pp60Src mediates insulin-stimulated sequestration of the beta(2)-adrenergic receptor: insulin stimulates pp60Src phosphorylation and activation.

Abstract

Insulin stimulates a rapid phosphorylation and sequestration of the beta(2)-adrenergic receptor. Analysis of the signaling downstream of the insulin receptor with enzyme inhibitors revealed roles for both phosphatidylinositol 3-kinase and pp60Src. Inhibition of Src with PP2, like the inhibition of phosphatidylinositol 3-kinase with LY294002 [2-(4-morpholynyl)-8-phenyl-4H-1-benzopyran-4-one], blocked the activation of Src as well as insulin-stimulated sequestration of the beta(2)-adrenergic receptor. Depletion of Src with antisense morpholinos also suppressed insulin-stimulated receptor sequestration. Src is shown to be phosphorylated/activated in response to insulin in human epidermoid carcinoma A431 cells as well as in mouse 3T3-L1 adipocytes and their derivative 3T3-F422A cells, well-known models of insulin signaling. Inhibition of Src with PP2 blocks the ability of insulin to sequester beta(2)-adrenergic receptors and the translocation of the GLUT4 glucose transporters. Insulin stimulates Src to associate with the beta(2)-adrenergic receptor/AKAP250/protein kinase A/protein kinase C signaling complex. We report a novel positioning of Src, mediating signals from insulin to phosphatidylinositol 3-kinase and to beta(2)-adrenergic receptor trafficking.

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Molecular Biology of the Cell
Vol. 13, 3943–3954, November 2002
pp60Src Mediates Insulin-stimulated Sequestration of
the ?2-Adrenergic Receptor: Insulin Stimulates
pp60Src Phosphorylation and Activation
Elena Shumay,* Xiaosong Song,* Hsien-yu Wang,†and Craig C. Malbon*‡
Departments of *Pharmacology and†Physiology and Biophysics, Diabetes and Metabolic Diseases
Research Center-Health Sciences Center, State University of New York at Stony Brook, Stony Brook,
New York 11794-8651
Submitted March 29, 2002; Revised August 14, 2002; Accepted August 21, 2002
Monitoring Editor: Guido Guidotti
Insulin stimulates a rapid phosphorylation and sequestration of the ?2-adrenergic receptor.
Analysis of the signaling downstream of the insulin receptor with enzyme inhibitors revealed
roles for both phosphatidylinositol 3-kinase and pp60Src. Inhibition of Src with PP2, like the
inhibition of phosphatidylinositol 3-kinase with LY294002 [2-(4-morpholynyl)-8-phenyl-4H-1-
benzopyran-4-one], blocked the activation of Src as well as insulin-stimulated sequestration of the
?2-adrenergic receptor. Depletion of Src with antisense morpholinos also suppressed insulin-
stimulated receptor sequestration. Src is shown to be phosphorylated/activated in response to
insulin in human epidermoid carcinoma A431 cells as well as in mouse 3T3-L1 adipocytes and
their derivative 3T3-F422A cells, well-known models of insulin signaling. Inhibition of Src with
PP2 blocks the ability of insulin to sequester ?2-adrenergic receptors and the translocation of the
GLUT4 glucose transporters. Insulin stimulates Src to associate with the ?2-adrenergic receptor/
AKAP250/protein kinase A/protein kinase C signaling complex. We report a novel positioning of
Src, mediating signals from insulin to phosphatidylinositol 3-kinase and to ?2-adrenergic receptor
trafficking.
INTRODUCTION
?2-Adrenergic receptors (?2ARs) are members of the super-
family of G protein-coupled receptors (GPCRs) and display
desensitization in response to ?2-adrenergic agonists as well
as counterregulation by several growth factor receptors with
intrinsic tyrosine kinase activity (Morris and Malbon, 1999).
Sequestration of the ?2AR plays a central role in agonist-
induced and insulin-induced regulation of ?-adrenergic sig-
naling (Lefkowitz, 1998; Morris and Malbon, 1999). ?2ARs
are sequestered in response to insulin and this loss of the
surface complement of receptors plays a critical role in the
counterregulatory physiological effects of insulin on cate-
cholamine action (Karoor et al., 1998). Although information
has been gained on agonist-induced trafficking of GPCRs
(Carman and Benovic, 1998; Gagnon et al., 1998), much less
is known about how counterregulation by tyrosine kinases
receptors influences GPCR trafficking.
?2ARs are substrates for insulin-stimulated tyrosine phos-
phorylation (Karoor and Malbon, 1998). In vivo, insulin
stimulates the phosphorylation on two major tyrosine resi-
dues, Y350 and Y364, both residues located in the C-terminal
cytoplasmic domain of the ?2AR (Karoor et al., 1995). Phos-
phorylation of the Y350 residue creates an SH2-binding site
to which several molecules can dock, including Grb2, the
p85 catalytic domain of phosphatidylinositol 3-kinase, and
the GTPase dynamin (Shih and Malbon, 1998). In vitro,
insulin stimulates the purified insulin receptor to phosphor-
ylate recombinant ?2-adrenergic receptor on these same res-
idues (Baltensperger et al., 1996; Doronin et al., 2000). The
phosphorylation of the ?2-adrenergic receptor impairs its
function, a blockade that requires Grb2 with an intact SH2
domain (Shih and Malbon, 1998). Although inducing a
rapid, profound sequestration of the ?2AR, ?-adrenergic
agonists and insulin display differences in the pathways by
which the sequestration occurs (Karoor et al., 1998). The
sequestration in response to insulin, but not ?-adrenergic
agonist, can be blocked with inhibitors of phosphatidylino-
sitol 3-kinase (PI3K), such as wortmannin or LY294002 [2-(4-
morpholynyl)-8-phenyl-4H-1-benzopyran-4-one) (Wang et
al., 2000).
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E02–03–0174. Article and publication date are at www.molbi-
olcell.org/cgi/doi/10.1091/mbc.E02–03–0174.
‡Corresponding author: E-mail address: craig@pharm.sunysb.edu.
Abbreviations used: pp60Src, phosphorylated, activated phos-
phoprotein 60-MrSrc; p60Src, phosphoprotein 60-MrSrc; and
?2AR, ?2-adrenergic receptor.
© 2002 by The American Society for Cell Biology 3943

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Recently, the nonreceptor tyrosine kinase Src has been
shown to be involved in ?-adrenergic agonist-induced de-
sensitization (Luttrell et al., 1996), associating with the ?2AR
and the scaffold protein gravin, also known as AKAP250
(Fan et al., 2001a,b), leading to its phosphorylation and ac-
tivation of G protein receptor kinases (Ruiz-Gomez and
Mayor, 1997; Sarnago et al., 1999). Whether Src functions in
insulin-stimulated counterregulation of the ?2AR is not
known. Investigation of the role of Src in this insulin-stim-
ulated response is the focus of the current studies. Herein,
we report that insulin activates Src, mediating signaling
from the insulin receptor to the level of PI3-kinase activation
and the trafficking of the ?2AR.
MATERIALS AND METHODS
Materials
The plasmid encoding the enhanced green fluorescent protein
(eGFP)-tagged human ?2AR (in pCDNA3) was obtained from Dr.
Jeffrey Benovic (Kimmel Cancer Center, Thomas Jefferson Univer-
sity, Philadelphia, PA). The pCDNA3GFP-GLUT4 expression vector
was a gift of Dr. Jeffrey Pessin (Department of Physiology and
Biophysics, University of Iowa, Iowa City, IA), and the expression
vector harboring the constitutively active Src Y527F mutant (CA-
Src) was generously provided by Dr. Joan Brugge (Department of
Cell Biology, Harvard Medical School, Boston, MA). To analyze and
characterize an expression of green fluorescent protein (GFP)-
tagged ?2AR, the following antibodies were used: anti-?2AR (CM02,
antipeptide antibody to the exofacial domain of the ?2AR; Wang et
al., 1989a) and anti-GFP rabbit polyclonal antibodies (Quantum
Biotechnologies, Montreal, Quebec, Canada); goat anti-rabbit anti-
body conjugated with horseradish peroxidase (Kirkegarrd and
Perry Laboratories, Gaithersburg, MD); and mouse anti-insulin re-
ceptor ? subunit (p95; Transduction Laboratories, Lexington, KY).
Cell Culture
Human epidermoid carcinoma A431 wild-type cells and A431
clones stably expressing either eGFP-tagged ?2AR or eGFP-tagged
GLUT4 glucose transporters were cultured in DMEM containing
10% fetal bovine serum, 100 U/ml penicillin, and 100 ?g/ml strep-
tomycin (George et al., 1988; Wang et al., 1991; Shih and Malbon,
1994, 1996; Shih et al., 1999). The mouse 3T3-L1 and 3T3-F442A cells
(American Type Culture Collection, Manassas, VA) were cultured
in the same medium with an addition of 1? nonessential amino
acids (Invitrogen, Carlsbad, CA) and were induced to the adipocyte
phenotype by the treatment with dexamethasone and 1-methyli-
sobutylxanthine, as described previously (Green and Kehinde, 1975;
Wang et al., 1992). Before analysis for insulin action, A431, 3T3-L1,
and 3T3-F442 cells were serum starved overnight (Karoor et al., 1995).
Assay of Phosphorylation of Src
A431 cells were stimulated with 100 nM insulin for the indicated
times, collected, and then lysed in a lysis buffer (150 mM NaCl, 5
mM EDTA, 50 mM NaF, 40 mM sodium pyrophosphate, 50 mM
KH2PO4, 10 mM Na-molybdate, 2 mM Na-orthovanadate, 20 mM
Tris-HCl, pH 7.4, 1% Triton X-100, 0.5% NP-40, 6 mM dithiothreitol,
10 ?g/ml aprotinin, 10 ?g/ml leupeptin, and 0.2 mM phenylmeth-
ylsulfonyl fluoride). Samples (50 ?g of protein/lane) were subjected
to electrophoresis on 10% polyacrylamide gels, the separated pro-
teins transferred to nitrocellulose, and the blots stained with anti-
bodies, as described previously (Jho et al., 1997). For these experi-
ments, the blots were stained with rabbit anti-phospho-Src (Y416)
antibodies purchased from Upstate Biotechnology (Lake Placid,
NY). These antibodies specifically recognize only the Y416 phos-
phorylated, activated form of Src family members. Other antibodies
used for immunoblotting of samples of cell lysates were anti-Src,
anti-Fyn, anti-Lck, anti-Lyn, and anti-Yes, each obtained from Santa
Cruz Biotechnology (Santa Cruz, CA).
Inhibitor and Morpholino Antisense Oligomer
Studies
A431 cells stably expressing eGFP-tagged ?2-adrenergic receptors
were pretreated for 30 min with one of the following inhibitors: the
PI3K inhibitor LY294002 (20 ?M), the mitogen-activated protein
kinase kinase (MEK) inhibitor PD98059 (2?-amino-3?-methoxyfla-
vone) (??M), or the inhibitor of nonreceptor Src family kinases PP2
(50 nM). After pretreatment with an inhibitor, cells were incubated
with or without 100 nM insulin for 30 min followed by live imaging
of receptor localization. The morpholino antisense oligomers were
designed and synthesized by Gene Tools (Philomath, OR). The
antisense sequence selected for Src was 5?-ATGGGTAGCAACAA-
GAGCAAGCC-3?, with the corresponding sense sequence used as a
control. Cells were treated according to the protocol provided by
Gene Tools. Treatment with the morpholinos was limited to 48 h,
because extending the period of treatment to ?72 h resulted in
significant cell death.
Src Activity Assay
Whole-cell lysates were prepared from either A431 or 3T3-L1 clones.
The phosphotransferase activity of Src kinase was determined in
immunoprecipitates from cell lysates by using Src assay kit (Upstate
Biotechnology), following the manufacturer’s instructions. The pre-
ferred Src substrate peptide KVEKIGEGTYGVVYK corresponding
to amino acids 6–20 of p34cdc2was used in these studies. The assay
is linear for incubation times to 30 min. Samples were assayed in
quadruplicate and the amount of incorporated label determined by
scintillation counting.
PI3K Activity Assay
Crude cell membrane fractions were resuspended in a reaction buffer
containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and
1 mM EGTA. An aliquot of membrane (100 ?g/50 ?l) was added to a
reaction mixture containing 13 mM MnCl2, 65 ?M phosphatidylinosi-
tol diphosphate, 0.2 ?g/?l phosphatidylserine, and 1 mM ATP (con-
taining 1 ?Ci of [?32P]ATP). The reaction was initiated by addition of
the protein sample and maintained at 30°C for 20 min. The reaction
was stopped with 2 N HCl and then an aliquot (160 ?l) of chloroform:
methanol (1:1) mixture was added. The samples were subjected to
centrifugation at 10,000 ? g for 1 min and the lower phase was
transferred to a new tube and washed by chloroform:methanol:0.1 N
HCl (1:1:1) twice. The lower phase was collected and dried under
vacuum. The samples were resuspended in 10 ?l of chloroform:metha-
nol (95:5) and then spotted onto the origin of a thin layer chromatog-
raphy (TLC) plate (Song et al., 2001). The plate was developed in
chloroform:methanol:25% NH4OH:H2O (100:70:25:15). The resolved
plate was dried and exposed to films Eastman Kodak, Rochester, NY)
or PhosphorImager (Molecular Dynamics, Sunnyvale, CA) cassette.
Radioligand Binding Studies
The number of ?2ARs was determined by radioligand binding by
using the high-affinity, ?-adrenergic antagonist [125I]iodocyanopin-
dolol (ICYP). Intact A431 cells were incubated with 0.5 nM ICYP
(PerkinElmer Life Sciences, Boston, MA) in the presence or absence
of 10 ?M propranolol at 23°C for 90 min. The incubation buffer
contained 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 150 mM
NaCl. The cells were collected under vacuum on GF/C membranes
(Whatman, Maidstone, United Kingdom) and washed rapidly three
times. The amount of ICYP bound to the washed cell mass retained
by the filter was quantified by use of gamma counting (George et al.,
1988).
E. Shumay et al.
Molecular Biology of the Cell 3944

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Epifluorescence of Receptor/GLUT4 Translocation
(Live Cell Microscopy)
Clones expressing either the eGFP-tagged ?2AR or the eGFP-tagged
GLUT4 were seeded onto coverslips of either four- or eight-chamber
slides. Confluent cells were serum deprived overnight, treated with
the drugs, and carefully washed three times with HBB buffer (1?
Hanks’ balanced salt solution, 0.1% bovine serum albumin, 10 mM
HEPES, pH 7.5; Invitrogen). Fresh DMEM medium without phenol
red and serum was added and cells were imaged rapidly in an
Figure 1.
action by sequestration of ?2ARs. A431 cells stably
transfected with an expression vector harboring
the cDNA for the human ?2AR tagged with en-
hance GFP were used. (A) Immunoblots of WT
cells and clones expressing ?2AR-GFP were
stained with antibodies to either ?2AR or GFP. (B)
Fluorescent images of live A431 cells that express
?2AR-GFP were collected by using epifluorescence
(inverted) microscopy after treatment of the cells
with either the ?-adrenergic agonist isoproterenol
(10 ?M, ?Iso) or insulin (100 nM, ?Ins) for 30 min
(bottom row). White arrows highlight receptors
localized to the cell membrane; yellow arrowheads
indicate the receptors in perinuclear zone. The
same experiments were performed, except that in
this case the cells were fixed and the images were
acquired by confocal microscopy (top row). (C)
Time course of ?2AR-GFP translocation after A431
cell stimulation with insulin. A431 cells were stim-
ulated with 100 nM insulin and imaged live on the
charge-coupled device camera as repetitive frames
were collected from the same spot over the time
period of 35 min.
Insulin counterregulates catecholamine
Src Mediates Insulin Signaling
Vol. 13, November 20023945

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Figure 2.
E. Shumay et al.
Molecular Biology of the Cell3946

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inverted fluorescent microscope (Nikon, Tokyo, Japan), fitted with a
40? objective (oil immersion). An acquisition of digital images was
performed using a cooled charge-coupled device Princeton camera
and WinView software (Roper Scientific, Inc., Trenton, NJ).
Confocal Microscopy
Clones were grown on glass slides, treated with drugs as indicated,
fixed with 2% paraformaldehyde in phosphate-buffer saline,
washed, and then treated with SlowFade Antifade kit (Molecular
Probes, Eugene, OR) to prevent photobleaching. The confocal mi-
croscopy was performed on Eclipse E600 microscope (Nikon) using
argon laser (488 nm). Images acquired with C-imaging system
(Compix, Cranberry Township, PA) operating SimplePCI software.
Data Presentation and Analysis
Unless otherwise noted, the values presented are mean values ?
SEM and are representative of multiple (at least three), independent
experiments. Adobe Photoshop5.5 and Illustrator 8.0 (Adobe Sys-
tems, Mountain View, CA) were used to prepare final images and
figures.
RESULTS
To facilitate study of ?2AR sequestration in response to
insulin, we stably transfected human epidermoid carcinoma
A431 cells, a well-characterized model of ?2-adrenergic re-
ceptor biology (Delavier-Klutchko et al., 1984; Kassis et al.,
1987; Lohse et al., 1990), with an expression vector harboring
eGFP-tagged human ?2AR (Fan et al., 2001a). Stable clones
were selected that expressed sufficient amounts of a GFP-
tagged version of the human ?2-adrenergic receptor (?2AR-
GFP) to enable high-quality epifluorescence microscopy, but
contributed ?10% to the endogenous level of the receptor
(Figure 1). Immunoblotting of wild-type (WT) and A431
clones (?2AR-GFP) stained with anti-?2AR antibodies re-
vealed the endogenous (Mrof 65 kDa) receptor in the WT
cells as well as the endogenous and the GFP-tagged versions
(Mrof ?95 kDa) in the A431 stable transfectants (Figure 1A).
Staining of the blots with anti-GFP antibodies, in contrast,
identified only the 95 kDa-Mr, GFP-tagged species in the
A431 clones and identified no immunoreactivity in blots
from the WT cells. Radioligand binding with the radiola-
beled, high-affinity ?-adrenergic antagonist [125I]iodocyan-
opindolol, revealed ?35,000 receptors/WT A431 cell and
?38,000 receptors/A431-transfected cell (our unpublished
data). The GFP-tagged human ?2AR has been characterized
fully and displays activation, desensitization, and sequestra-
tion in response to agonist, as does the native ?2AR (Kallal
et al., 1998; Fan et al., 2001b).
We made use of both epifluorescence and confocal micros-
copy to examine the effects of insulin on the localization of
the ?2AR (Figure 1B). Treatment with insulin (100 nM, ?Ins)
provoked a substantial sequestration of ?2AR to perinuclear
regions of the cell (yellow arrowheads) away from the cell
membrane (white arrows), within 15 min of the addition of
insulin. Challenge with the ?2-adrenergic agonist isoproter-
enol (10 ?M, ?Iso) was examined as a control. ?2ARs were
observed to sequester in the same manner in response to
stimulation by either insulin or by isoproterenol. The results
of confocal microscopy were in agreement with the epiflu-
orescence data, revealing a significant translocation of ?2AR
from the cell membrane to a perinuclear locale in response to
homologous (agonist) and heterologous (insulin) stimula-
tion. A time course of ?2AR localization in response to 100
nM insulin extended to 35 min revealed the profound redis-
tribution of ?2AR stimulated by insulin (Figure 1C). In the
absence of insulin, the majority of the ?2AR is localized to
the cell membrane. Within 30–35 min of insulin treatment
the bulk the receptor is now found densely packed within a
perinuclear region of the cell. Washout of the insulin was
followed by a full recovery of the ?2AR to the cell membrane
within 60 min, trafficking back from the perinuclear region
of the cell (our unpublished data). ?2ARs display a high-
level of recycling after internalization, with little discernable
degradation over the period of a few hours (Wang et al.,
1989b). Significant degradation (20–30%) of ?2AR is only
detected after 24 h of chronic agonist treatment (Shenoy et
al., 2001).
To unravel the details of the downstream signaling from
insulin to the sequestration of ?2ARs, we first investigated
roles of three likely signaling enzymes: PI3-kinase, the Src
family of nonreceptor tyrosine kinases, and the mitogen-
activated protein kinase cascade. Using selective inhibitors
of each enzyme, we investigated whether inhibition would
promote blockade of insulin-stimulated sequestration of
?2ARs (Figure 2A). A431 clones were treated with insulin
and the sequestration of GFP-tagged ?2ARs examined by
epifluorescence microscopy at 30 min postchallenge. The
PI3-kinase inhibitor LY294002 effectively blocked the se-
questration of ?2AR induced by insulin. The ability of iso-
proterenol to induce the agonist-specific sequestration of
?2AR, unlike insulin action, was unaffected by inhibition of
PI3-kinase. Treatment with the PD98059 inhibitor of MEK in
the mitogen-activated protein kinase pathway, in contrast to
the effects of LY294002, did not alter the ability of insulin to
sequester ?2AR. Previously, it was shown that the nonre-
ceptor tyrosine kinase Src plays a role in agonist-specific
desensitization and internalization of ?2ARs, so the effects of
the PP2 inhibitor of Src family kinases were tested on insu-
lin-induced sequestration of ?2ARs. We found that PP2 was
Figure 2 (facing page).
phosphotyrosine kinases, and depletion of Src block insulin-in-
duced sequestration of ?2AR. (A) A431 clones were untreated (con-
trol) or treated with the PI3-kinase inhibitor LY294002, Src kinase
inhibitor PP2, or the MEK inhibitor PD98059 and then stimulated
with either isoproterenol (10 ?M, ?Iso) or insulin (100 nM, ?Ins).
After 30 min with the hormones, the cells were examined by epi-
fluorescence microscopy. White arrows highlight cell membrane-
localized receptors; yellow arrowheads highlight perinuclear-local-
ized receptors, sequestered from the surface. (B) A431 cells were
treated with morpholinos antisense to Src for 48 h to deplete cellular
levels of Src as well as sense morpholinos as a control. Cells were
then stimulated either with 100 nM insulin (?Ins) or without insulin
(control). After 30 min with the hormones, the cells were examined
by epifluorescence microscopy. White arrows highlight cell mem-
brane-localized receptors; yellow arrowheads highlight perinuclear-
localized receptors, sequestered from the surface. (C) Immunoblot
of total cell lysates of A431 cells that were untreated (control) or
treated with morpholinos either antisense (antisense) or sense
(sense) to Src. The lysates were subjected to SDS-PAGE, the resolved
proteins transferred to nitrocellulose blots, and stained with an
anti-Src antibody. The blots were stained with antibody against the
insulin receptor ?-subunit (p95 IR?) to establish equivalence of
sample loading.
Inhibitors of PI3-kinase, inhibitors of Src
Src Mediates Insulin Signaling
Vol. 13, November 2002 3947