(Received for publication, July 26, 1995; and in revised form, August 23, 1995)
From the
Insulin stimulates a loss of function and increased
phosphotyrosine content of the -adrenergic receptor in
intact cells, raising the possibility that the
-receptor itself is a substrate for the insulin
receptor tyrosine kinase. Phosphorylation of synthetic peptides
corresponding to cytoplasmic domains of the
-adrenergic receptor by the insulin receptor in
vitro and peptide mapping of the
-adrenergic
receptor phosphorylated in vivo in cells stimulated by insulin
reveal tyrosyl residues 350/354 and 364 in the cytoplasmic, C-terminal
region of the
-adrenergic receptor as primary targets.
Mutation of tyrosyl residues 350, 354 (double mutation) to
phenylalanine abolishes the ability of insulin to counterregulate
-agonist stimulation of cyclic AMP accumulation. Phenylalanine
substitution of tyrosyl reside 364, in contrast, abolishes
-adrenergic stimulation itself.
The counterregulatory effects of insulin and catecholamines on
carbohydrate and lipid metabolism are well known, whereas the molecular
details of insulin regulation of G-protein-linked pathways remain
unknown. Upon ligand binding, the insulin receptor displays tyrosine
kinase activity which is critical to signal propagation(1) .
G-protein-linked receptors (like the -adrenergic
receptor,
AR),
in contrast, activate
adenylyl cyclase via G
and are phosphorylated during
agonist-induced desensitization(2, 3) . We
demonstrated recently that the well known counterregulatory actions of
insulin included loss of function and increased phosphorylation of the
-adrenergic receptor(4) . In the current study
the structural basis for these counterregulatory effects of insulin
exerted on the
-adrenergic receptor is explored.
In an effort to explore the site(s) for insulin-stimulated
phosphorylation of the AR, we prepared synthetic
peptides to corresponding to each cytoplasmic regions of the
AR that harbors candidate tyrosyl residues, i.e. Tyr-70, Tyr-132, Tyr-141, Tyr-350, Tyr-354, and Tyr-364, and
analyzed their potential as substrates for rIR (Fig. 1). The
AR peptides were reconstituted with rIR in the absence
(not shown) or presence of insulin (100 nM). For the in
vitro assay, no labeling of peptides by rIR was observed in the
absence of insulin. Insulin-stimulated phosphorylation of the peptides
by the rIR was compared, after electrophoretic separation of the
labeled products from the rIR (Fig. 2A). Insulin
stimulated rIR-catalyzed phosphorylation of peptides L339 (Tyr-350 and
Tyr-354), T362 (Tyr-364), and to a lesser extent peptides Y132 (Tyr-132
and Tyr-141) and I135 (Tyr-141).
Figure 1:
Topological
model of -adrenergic receptor, highlighting candidate
tyrosyl residues for insulin-stimulated phosphorylation. A,
model of
AR organization and location of synthetic
sequences used as substrates for insulin-stimulated rIR-catalyzed
phosphorylation of the
AR and as precursors for
tryptic fragments used as markers for peptide mapping of the labeled
tyrosyl residues. B, synthetic peptides used to map
cytoplasmic tyrosyl residues for phosphorylation by activated tyrosine
kinase growth factor receptors. Six sequences were selected to be used
as substrates for insulin-stimulated rIR-phosphorylation. The peptides
were designed as substrates for phosphorylation as well as a source of
markers for reverse-phase HPLC and two-dimensional mapping of tryptic
fragments. The derivative peptide fragments are
displayed.
Figure 2:
Synthetic peptides corresponding to
cytoplasmic sequences of the C terminus of the AR that
possess Tyr-350/Tyr-354 and Tyr-364 (L339 and T362, respectively) are
preferred substrates for phosphorylation by rIR. A,
insulin-dependent phosphorylation of
AR-peptides by
the rIR. rIR
,
-subunit of the recombinant IR. B, dose-response-relationship of
AR peptide
phosphorylation by the rIR. The synthetic peptides were incubated with
rIR in the presence of insulin (100 nM) at concentrations
indicated. Phosphorylation of the peptides was quantified and computed
as picomoles of phosphate/tyrosyl residue.
Since peptide L339 contains both Tyr-350 and Tyr-354, it was important to explore if one site, or the other, or both were phosphorylated in vitro by rIR in the presence of insulin. When L339 peptide analogs that carried phenylalanine substitutions at either Tyr-350 or Tyr-354 were reconstituted with rIR in the in vitro system, both analogs served equally well as substrates for insulin-stimulated phosphorylation (data not shown). Elimination of the YIA sequence of Y132 to create peptide I135 reduced insulin-stimulated, rIR-catalyzed phosphorylation of the residual peptide, suggesting either that Tyr-132 is the site of phosphorylation or that the YIA sequence is required for recognition/phosphorylation of the Tyr-141. The absence of labeling of the E122 peptide containing Tyr-132 and the detection of some label in tryptic fragments containing Tyr-141 supports the latter interpretation. Phosphopeptides from peptides R62 (Tyr-70) and E121 (Tyr-132) could not be detected, although the presence of the unphosphorylated peptides in the gel could be detected by silver staining. Phosphoamino acid analysis of the labeled peptides was performed, and for all phosphopeptides, labeling was confined to phosphotyrosine (not shown). These data demonstrate that residues Tyr-350, Tyr-354, and Tyr-364 (and to a lesser extent Tyr-141) are phosphorylated by the rIR tyrosine kinase in vitro.
The
efficacy of peptide phosphorylation was assessed by comparing the
amount of phosphate incorporated into each peptide by
insulin-stimulated rIR at various concentrations of peptides (Fig. 2B). Peptide L339 was clearly the best substrate
for the rIR. The ED for insulin-stimulated phosphorylation
of L339 peptide was
100 µM. At 3 mM concentrations of peptide, saturation of rIR-catalyzed
phosphorylation for the other peptides was not achieved, precluding the
calculation of ED
values for the other peptides. The rank
order of insulin-stimulated phosphorylation for the synthetic peptides
employed at 1 mM, from best to worst substrate, was L339
(Tyr-350 and Tyr-354) > T362 (Tyr-364) > Y132 (Tyr-132) I135
(Tyr-141). Phosphorylation of peptides R62 and E122, containing Tyr-70
and Tyr-132, respectively, was not detected.
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 (Fig. 1, A and B). Maps of tryptic digests might permit analysis of the sites
phosphorylated on the AR in response to insulin in
vivo (Fig. 1). Tryptic digests of peptides phosphorylated in vitro by rIR in response to insulin provided markers for
HPLC analysis (Fig. 3, A and B). The retention
times for the tryptic fragments subjected to HPLC separation agreed
well with the retention times calculated from the sequence information
(not shown).
Figure 3:
Reverse-phase HPLC phosphopeptide mapping
of the -adrenergic receptor demonstrates insulin-stimulated
rIR-catalyzed phosphorylation of tyrosyl residues 350, 354 and 364 in vivo. C-F, insulin promotes phosphorylation of the
-adrenergic receptor in metabolically labeled
DDT
MF-2 smooth muscle cells. Tryptic peptides of in
vitro labeled L339 (harboring Tyr-350 and Tyr-354) and of T362
(harboring Tyr-364) were employed as standards (panels A and B, respectively). Cells metabolically labeled with
[
P]orthophosphate (see ``Materials and
Methods'') were incubated for 20 min without (panels C and D) or with (panels E and F) 100
nM insulin. After lysis of the cells, the
AR
was immunoprecipitated, and the phosphorylated receptor isolated, and
then digested with trypsin. Reverse-phase HPLC analysis of the tryptic
fragments was performed as described under ``Materials and
Methods.'' Chromatograms from two separate experiments,
representative of five independent experiments, are displayed. The
label in fraction 30 of panel F was observed on occasion, but
contained no phosphotyrosine.
In vivo, metabolic labeling of
DDTMF-2 smooth muscle cells in culture with
[
P]orthophosphate revealed the phosphotyrosine
content of
AR to increase from 0.86 ± 0.10
(basal) to 1.76 ± 0.39 (n = 4) mol/mol receptor
in response to insulin (20 min, 100 nM). Some phosphotyrosine
was found in tryptic fragments harboring Tyr-350 and Tyr-354 of
AR isolated from cells in the absence of stimulation
by insulin (Fig. 3, C and D). In the presence
of insulin, increased phosphorylation of the
AR was
observed, confined largely to Tyr-350, Tyr-354, and Tyr-364 (Fig. 3, E and F). Insulin-stimulated
phosphorylation displayed two patterns in which labeling occurred
either at both Tyr-350/Tyr-354 and Tyr-364 (Fig. 3F) or
more prominently at Tyr-350/Tyr-354 with reduced labeling of Tyr-364 (Fig. 3E). Other peaks occasionally observed in the
HPLC profiles (e.g. fraction 30, Fig. 3D) were
subjected to phosphoamino acid analysis and found to contain no
phosphotyrosine. High voltage electrophoresis followed by thin-layer
chromatography of the tryptic fragments confirmed the identity of the
HPLC peaks (Fig. 4, A and B) and provided
additional markers for analysis of phosphopeptides derived from
insulin-stimulated rIR-catalyzed phosphorylation of receptor peptides (Fig. 4, C and D). The two-dimensional
analysis confirmed the results of reverse-phase HPLC, establishing that
the predominant sites of insulin-stimulated phosphorylation are
Tyr-350/Tyr-354, and to a lesser extent Tyr-364.
Figure 4:
Two-dimensional phosphopeptide mapping of
the -adrenergic receptor demonstrates insulin-stimulated
phosphorylation of tyrosyl residues 350, 354, and 364 in vivo. A-D, insulin promotes phosphorylation of the
-adrenergic receptor in metabolically labeled
DDT
MF-2 smooth muscle cells. Tryptic peptides of in
vitro-labeled L339 (harboring Tyr-350 and Tyr-354) and of T362
(harboring Tyr-364) were employed as standards (panels A and B, respectively). Cells metabolically labeled with
[
P]orthophosphate (see ``Materials and
Methods'') were incubated for 20 min without (data not shown) or
with (panels C and D) 100 nM insulin. After
lysis of the cells, the
AR was immunoprecipitated, and
the phosphorylated receptor isolated, and then digested with trypsin.
High voltage electrophoresis and thin layer chromatography of the
tryptic fragments was performed as described under ``Materials and
Methods.'' Analyses from two separate experiments are
displayed.
Site-directed
mutagenesis of the tyrosyl residues was performed to test independently
the role of Tyr-350/Tyr-354 and Tyr-364 in the counterregulatory
effects of insulin on the AR. Tyr-350/Tyr-354 (double
substitution) or Tyr-364 were mutated to phenylalanine and the mutant
receptor expressed in CHO cells. The
-adrenergic agonist
isoproterenol (1 µM) stimulated cyclic AMP accumulation in
CHO cells expressing wild-type and Y350F/Y354F mutant receptors, but
not the Y364F mutant
AR (Table 1). Tyrosine to
phenylalanine substitution of residues 350 and 354 abolishes the
ability of insulin to counterregulate
-agonist-stimulated cyclic
AMP accumulation in CHO cells. The Y364F mutation, in contrast,
abolishes isoproterenol-stimulated cyclic AMP accumulation itself.
These data, gathered from an independent approach, clearly highlight a
critical role of Tyr-350/Tyr-354 in expression of the counterregulatory
actions of insulin upon
AR.
Our results illuminate
cross-regulation among two major transmembrane signaling
pathways(4) . The recent report that bradykinin B2 receptors
isolated from WI-38 human lung fibroblasts in culture can be detected
by anti-phosphotyrosine antibodies (16) provides additional
evidence to support cross-talk from tyrosine kinase to G-protein-linked
receptors (4) . By study of rIR-catalyzed phosphorylation of
AR peptides in vitro, by peptide mapping of
AR phosphorylated in cells stimulated by insulin in vivo, and by site-directed mutagenesis studies,
AR tyrosyl residues 350, 354, and 364 are shown to be
sites of insulin-stimulated phosphorylation. The peptide sequences
flanking Tyr-364 suggest a growth factor tyrosine kinase recognition
motif(17) , which agrees well with our data implicating this
residue as a phosphorylation site for the IR tyrosine kinase.
Interestingly, the best peptide substrate, peptide L339, harbors
tyrosyl residue 350, which lies in a sequence motif (Tyr-Gly-Asn-Gly)
with similarity to motifs known to interact with Caenorhabditis
elegans sem5 Src homology 2 (SH2) domains when
phosphorylated(18) . A common feature of these motifs is an Asn
residue at position +2 from the tyrosine residue, while residues
+1 and +3 contain aliphatic side chains. It is tempting to
speculate that residue Tyr-350, once phosphorylated by IR tyrosine
kinase, constitutes a potential binding site for SH2-containing
proteins such as the mammalian homolog of sem5, GRB2.
AR and IR co-exist in a number of mammalian
tissues, including skeletal muscle and liver. How tyrosine kinase
receptor-catalyzed phosphorylation of G-protein-linked receptors, like
the
AR, contributes to physiological regulation
remains an important question, derivative of exciting results presented
herein. The data suggest that phosphorylation or mutagenesis of the
tyrosyl residues in this domain(350-364) impairs
AR function. Mutation of Tyr-350/Tyr-354 to alanine
has been shown to alter
AR coupling to
G
(19) , much like the Y364F mutations (this study).
Phosphorylation of Tyr-350/Tyr-354 by the IR is shown to impair G
coupling ( (4) and this study), but further analysis of
the multi-site phosphorylation of this receptor will be required making
use of the mutant cell lines developed herein. Taken together these
data provide strong evidence that this region is a regulatory domain of
the
AR involved in G-protein coupling and that it is
subject to covalent modification by counterregulatory, tyrosine kinase
receptors.