Identification of skeletal muscle protein-tyrosine phosphatases by amplification of conserved cDNA sequences

Vol. 178, No. 3, 1991 August

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1291-l 297

15, 1991

IDENTIFICATION OF SKJWXTALMUSCLEPROmIN-TYROSINEPHOSPHATASES BYMPLIFICATIONOF CONSERVED cDNASEQUENCES Wei-Ren

Zhang

and Barry

J.

Goldstein

Research Division, Joslin Diabetes Center and Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02215 Received

June

10,

1991

SUNNARY: Specific protein-tyrosine phosphatase (PTPase) enzymes that regulate signal transduction by the insulin receptor in target tissues have not been identified. We evaluated the expression of PTPase homologs in skeletal muscle since this tissue is the major site of insulin-mediated glucose disposal in vivo. A rat skeletal muscle cDNA pool was prepared with a set of degenerate oligonucleotide primers and PTPase cDNA sequences were amplified using pairs of "guess-mers" that were deduced from highly conserved residues within the known catalytic domains of these enzymes. Sequences encoding three "receptor-like" transmembrane PTPases were identified and two of these (known as LAR and LRP) were confirmed to be expressed in muscle by subsequent cDNA library screening and Northern blot The expression of the LAR and LRP PTPases in skeletal muscle analysis. suggests that these enzymes might have a role in the regulation of insulin action in muscle and other insulin-sensitive tissues. 0 1991 Academic Press, Inc.

Protein-tyrosine important

phosphatases

role

in the

insulin-stimulated catalyzing

the

dephosphorylation

receptor

regulate

signal

insulin

Characterization help

in this the

of insulin

action

identify

and other

pathophysiology

However, is

the

tissue

resistance

observed

of the major

PTPase

candidate

PTPases

insulin-sensitive of insulin-resistant

insulin

receptor

that

of the

of insulin-mediated II

diabetes

expressed only

but

disease

for in

regulate

also

might

as by

enzymes(s)

have not

responsible

not

tissues,

the as well

PTPase tissues

in Type enzymes

an

proteins

specific

site

largely

have

by reversing

in target

major

is

3.1.3.48)

substrate the

by insulin

muscle

and this

of the of cellular

(1,2).

transduction

in vivo,

peripheral would

kinase

Skeletal

identified.

; E.C.

autophosphorylation

insulin

disposal

regulation

(PTPases

that

been glucose

the mellitus

skeletal

(3). muscle

insulin be involved

action in

states.

The abbreviations used are; PTPase, protein-tyrosine phosphatase; LCA, leukocyte common antigen; LAR. LCA-related; LRP, LCA-related phosphatase; EDTA, ethylenediamine tetraacetic acid; SDS, sodium dodecyl sulfate.

Vol.

BIOCHEMICAL

178, No. 3, 1991 A number

family

of full-length

of related

duplicated

that

are

distinguished

overall

enzyme

transmembrane

regions

transmembrane

structure,

antigen

CD45),

(LCA;

and two (5316)s (for LCA-related tissue

only

expression that

are

Sequencing

represent

limited

called

or tandemly

contain have

a series

also

PTPase

both

of which

1B and lack

a receptor-like enzymes

leukocyte

to hematopoietic LAR (for

PTP-a)

of

been

subcellular

tandem-domain is

single

single-domain

have

PTPases,

also

blot

in crude in the

in

this

skeletal

literature

any of the skeletal

in

which

conserved amplification analysis

major

PTPase

muscle (2,17).

recently

muscle. tissue,

cloned

In order

have

common

cells

LCA-related)

and LEP

a wider

primers

residues

within

products revealed homologs

that

extracts

has been

Furthermore,

the

PTPase amplification PTPase

followed

been

enzymes

of cDNA with that

catalytic

by cDNA library

two transmembrane

in skeletal

PTPase

and "guess-mers" the

potential

cDNAs has not

to identify

we performed

oligonucleotide

highly

of the

and Northern LEP,

briefly

expressed from

the

similar

that

PTPases

a

(9-12).

of degenerate

deduced

acids

the

PTPases

comprise laboratories

of

cDNA library,

expression

phosphatase;

examined

series

includes

including whose

consists

and presumed

a T-cell Other

structurally

of mRNA for

directly

from

enzyme activity

described

structure

(6-8).

distribution PTPase

PTPases

that

by several

The cloned

of PTPases

isolated

sequences

reported

300 amino

conserved.

One class

a related

of these

of approximately highly

by their

localization.

PTPase

have now been

domain

segments

residues

and partial

proteins

The catalytic

(4-15).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PTPases,

a

were domains. screening LAB and

muscle.

METHODS ANDMATERIALS Pre rrationa Skeletal muscle was obtained from male Sprague-Dawley rats weighing 150-175 g (Taconic Farms, Germantown, NY) that had free access to food and water prior to sacrifice. Total RNA was prepared by homogenization in guanidinium thiocyanate, extraction with phenol/chloroform and precipitation from isopropanol (18). The polyadenylated mRNA fraction was enriched by chromatography over oligo-d(T) cellulose as described (19). Amnlification of cDNA: Complementary DNA was prepared by reverse transcription of muscle mRNA as described previously (20), but using polyadenylated RNA and a set of antisense degenerate oligonucleotide primers (Figure 1) that were derived from highly conserved residues that occur in all of the available cloned mammalian PTPase catalytic domains (10). An aliquot of the cDNA pool was then amplified with T. aquaticus DNA polymerase (Perkin Elmer-Cetus) in a reaction containing 25 pmol each of paired sense and antisense "guess-mer" primers (Figure 1) at reduced stringency using thermal cycling conditions of 37'C for 60 set, 72'C for 120 set and 94OC for 30 sec. for a total of 30 cycles. Amplification products were purified by electrophoresis through a 5% polyacrylamide gel and electroeluted prior to treatment with Klenow DNA polymerase to blunt the ends (21). After phosphorylation with T4 polynucleotide kinase, the cDNA fragments were 1292

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BIOCHEMICAL

AND

BIOPHYSICAL

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subcloned into the dephosphorylated SmaI site of the pBluescriptI1 plasmid vector (Stratagene). The cDNA inserts were then sequenced with T3 and T7 primers using modified T7 DNA polymerase (US Biochemical) as described by the manufacturer. Seauence Analvsis: the Molecular Biology Institute and Harvard

PTPase homologies were identified using programs of Computer Research Resource at the Dana Farber Cancer School of Public Health, Boston, MA.

cDNA Library Screening: One million phage plaques of a rat skeletal muscle cDNA library in XgtlO (Clontech) were hybridized (21) at reduced stringency in a solution containing 40% (v/v) formamide, 0.1% (w/v) Ficoll, 0.1% (w/v) polyvinylpyrrolidone, 0.1% (w/v) bovine serum albumin, 5 mM EDTA, 0.1% (w/v) SDS, 100 ug@l denatured salmon sperm DNA, 750 mM NaCl, 50 mM NaH2P04, pH 7.4, at 37 C Eith the labeled rat LRP cDNA insert (22). Filters were washed at 50 C in buffer containing 75 mM NaCl and 0.1% (w/v) SDS in 7.5 mM Na Citrate, pH 7.0. Inserts from positive plaques were subcloned into p ? asmid vectors and sequenced. Northern Blot Analvsis: Polyadenylated 1% agarose/2.2M formaldehyde gel employing previously (23). After capillary transfer, cDNA fragments that were labeled by random

RNA was fractionated in a RNA size markers as described the filter was hybridized with priming (22).

RESULTSANDDISCUSSION To identify first

PTPase

prepared

degenerate

antisense

cDNA synthesis cloned

from

as well in muscle.

that

are

muscle

oligonucleotide

on mRNA templates

PTPases

be expressed

homologs

a cDNA pool

as novel

expressed

primers PTPase

enzymes

3'-

5A:

5'-at

Q(t,c) ac(a,g)

-(H)-(C)-(S)-(A)-(G)-(v,i)-(G)-(R)(t,a)(g,c)(c,t) gc(t9g.a) (a.t)(c.g)(g,a) c.s(a,c,t)

-(F)-(W)-(r)-(M)-v,i-(W)ttc tgg agg atg

51-x

-(F)-(W)-(k)-(M)-v,i-(W)ttc tgg aaa atg

which

this

amplified

will

of

prime

previously

family in

we

a set

that

might

separate

SET:

gg(t,c) cc(a,g)

(g,a)t(g,t) (c,tMc,a)

ggg cg ccc gc

-5'

-(H)-(C)-(S)-(A)-(G)-(v)-(G)gtg

cat tgc agt get ggt gtg ggc

tgg-3' 3A:

5B:

l),

within

PRIMER

muscle,

RNA using

to each of the

was then

ERATE ANTISENSE

ca(t,c) .gt(a,g)

(Figure

corresponding

The cDNA pool

in skeletal

polyadenylated

3'-gta

acg tea

cga cca

cat

ccg-5'

-(H)-(C)-(s)-(A)-(G)-(v)-(G)atg

cat tgc cgt get ggt gtg ggt

tgg-3' 3B:

3'-gtg

acg

tea

cga cca

cat

cca-5'

Fieure 1. Primers used for cDNA synthesis and amplification. The degenerate primer set was made from the amino acid sequence indicated by the Residues in capital letters are fully conserved in all single-letter code. of the cloned enzymes within this gene family (10). Below the amino acid sequence the derivation of the antisense DNA oligomers is indicated; degenerate residues are shown in parentheses. The "guess-mers" are similarly derived from conserved upstream and downstream regions of the PTPase The 5A and 5B primers correspond to the sense strand as catalytic domain. shown; the 3A and 3B primers correspond to the antisense strand of the cDNA encoding the indicated peptide sequence.

1293

Vol.

BIOCHEMICAL

178, No. 3, 1991

reactions

using

the 4 possible

and 3B) oligonucleotide were

designed

where

third

the

they

position

a mismatch

non-denaturing

of cDNA amplification subclones.

as potential

analysis

(Figure

(clone

LRP (11,12) been

clone ml.RP: 5A3Bl: mLRP:

clone rLAR: 5A3B2: rLAR:

clone rLCA: 5B3B: rLCA:

that

1). codon

that remained used

subcloned

the

reported.

to represent A second

their

the

size as

further with

deduced

the

rat

with amino

homolog,

reactions.

the acid

whose

product

identification

the

products

obtained

its

amplification

expected

and isolated

amplification

amplification

One of the products in

within

hybridization in

After

of 6 discrete

electroeluted after

oligonucleotide,

DNA polymerase.

a total

migrated were

cells

specifically

3' end of the

electrophoresis,

primers

in mammalian

that

by the

(3A

amplification

usage

sequences at the

by colony were

was identical

and appears

previously

products

These

(Figure

extension

products

of the

2).

5A3Bl)

gel

RESEARCH COMMUNICATIONS

(5A and 5B) and antisense

optimal

conserved

primer

sequences

primers

Sequence

for

350 to 400 bases Four

PTPase

oligonucleotide

of sense

degeneracy

polyacrylamide

of approximately

performed

encoded hinder

range

pairs

rules

codon

might

bands plasmid

pairs

"guess-mers'

by using

In addition,

(24). avoided

AND BIOPHYSICAL

was then 5A and 3B primer sequence

to mouse

sequence

has not

of the

5A/3B

primer

EQNTATIVMV TNLKERKECK CAQYWPDQGCWTYGNVRVSVEDVTVLVDYT IIIllIIllI IIllllIIll llIIllllII IIllIllIlI ltlllllltI ., .MIW EQNTATIVMV TNLKERKECK CAQYWPDQGC WTYGNVRVSVEDVTVLVDYT

SA3Bl:

VRKFCIQQVG DVTNRKPQRL ITQFHFTSWP DFGVPFTPIG MLKFLKKVKA CNPQYAGAIW IIIIIllllI llIIllIIII llIIIIIIII IIIllllllI IIIIIllIII lllllllllll VRKFCIQQVG DVTNRKPQRL ITQFHFTSWP DFGVPFTPIG MLKFLKKVKA CNPQYAGAIWHCS...

5A3B2:

EQRTATWMM TRLEEKSRVK CDQYWPARGTETYGLIQVTL VDTVELATYT IIlIIIIlII llIIIIIIll IIIllIIIII IIllllIIll lIIIIllllI . . .MVW EQRTATWMM TRLEEKSRVK CDQYWPARGTETYGLIQVTL VDTVELATYT

MRTFALHKSG SSEKRELRQF QFMAWPDHGVPEYPTPIIAFL RRVKACNPLD AGPMVV llIIllllll llllIllllI llIItIllll IIIIIIIIIII llllllllll llllll MRTFALHKSG SSEKRELRQF QFMAWPDHGVPEYPTPILAFL RRVKACNPLD AGPMVVHCS

5B3B:

EQKATVIVMV TRCEEGNRNKCAEYWPCMEEGTRTFRDWV TINDHKRCPD IIIIlllIlI IIIIllIIII llIIII1llI IIIIIIIIll llIIIllIII .MIW EQKATVIVMV TRCEEGNRNK CAEYWPCMEEGTRTFRDVW TINDHKRCPD

YIIQKLSIAH KKEKATGREV THIQFTSWPD HGVPEDPHLL LKLRRRVNAF SNFFSGPtW IIIIIllIII llllllIIII llllIllIll lIllIllIIl IllllIIItI lllllll II YIIQKLSIAH KKEKATGREV THIQFTSWPD HGVPEDPHLL LKLRRRVNAF SNFFSGPIWHCS...

Figure 2. Comparison of the deduced amino acid sequences for three PTPase homologs isolated from rat skeletal muscle with the proximal conserved PTPase domain of previously reported PTPases. Upper panel, alignment of the clone 5A3Bl sequence with the s?quence of mouse LRP (11,12); middle panel, alignment of 5A3B2 with rat LAR ; lower panel, alignment of 5B3B with rat LCA (25).

Vol. 178, No. 3, 1991 pair

(clone

that

of clone

5A3B2)

has been

liver

cDNA library'. third

LAR.

migrated

5A3Bl.

which the

BIOCHEMICAL

a segment

PTPase

million

homologs

plaques plaque whose

lower liver

sequence

LAR, in a rat

generated

corresponded primer

to rat

pair,

of the

was found

rat

LCA PTPase

expression

of these

than

less

Since

.

LRP sequences

(Figure

by using

tissues,

placenta,

with similar

A definite

was observed

the

is

not

screening

the

spleen

and not

with

previous

in agreement

the

for

Northern

in spleen,

tissue.

in the

brain, muscle,

LCA (clone

other

studies

kidney

tissues

1 Manuscript

tissue

that

was used

in preparation.

1295

confirming

indicating lineage

to prepare

Northern

5B3B), (not

that

expression

shown). the

and

(11,12).

When an identical

rat

hematopoietic

in each of the

mouse tissues

in skeletal

for

muscle

analysis

16). Detection of mRNA encoding the LCA PTPase by the of cDNA amplification may have resulted from peripheral muscle

LRP

skeletal

reference

in the

LRP

containing

restricted

leukocytes

of the

in a

that

of LCA is technique

to cells

suggested

LRP in several

in this

of

of LRP mRNA among several

expression

cDNA insert

in

expression

3.0 kb was detected

was observed

LRP PTPase

been detected

of LAR transcripts

by screening expression

for

plaque

plaques

or approximately

no plaques

as a probe

abundant

the

cDNA probe.

insert

reported

with phage

relative

also

since

LAR, cognate

detected

signal

with in

than its

content

relative

most

of the only

for

the million,

the

to data

was re-probed

that

of approximately

hybridization

expression

have

5A3Bl

A transcript

3).

would

the

were the

LAR.

cDNA domain

observed

with

we assessed

tissues

a single additional

of 1 per

in muscle

screened

only

suggested

one

Interestingly,

Since

on the order

obtained

were

screening,

The library

Initially,

stringency.

to rat

result

we have

abundant were

cDNA library,

result

this

methods.

cDNA library

at relaxed

LAR PTPase

is

relative

by several

corresponded

muscle

the

muscle

5A3Bl)

of the

cDNA library'.

mRNA is

the

to rat

pair

also

5B/3B

domain

from

by cloning

primer

clone the

catalytic

and secondary

screening,

cDNA inserts

blot

5A/3A

with

tissue

skeletal

(clone

segments

library

7-fold

rat

laboratory

of this

we analyzed

in muscle

of a rat

LAR in skeletal

rat

5A3B2 corresponded

the

obtained

conserved

purification

containing

rat

sequence

experiments,

LRP cDNA insert

remained the

of the

with

distinct

(25).

three

after

the

cDNA product,

as a band

in our

Amplification

In subsequent

rat

of clone

characterized

cDNA product;

homolog

electrophoresis

The sequence

previously

The fourth

to encode

on gel

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This expression

(reviewed

in

sensitive blood

the RNA template.

Vol.

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BIOCHEMICAL

3, 1991

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BIOPHYSICAL

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COMMUNICATIONS

SPMLKFB

- 3.0 kb

rLRP Fieure 3. Northern blot LRP cDNA probe. Polyadenylated (P), muscle (M), liver (L), fractionated by electrophoresis

analysis of mUNA from rat tissues using the RNA (20 pg) from rat spleen (S), placenta kidney (K), fat (F) and brain (B) was in a 1% agarose/2.2 M formaldehyde gel.

rat

After transfer to a nitrocellulose filter, the blot was hybridized with labeled cDNA insert from clone 5A3B1, which corresponds to the proximal PTPase

domain

These

results

evaluate

the

reported

for

sensitivity

further

demonstrates of PTPase

human liver

which extract

skeletal

LRP.

expression

muscle

activity, in

rat

of this

skeletal a liver

of

this

has been (2).

muscle

specialized

metabolic

transduction

through

the utility

homologs

of cDNA amplification

in specific

(1.5) and an invertebrate

technique

since

the

is

estimated

to be approximately

that

functions the

low

of the

they

are

of this

insulin

action

The

when applied content

10% of the in regulating

which

to

of PTPase

LAR and LRP PTPase

involved

tissue

as recently

(14).

advantageous

has a relatively

The identification suggests

species

particularly

tissue

tissues,

to

activity

in

homologs the highly

may include

signal

pathway.

ACKNOWLE-S

These studies were supported by a Research American Diabetes Association to Dr. Goldstein DERC grant DK36836. The degenerate primer set Metabolic Biosystems/California Biotechnology,

and Development and the Joslin was generously Inc., Mountain

Award from the Diabetes Center provided by View, CA.

REFERENCES 1.

2. 3. 4. 5. 6.

Goldstein, B.J., Meyerovitch, J., Zhang, W.R., et al. (1991) Adv. Prot. Phosphatases 6, 1-17. Sale, G.J. (1991) Adv. Prot. Phosphatases 6, 159-186. DeFronzo, R.A. (1988) Diabetes 37, 667-687. Charbonneau, H., Tonks, N.K., Kumar, S., et al. (1989) Proc. Natl. Acad. Sci. (USA) 86, 5252-5256. Tonks, N.K., Charbonneau, H., Diltz, C.D., et al. (1988) Biochemistry 27, 8695-8701. Guan, Sci.

K.L., (USA)

Haun, R.S., 87, 1501-1505.

Watson,

S.J.,

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