Tyrosine kinase activity of brain insulin and IGF-1 receptors

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

January 29, 1986

Pages 532-538

TYROSINE KINASE ACTIVITY OF BRAIN INSULIN AND IGF-] RECEPTORS

William L. Lowe, Jr. and Derek LeRoith

Diabetes Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Building i0, Room 8S-243, Bethesda, Maryland

Received November 20, 1985

Summary: Lectin-purified rat brain preparations demonstrate specific [125I]insulin and [125I]-IGF-1 binding. Insulin-stimulable tyrosine kinase activity as measured by exogenous substrate phosphorylation was present in brain and liver lectin purified preparations with the A kinase activity/B/F of brain -2.5 fold greater than that of liver. Insulin-stimulable tyrosine kinase activity was abolished in liver but decreased by only -50 percent in brain after immunodepletion with antiserum which recognizes insulin but not IGF-I receptors. Insulin and IGF-I dose responses for phosphorylation of the immunodepleted brain preparations suggested that the remaining tyrosine kinase activity was IGF-I receptor mediated. Thus, functional IGF-I receptors are present in rat brain, and the doses of insulin typically used to evaluate insulin receptor tyrosine kinase activity will stimulate IGF-I receptor tyrosine kinase activity as well. © 1986 Academic Press, Inc.

Insulin and IGF-I receptors which possess tyrosine-specific demonstrated

are members of a family of proteins kinase activity

that insulin and IGF-I receptors

lation as well as phosphorylating

and divalent

similarity,

cation requirements

tyrosine kinase activity are essentially

Previous studies have

are capable of autophosphory-

synthetic polymers

In addition to their apparent structural specificities

(I).

containing tyrosine

the synthetic polymer

for insulin and IGF-I receptor

identical

(6).

Previous studies have documented the presence of insulin receptors insulin-stimulable

and

tyrosine kinase activity in the brain (7,8), while others

have demonstrated specific IGF-I binding in brain (9,10). that insulin-stimulable compared to liver.

(2-5).

We demonstrate here

tyrosine kinase activity is greater in rat brain as

Thus, we have attempted to determine whether

IGF-I stimul-

able tyrosine kinase activity was present in brain and whether insulin was capable of stimulating

IGF-I receptor tyrosine kinase activity in brain as a

possible explanation for the difference

between brain and liver insulin-

stimulable tyrosine kinase activity.

0006-291X/86 $1.50 Copyr@ht © 1986 by Academ~ Pre~, Inc. Aft r@h~ofreproduction m any form reserveK

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Vol. 134, No. 2, 1986

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Materials and Methods: Solubilization and Wheat Germ Agglutinin Chromatography. Adult male Sprague-Dawley rats of ~200 gm body weight were sacrificed by decapitation, and livers and brains were excised and pooled. Crude membrane preparations were prepared by differential centrifugation (7), were solubilized with Triton X-IO0 and were purified over wheat germ agglutinin-agarose ( M i l e s Laboratories, Elkhart, IN) (11). Solubilized receptor binding assays were performed as described previously (11) except that the assay buffer used was 50mM HEPES, 0.15M NaC1, I% BSA and I mg/ml bacitracin, pH 8.0 and that incubations were for 18 hours at 4°C. Artificial Substrate Phosphorylation. This was performed using a modification of methods described previously (8). Lectin-purified receptor preparations and 200 ug of artificial substrate were preincubated in the absence or presence of varying concentrations of porcine insulin or IGF-I (AmGen Biologicals, Thousand Oaks, CA). After 30 min at 22°C phosphorylation was initiated by the addition of reaction mix to give final concentrations of 50~M [¥-32p]ATP (specific activity ~3~Ci/nmol), ImM CTP, ImM sodium vanadate and 20mM MgCI 2. The reaction was terminated after 30 min as described. The counts of 32p incorporated in the absence of artificial substrate were subtracted from counts in the presence of artificial substrate to yield actual substrate phosphorylation. One unit of kinase activity was defined as incorporation of Ipmol of phosphate into I u g of artificial substrate. The component of 32p incorporation into artificial substrate due to hormone stimulation was termed A kinase activity and was calculated by subtracting the basal kinase activity from the stimulated kinase activity. To normalize the A kinase activity of lectinpurified preparations prepared separately or from different tissues, the A kinase activity was divided by the B/F of the lectin-purified preparation in the presence of tracer [125I]-insulin. Since the affinities of the insulin receptors in brain and liver lectin-purified preparations were essentially identical, B/F was thought to be an accurate reflection of the receptor number of the preparation (data not shown). Immunodepletion of Lectin-Purified Preparations. 500 u1 of anti-insulin receptor antiserum (BI0) or control serum was incubated for 3 hours on ice with 1.5 ml of packed gel of Protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden) rehydrated in 50mM HEPES, 150mM NaC1, 0.1% Triton X-IO0, pH 7.6. The gel was then washed extensively with the HEPES buffer, followed by incubation of 400 U1 of lectin-purified receptor preparation with the packed gel for 18 hours at 4°C. The suspension was centrifuged, and the supernatant was collected and used for exogenous substrate phosphorylation assays.

Results:

Specific binding of [125I]-insulin

purified preparations

was present.

and IGF-I demonstrated (Fig.

and [125I]-IGF-1

to brain lectin

Specificity studies using unlabeled insulin

the expected ability of each to displace labeled hormone

I). Insulin-stimulable

lectin-purified

tyrosine kinase activity was present in brain and liver

preparations

as measured

ous substrate poly(Glu,Tyr),4:1 Under these conditions,

by the phosphorylation

using I0-7M porcine insulin

of the exogen-

(Fig. 2, left).

the A kinase activity/B/F was consistently

greater in brain as compared to liver

~2.5-fold

(Fig. 2, left). Similar results were seen

533

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I00 ~ z

100

a z

90

s0 rn

7O •

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o c.)

ca ~60

°50

~50

~.- E ~40 -i"

E

60

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z

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10 _

I I ' ,Htd

103

1

104

10

102

. ....... I

103

HORMONE CONCENTRATION (ng/ml)

Figure I: Binding to lectin-purified brain preparations. Tracer concentrations of [IzgI]-IGF-I (left) or [IzbI]-insulin (right) were incubated with lectin-purified brain preparation (-10 pg) and different concentrations of unlabeled IGF-I(A) and insulin (0). The percentage of maximal specific binding of labeled hormone is plotted against ng/ml of unlabeled peptide. The dashed lines represent the concentration of unlabeled hormone which displaces 50 percent of labeled hormone.

in the presence tive exogenous substrate

of other

divalent

substrate

(Fig.

poly(Glu,Tyr),4:1

cation mixtures

2, left).

was linear

as with an alterna-

32p incorporation

over

!

as well

into the exogenous

40 min in the absence

or presence

I

,05

i

u_ ~,04

'14i ADDITION: ,121 ED NONE ~__,lOI I lO-7M INSULIN

TISSUE: E]LIVER mBRAIN

~ ,03

I I

=<,o2 <1

,01

BIO CONTROL LIVER a-b-~

BIO

CONTROL BRAIN

o,-~--~; E c j

~

c~

c,4

Figure 2: (Left): Phosphorylation of exogenous substrate. Lectin-purified brain and liver preparations were incubated with IO-(M insulin and the indicated exogenous substrate (30 min, 22°C). Phosphorylation was initiated by the addition of [¥-32p]ATP and the indicated concentrations of divalent cations for 30 min at 22°C. (Right): Effect of immunodepletion with control or B-IO IgG on insulin-stimulated tyrosine kinase activity. Lectin-purified preparations were immunodepleted as described under "Methods." Immunodepleted supernatants were incubated without or with I0-7M insulin and poly(Glu,Tyr),4:1. [~-32p]ATP and MgCI 2 were added for 30 min.

534

104

VOI. 1 3 4 , NO. 2, 1 9 8 6

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

04

.03

.02

>.- .01

~

> I-¢..D <

I//l

i

0

lll,llll

CONTROL B10

i

, ,...I

1

i

i ip,,,,l

I0

I00

IGF-I CONCENTRATION (nM) z .04 < v' ,~

CONTROL

.02

B10

.01 t i i I .... 1

I.; .........

0

I

1

~. ,,.I

10

........

I

100

INSULIN CONCENTRATION (riM)

Figure 3: Effect of immunodepletion with control or B-tO IgG on exogenous substrate phosphorylation. Brain lectin-purified preparations were immunodepleted as described under "Methods". Immunodepleted supernatants were incubated without or with increasing doses of IGF-I (top) or insulin (bottom) and poly(Glu,Tyr),4:1. [7-32p]ATP and MgCI 2 were added for 30 min. Dashed lines indicate the concentration of hormone giving half maximal stimulation of phosphorylation.

of I0-7M insulin in both brain and liver, and the g kinase activity/B/F of brain was -2.5-fold greater than that of liver at all time points

(data not

shown). Varying the concentrations of MgC12 from 10mM to 45mM in the reaction mix resulted in a linear increase of 32p incorporation into poly(Glu,Tyr),4:1 in brain and liver with preservation of the difference in A kinase activity/B/F noted above

(data not shown).

When the brain and liver lectin-purified preparations were immunodepleted with anti-insulin receptor antiserum B-IO which immunoprecipitates insulin receptors but not IGF-I receptors

(4), essentially no insulin-stimulable

tyrosine kinase activity was present in liver preparations whereas only -50% of insulin-stimulable tyrosine kinase activity in the brain preparation was removed (Fig. 2, right).

Since the specific binding of [125I]-insulin was

decreased by 95 percent in immunodepleted as compared to control brain preparations (data not shown), saturation of the antibody by the brain preparation was 535

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,14

.12

ADDITION: [ ] INSULIN • IGF-I • INSULIN + IGF-I

>~- .10

.08 < z v <1 .06

.04

.02

InM

10nM 1~nM HORMONE CONCENTRATION

Figure 4: Additivity of insulin and IGF-I stimulated tyrosine kinase activity. Brain lectin-purified preparations were incubated without or with increasing concentrations of insulin or IGF-I alone or insulin and IGF-I together and with poly(Glu,Tyr),4:1 for 30 min at 22Oc. [¥-32p]ATP and MgCI 2 were added for 30 min.

unlikely.

Following immunodepletion of the brain preparation with anti-

receptor antiserum B-IO, stimulation of exogenous substrate phosphorylation by I0-9M insulin was markedly reduced, whereas I0-9M IGF-I stimulation of exogenous substrate phosphorylation was similar with the immunodepleted and control

(non-immunodepleted)

preparations

(Fig. 3). This suggests that the

residual tyrosine kinase activity in immunodepleted brain preparations stimulated with I0-7M insulin represents IGF-I receptor tyrosine kinase activity. When insulin and IGF-I were added to brain preparations individually or together to stimulate exogenous substrate phosphorylation, an additive response was seen with I0-9M hormone whereas essentially no additivity was present at I0-7M hormone (Fig. 4). In the liver preparation, which is thought not to have IGF-I receptors

(12), there was only a small response of exogenous substrate

phosphorylation to I0-7M IGF-I as compared to the response to I0-7M insulin (data not shown).

Discussion Previous studies have documented the presence of [125I]-IGF-1 the brain (9,10).

binding in

In this study, we have demonstrated that IGF-I receptors

present in the adult rat brain are functional as measured by their ability to

536

Vol. 134, No. 2, 1986

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

specifically bind IGF-I and to phosphorylate strates.

Since IGF-I and insulin receptors

exogenous tyrosine-containing phosphorylate

substrates and have similar divalent cation requirements

sub-

similar exogenous (6), differentiating

between their respective

kinase activities

receptors

The use of an anti-insulin receptor antiserum which

is difficult.

apparently recognizes demonstrate

insulin receptors

in tissues which contain both

but not IGF-I receptors allowed us to

the presence of both insulin and IGF-I receptor tyrosine kinase

activity in brain lectin-purified

preparations.

When stimulated with I0-7M insulin, the A kinase activity/B/F preparations was greater than that of the liver preparations tions studied.

under all condi-

The normalization with B/F used in the calculations

insulin receptors

in the preparation,

since tracer concentrations

insulin probably do not measure IGF-I receptors. brain preparations

stimulated with I0-7M insulin, however,

contains both insulin and IGF-I receptors,

reflected

of [125I]-

The A kinase activity of the

the kinase activity of both insulin and IGF-I receptors.

IGF-I receptors

of the brain

apparently reflected Since the brain

whereas the liver does not contain

(12), the stimulation of brain IGF-I receptor tyrosine kinase

activity by I0-7M insulin is the likely explanation for the discrepancy A kinase activity/B/F

in the

of the brain and liver preparations.

These observations

are of importance when quantitation of

receptor tyrosine kinase activity is attempted.

insulin

Previous quantitative

of insulin receptor tyrosine kinase activity in altered physiological

studies states

have used the binding activity of the preparation and the stimulation of phosphorylation comparisons

of exogenous substrates with I0-7M insulin to make their

(13,14).

Our results indicate that when quantitative

performed in tissues which contain IGF-I receptors receptors,

studies are

in addition to insulin

adequate controls should be performed to ensure that the observed

effects are due to alterations

in insulin receptors as opposed to IGF-I

receptors.

Acknowledgements We would like to thank Drs. George Grunberger, Simeon Taylor, Jesse Roth and Richard Comi for helpful discussions and advice as well as Ms. Violet Katz and Deborah Hubbard for their secretarial assistance. References I.

Sefton, B.M., and Hunter, T. (1984) in: Advances in Cyclic Nucleotide and Protein Phosphorylation Research, Vol. 3, 195-225, Raven Press, New York.

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