino acids 679-692 (EGFR-14). Moreover, because the addition of either EGFR-13 or EGFR-14 effectively decreased the association between EGF receptor and Gs
,
it would appear that the loss of contact of Gs
at one of
the two sites on the EGF receptor diminishes the affinity for the other
site. Alternatively, in the presence of one of the peptides corresponding to the EGF receptor juxtamembrane domain, the
conformation of the Gs
is altered so that it loses the
ability to bind at any point on the juxtamembrane region of the EGF
receptor. The identification of the critical amino acid residues
involved in association with the Gs
will be facilitated
by future experiments involving site-directed mutagenesis of the
receptor.
In conclusion, the studies described herein demonstrate that the
cytosolic, juxtamembrane region of the EGF receptor encompassing sequences corresponding to EGFR-13 and EGFR-14 (48 amino acids; Arg645-Lys692) is important for the direct,
and stoichiometric, association with Gs
. Since the first
13 amino acids in this region (Arg645-657) are also
important for activation of Gs (11) and because the EGF
receptor protein tyrosine kinase can phosphorylate Gs
on
tyrosyl residues (12), it is possible that association of Gs
with the juxtamembrane region of the EGF receptor is
important for its activation. Further support for the latter contention is derived from the observation that expression of the constitutively active form of Gs
in the two-hybrid assay (Fig. 2) or
the addition of GTP
S (Fig. 5) decreases the interaction of EGF
receptor with Gs
. Another interesting finding from the
experiments described herein is that the region on Gs
that is involved in association with the EGF receptor is different from
the region (C terminus), which is important for activation of
Gs
by receptors in terms of mediating signals to
adenylyl cyclase (8). The definition of the precise regions on
Gs
which associate with the EGF receptor forms the
subject of future experiments.
We are greatly indebted to the following
individuals for providing the indicated reagents and cDNAs: Dr.
Alfred G. Gilman (University of Texas Southwestern Medical Center,
Dallas, TX) for the BL21(DE-3) for the strain of E. coli
transformed with plasmid pQE60 containing cDNA for expression of
Gs; Dr. Gordon Gill (University of California at San
Diego, CA) for the EGF receptor cDNA; Dr. Heidi E. Hamm (University
of Illinois College of Medicine, Chicago) for the peptides
corresponding to N and C terminus of Gs
; and Dr. Graeme
Milligan (University of Glasgow, Scotland) for providing us with
anti-Gs
antiserum, CS1.