(Received for publication, November 2, 1995)
From the
G-protein-linked receptors and intrinsic tyrosine-kinase growth
receptors represent two prominent modalities in cell signaling.
Cross-regulation among members of both receptor superfamilies has been
reported, including the counter-regulatory effects of insulin on
-adrenergic catecholamine action. Cells stimulated by insulin show
loss of function and increased phosphotyrosine content of
-adrenergic receptors. Phosphorylation of tyrosyl
residues 350/354 of
-adrenergic receptors is
obligatory for counter-regulation by insulin (Karoor, V.,
Baltensperger, K., Paul, H., Czech, M., and Malbon, C. C.(1995) J.
Biol. Chem. 270, 25305-25308), suggesting the hypothesis
that G-protein-linked receptors themselves may act as substrates for
the insulin receptor and other growth factor receptors. This hypothesis
was evaluated directly using recombinant human insulin receptor,
hamster
-adrenergic receptor, and an in vitro reconstitution and phosphorylation assay. Insulin is shown to
stimulate insulin receptor-catalyzed phosphorylation of the
-adrenergic receptor. Phosphoamino acid analysis
establishes that insulin receptor-catalyzed phosphorylation of the
-adrenergic receptor in vitro is confined to
phosphotyrosine. High pressure liquid chromatography and
two-dimensional mapping reveal insulin receptor-catalyzed
phosphorylation of the
-adrenergic receptor at
residues Tyr
/Tyr
,
Tyr
/Tyr
, and Tyr
, known sites
of phosphorylation in response to insulin in vivo.
Insulin-like growth factor-I receptor as well as the insulin receptor
displays the capacity to phosphorylate the
2-adrenergic receptor in vitro, establishing a new paradigm, i.e. G-protein-linked receptors acting as substrates for intrinsic
tyrosine kinase growth factor receptors.
Protein phosphorylation plays a prominent role in the regulation
of cell signaling. A populous family of G-protein-linked receptors
mediate activation of a diverse class of effectors, such as adenylyl
cyclase, phospholipase C, and various ion channels, via a less populous
class of G-proteins(1) . These G-protein-linked receptors share
many features, including regulation via protein
phosphorylation(2, 3) . Insulin counter-regulates the
action of -adrenergic catecholamine stimulation, at a point
proximal to the
-adrenergic receptor(4) . Cells stimulated
by insulin show increased phosphotyrosine content and loss of function
of
-adrenergic receptors (4) . The insulin
receptor, upon ligand binding, expresses intrinsic tyrosine kinase
activation(5) , raising the intriguing hypothesis that
G-protein-linked receptors and intrinsic tyrosine kinase growth
receptors may interact directly, the former a substrate for the latter.
Recently, we have deduced structural information on sites of
phosphotyrosine labeling in vivo(6) . In the current
work, we directly test the hypothesis that the
-adrenergic receptor is a substrate for growth factor
receptor tyrosine kinase, using recombinant receptors and a defined
reconstitution assay in vitro. The results demonstrate that
growth factor tyrosine kinase receptors (e.g. insulin receptor
and the IGF-I (
)receptor) can directly phosphorylate a
G-protein-linked receptor.
In cultured smooth muscle cells, insulin impairs the function
and increases the phosphotyrosine content of AR (4) . To address the possibility that a G-protein-linked
receptor might serve as a substrate for an intrinsic tyrosine kinase
growth receptor, we reconstituted the two receptors in vitro.
rIR was isolated and partially purified from a Chinese hamster ovary
cell line (CHO-T) that stably overexpresses the human insulin
receptor(10) . This rIR preparation reduced levels of uncleaved
proreceptor and showed marked increase of rIR
-subunit
phosphorylation upon insulin stimulation (Fig. 1, lanes 1 and 2). Marked insulin-stimulated phosphorylation of the
r
AR (M
, 45,000) as well as of the rIR
-subunit (M
, 86,000) was observed following
reconstitution of purified r
AR expressed in
baculovirus-infected Sf9 cells and rIR purified from CHO-T cells in the
presence of detergent (Fig. 1, lanes 3 and 4).
No phosphorylation of r
AR was evident in reconstitutions performed
in the absence of rIR. Phosphorylation of some incompletely processed
proreceptor (M
, 180,000) present in the IR
preparations was observed occasionally as shown here.
Insulin-stimulated phosphorylation of r
AR was observed in
reconstitutions with lectin-purified IR from either CHO-T cells or a
second source, SV40-transformed African green monkey kidney COS-1
cells, which were transiently transfected with the human insulin
receptor cDNA (not shown).
Figure 1:
Direct phosphorylation of rAR
catalyzed by the insulin receptor tyrosine kinase in vitro in
response to insulin stimulation. Recombinant human insulin receptor (5
µl) from CHO-T cells (lanes 1-4) was reconstituted
with r
AR (lanes 3 and 4) (20 µl) in the
absence (lanes 1 and 3) or the presence (lanes 2 and 4) of insulin (INS; 100 nM) and
incubated for 30 min at 22 °C with
[
-
P]ATP (5 µM) as described
under ``Materials and Methods.'' Phosphorylated proteins were
then separated by SDS-PAGE and visualized by autoradiography of the
dried gel. proIR, proinsulin
receptor.
Phosphoamino acid analysis of the
rAR and rIR products from an in vitro phosphorylation
reaction revealed phosphotyrosine labeling of the r
AR, whether
performed with rIR expressed in CHO-T (Fig. 2, lanes 1 and 2) or COS-1 (lanes 3 and 4) cells.
The rIR
-subunit was predominantly phosphorylated on tyrosine
residues (Fig. 2, lanes 5 and 6), providing an
internal control. Some phosphoserine in the rIR
-subunit was
detected, which is due to low level serine kinase activity of the IR
tyrosine kinase(9) . Similarly, upon overexposure of the
autoradiography, some phosphoserine could be detected in phosphorylated
r
AR (not shown), which is consistent with the hypothesis that the
insulin receptor directly interacts with and phosphorylates the
r
AR.
Figure 2:
Phosphoamino acid analysis of rbAR
phosphorylated in vitro by rIR reveals increased
phosphotyrosine content. Lanes 1 and 2, rAR
phosphorylated by rIR expressed in CHO-T cells. Lanes 3 and 4, r
AR phosphorylated by rIR expressed in COS-1 cells. Lanes 5 and 6,
-subunit of rIR expressed in
CHO-T cells. The migration of unlabeled phosphoamino acids is
demarcated in the margin: Tyr(P), phosphotyrosine; Thr(P), phosphothreonine; Ser(P),
phosphoserine.
We explored whether the -adrenergic
receptor was a substrate for a second growth factor-activated tyrosine
kinase receptor, the IGF-I receptor, which is structurally closely
related to the IR(14) . We assayed the ability of insulin to
stimulate phosphorylation of r
AR in vitro in
reconstitution studies with IGF-IR purifed from human osteogenic
sarcoma cell extracts (Fig. 3, lanes 2-5) and
compared it with the phosphorylation of r
AR by rIR (Fig. 3, lane 1, and Fig. 1). The IGF-1R phosphorylated r
AR
in response to stimulation by high concentrations (1 µM)
of insulin (lane 3). IGF-1R-dependent phosphorylation of the
45,000 M
protein was absent in the reconstitutions
devoid of r
AR (lanes 4 and 5). A small amount of
phosphorylation was observed in the lectin-purified extract rich in
IGF-IR in the absence of hormonal stimulation (lane 3), which
may reflect phosphorylation catalyzed by platelet-derived growth factor
receptor, which was activated (autophosphorylated) in this fraction
(see *, Fig. 3, lanes 2-5). These observations
demonstrate that tyrosine kinase growth factor receptors, in addition
to the IR, can catalyze specific phosphorylation of r
AR in
response to insulin.
Figure 3:
Direct phosphorylation of the rAR in vitro by the IGF-I receptor as well as insulin receptor.
Recombinant IR from CHO-T cells (lane 1) or the glycoprotein
fraction from human osteogenic sarcoma cells (lanes
2-5), which is highly enriched in IGF-1 receptor were
reconstituted as described above (Fig. 1) in the presence of
[
-
P]ATP with (lanes 1-3) or
without (lanes 4 and 5) r
AR in the absence (lanes 2 and 4) or the presence of insulin (INS; lane 1, 100 nM insulin; lanes 3 and 5, 1 µM insulin to stimulate the IGF-1R
tyrosine kinase activity). Phosphoproteins were separated by SDS-PAGE
and visualized by autoradiography of the dried gel. The extent of the
phosphorylation of r
AR catalyzed by rIR and IGF-IR was quantified
by excising gel pieces containing the tyrosine kinase receptors and
r
AR. *, represents the phosphorylated/activated form of the
platelet-derived growth factor receptor, as established by
immunoblotting of the gels with anti-platelet-derived growth factor
antibody (not shown).
In an effort to explore the site(s) for
insulin-stimulated, rIR-catalyzed phosphorylation, recently we prepared
synthetic peptides corresponding to cytoplasmic regions of the
AR that harbors tyrosyl residues, i.e. Tyr
, Tyr
, Tyr
,
Tyr
, and Tyr
(6) , and analyzed
their potential as substrates for rIR. For the in vitro assay(6) , no labeling of peptides by rIR was observed in
the absence of insulin. Insulin (10 nM)-stimulated
rIR-catalyzed phosphorylation of
2-adrenergic receptor peptides
was found prominently in peptides L339 (Tyr
and
Tyr
), T362 (Tyr
), and to a lesser extent
peptides Y132 (Tyr
and Tyr
), and I135
(Tyr
).
The synthetic peptides were designed not only
to probe all cytosolic tyrosyl residues available for phosphorylation
by IR but also to provide a source of tryptic fragments in which the
candidate sites for tyrosine kinase phosphorylation were
imbedded(6) . Maps of tryptic digests might permit analysis of
the sites phosphorylated on the AR in response to
insulin in vitro (Fig. 4). Tryptic digests of peptides
phosphorylated in vitro by rIR in response to insulin provided
markers for HPLC analysis (Fig. 4, A-C). The
retention times for the tryptic fragments subjected to HPLC separation
agreed well with the retention times calculated from the sequence
information (not shown). For tryptic digests of phosphorylated I135
peptide, uncleaved peptide was detected routinely (*, Fig. 4A). Tryptic digests of phosphopeptide Y132
display the same mobility as the fragments of I135 (not shown), i.e. fraction 4 (Fig. 4A). The Y132 peptide
was shown previously to be the preferred substrate for
insulin-stimulated, rIR-catalyzed phosphorylation(6) . Using
these labeled standards, we established that
AR
reconstituted in vitro with rIR in the presence of insulin was
phosphorylated predominantly on peptides harboring residues
Tyr
/Tyr
,
Tyr
/Tyr
, and Tyr
(Fig. 4D). Phosphorylation of the
r
AR by rIR in vitro was not detected in the
absence of insulin.
Figure 4:
Reverse-phase HPLC mapping of the
-adrenergic receptor tryptic digests demonstrates
insulin-stimulated rIR-catalyzed phosphorylation of tyrosyl residues
350, 354, and 364 in vitro. A, HPLC analysis of
tryptic fragments from peptide I135 (ITSPFKYQSLLTKNKAR) harboring
Tyr
following in vitro, insulin-stimulated
rIR-catalyzed phosphorylation, B, HPLC analysis of tryptic
fragment of L339 (AYGNGYSSNSNGK) harboring
Tyr
/Tyr
following in vitro insulin-stimulated rIR-catalyzed phosphorylation of L339; C, HPLC analysis of tryptic fragment T362 (TDYMGEASGCQLGQEK)
harboring Tyr
following in vitro insulin-stimulated rIR-catalyzed phosphorylation. D, HPLC
analysis of tryptic fragments of r
AR following
reconstitution with and insulin-stimulated phosphorylation in vitro by rIR. *, represents phosphopeptide I135 resistant to tryptic
digestion.**, represents a small and variable amount of
disulfide-bridged tryptic peptide of Tyr
, reflecting the
presence of a cysteinyl residue in the fragment. The standards for
tryptic digests of L339 and T362 reported earlier (6) are
included for reference.
High voltage electrophoresis followed by
thin-layer chromatography of the tryptic fragments (Fig. 5, A-C) provides additional markers for analysis of
phosphopeptides derived from the AR phosphorylated by
insulin-stimulated rIR using the in vitro, reconstitution
assay (Fig. 5D). The two-dimensional analysis extends
the results of reverse-phase HPLC, establishing that the predominant
sites of insulin-stimulated phosphorylation catalyzed by the rIR in
vitro are Tyr
/Tyr
,
Tyr
/Tyr
, and Tyr
(Fig. 5D). Peptides harboring Tyr
and/or Tyr
are substrates for rIR-catalyzed
phosphorylation in response to insulin stimulation, the peptide
harboring both Tyr
and Tyr
being the
preferred substrate(6) . The analysis for I135 only is
displayed (Fig. 5A), being representative of tryptic
fragments from Y132 also, which behave identically with those of I135
in both the HPLC and two-dimensional analyses (not shown).
Two-dimensional analysis of tryptic fragments of
AR
phosphorylated by rIR in vitro revealed labeling of
Tyr
/Tyr
, as shown here. Thus, in vitro phosphorylation of the
AR by the IR tyrosine
kinase is confined to Tyr
/Tyr
,
Tyr
/Tyr
, and Tyr
.
Figure 5:
Two-dimensional phosphopeptide mapping of
the -adrenergic receptor demonstrates insulin-stimulated
rIR-catalyzed phosphorylation of tyrosyl residues 350, 354, and 364 in vitro. A, high voltage electrophoresis and
thin-layer chromatography two-dimensional analysis of tryptic fragment
I135 (ITSPFKYQSLLTKNKAR) harboring Tyr
following in
vitro insulin-stimulated rIR-catalyzed phosphorylation. B, high voltage electrophoresis and thin-layer chromatography
two-dimensional analysis of tryptic fragment of L339 (AYGNGYSSNSNGK)
harboring Tyr
/Tyr
following in vitro insulin-stimulated rIR-catalyzed phosphorylation. C, high
voltage electrophoresis and thin-layer chromatography two-dimensional
analysis of tryptic fragment T362 (TDYMGEASGCQLGQEK) harboring
Tyr
following in vitro insulin-stimulated
rIR-catalyzed phosphorylation. D, high voltage electrophoresis
and thin-layer chromatography two-dimensional analysis of tryptic
fragments of r
AR following reconstitution with and
insulin-stimulated phosphorylation by rIR in vitro. The
standards for tryptic digests of L339 and T362 reported earlier (6) are included for reference.
In the
current work we exploited the ability to prepare rIR, rbAR, and
IGF-IR-enriched fractions from human osteogenic sarcoma cells to
directly test the hypothesis that a G-protein-linked receptor can act
as a substrate for an intrinsic tyrosine-kinase growth factor receptor.
The following observations provide compelling evidence in support of
this hypothesis: (i) insulin stimulates rIR-catalyzed phosphorylation
of the rAR in a reconstituted, in vitro assay; (ii) insulin-stimulated phosphorylation of the
r
AR in vitro is confined to tyrosyl residues;
(iii) the sites phosphorylated in vitro in response to insulin
are those phosphorylated in vivo, as determined by structural
analysis of
AR isolated from metabolically labeled
cells; (iv) insulin at high concentrations stimulates in vitro phosphorylation of r
AR catalyzed by purified
IGF-IR; and (v) mutagenesis of these tyrosyl residues of the
AR in vivo results in loss of
insulin-stimulated counter-regulation of
AR
function(6) . In addition, the bradykinin receptor has been
shown to be phosphorylated on tyrosyl residues in response to
serum(15) , and the angiotensin II AT1 receptor has been
reported to be a substrate for phosphorylation by the src family of tyrosine kinases(16) . Based upon these
observations we propose a new paradigm in which two prominent pathways
in cell signaling cross-talk to each other at the most proximal point,
receptor to receptor.
Interestingly, the sites on the
AR phosphorylated by the IR and IGF-IR include a well
known motif for tyrsoine kinase growth factor receptors at
Tyr
(17) , a prominent GRB2 site at
Tyr
(6, 18) , and a potential SHC binding
site at Tyr
(19) . The co-migration of the tryptic
fragments harboring Tyr
and Tyr
preclude
definition of the contribution of each to phosphotyrosine labeling by
the IR, either in vitro (present study) or in
vivo(6) . Insulin-stimulated, IR-catalyzed phosphorylation
of peptide Y132, which harbors Tyr
/Tyr
,
however, was prominent, whereas that for peptide I135, which lacks
Tyr
, was decidely poor(6) . Further analysis of
this site will be required, because phosphorylation of Tyr
creates a Shc binding site, and this site is conserved among many
G-protein-linked receptors, including receptors for neuropeptide Y,
tachykinin, A2-adenosine, thyrotropin releasing factor, and serotonin
(Genebank). Although speculation, the full range of adaptor molecules
known to play a prominent role in tyrosine kinase receptor signaling
may be made available to G-protein-linked receptors, once
phosphorylated by a tyrosine kinase activated in response to a growth
factor.