J. Biol. Chem. 266, pp. 8302-8311,1991

1
J. Biol. Chem. 266, pp. 8302-8311,1991 Purificatio
n and Partial Sequence Analysis of pp185, the
Major Cellular Substrate of the Insulin Receptor
Tyrosine Kinase Paul L. Rothenberg, William S.
Lane, Avraham Karasik, Jonathan Backer, Morris
White, C. Ronald Kahn The Department of
Medicine, Brigham and Womens Hospital, Harvard
Medical School, Boston, MA
FaO hepatoma cells /- insulin (1
mM), 1 min (1) SDS(100oC) extraction/TCA
ppt or (2) Liquid N2 quick-freezing
Triton X-100 extraction IP by anti-PY
antibody SDS-PAGE Western blot by
anti-PY antibody
pp185
INR(b)
2
Rats /- insulin (from portal
vein) 1 min or various time points
Tissue removed SDS/TCA extraction
IP by anti-PY antibody SDS-PAGE
Western blot by anti-PY antibody
Liver
pp185
INR(b)
Fat pads
TCA ppt/washed by organic solvent/ lypholized/0.1
M NaOH solubilized/ filtered with 0.45 mM membrane
pp185
INR(b)
3
Rats insulin infusion (from portal
vein) (0.5 min at 1 ml/min)
Liver removed SDS/TCA extraction IP
by anti-PY antibody SDS-PAGE Western
blot by anti-PY antibody
Half maximum stimulation 1-5 x 10-8 M
insulin
4
Rats insulin infusion (from portal
vein) (0.5 min at 1 ml/min)
Liver removed SDS/TCA extraction IP
by anti-PY antibody Eluted by pNPP Sup.
ppt. SDS-PAGE Western blot by anti-PY
antibody silver stained
5
50 Rats (starvation for 2-3 days)
/-insulin infusion (from portal vein)
(0.5 min at 1 ml/min) Liver removed
SDS/TCA extraction IP by anti-PY
antibody Eluted by pNPP Sup. ppt.
SDS-PAGE Transferred to NC membrane
trypsin digested RPC18 HPLC Amino acid
sequencer

insulin
-
6
CPS carbamyl phosphate synthase as
major contaminant in pp185 fraction
7
Production of anti-peptide 80 Ab
IP aPY aPY aPep80
aPep80 WB aPY aPep80 aPep80 aPY
- - - -
(insulin)
Rats insulin infusion (from portal
vein) (0.5 min at 1 ml/min)
Liver removed SDS/TCA extraction IP
by anti-PY or anti-pep 80 antibody
SDS-PAGE Western blot by anti-PY or
anti-pep 80 antibody
8
Nature. 1991 Jul 4352(6330)73-7. Structure of
the insulin receptor substrate IRS-1 defines a
unique signal transduction protein.Sun XJ,
Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden
PA, Cahill DA, Goldstein BJ, White MF.Joslin
Diabetes Center, Department of Medicine, Harvard
Medical School, Boston, Massachusetts, USA

IRS-1 cloning strategy (1)Two cDNA
libraries (2)Screened by 32P-oligonucleotide
probes made from peptide 80 and 138 (3)Picked up
positive clones and sequence determination (C18,
C19 P2-2) (4)Screened further by probes derived
from C18 P2-2 (14 clones were
obtained) (5)Sequencing and overlapping DNA
sequences of 14 clones
9
IRS-1 cDNA (1) Hydrophilic protein, 131 kDa (2)
9 tryptic peptides in DNA sequence (3) No SH2/SH3
domains (4) Contain ATP-binding domain
(GXGXXG) (5) No protein kinase activity (6) mRNA
9.5 kb
10
Synthetic YMXM-containing peptide can be
phosphorylated by purified INSR, Km50 mM
6 YMXM motif 3 YXXM motif 1 EYYE motif
11
CHO/IR cells CHO/IRS-1 CHO/Neo
metabolic 32P-labelling /- insulin (100 nM,
1 min) cell extractes IP by
PY-Ab SDS-PAGE/autoradiography
IRS-1 expressed in CHO cells as 185 kDa
PY-containing protein
12
CHO/IR or CHO/IRF960 or CHO/IRD960 cells
metabolic 32P-labelling /- insulin (100 nM,
1 min) cell extractes IP by PY-Ab or
IRS-1 Ab SDS-PAGE/autoradiography cut out
32P-labelled pp185 and IRS-1 Phosphoamino acid
analysis
Y960 the aa required for insulin signaling
13
CHO/IR cells /- insulin (100 nM, 10 min)
cell extractes IP by IR Ab or PY Ab or
IRS-1 Ab PI3 kinase assay in the IPP
14
Role of IRS-1 in insulin-mediated signal
transduction