Vol.

170,

August

No.

3. 1990

BIOCHEMICAL

BIOPHYSICAL

AND

RESEARCH

COMMUNICATIONS

Pages

16, 1990

FUNCTIONAL GALACTOSYL RECEPTORS ON ISOLATED ARE HETERO-DLIGOMERS Janet

A. Oka,

Department The University Received

June

25,

Maryanne

C.S.

Herzig,

1308-1313

RAT HEPATOCYTES

and Paul

H. Weigel

of Human Biological Chemistry and Genetics Texas Medical Branch, Galveston, Texas 77550

1990

Several lines of indirect evidence have supported the conclusion that rat hepatic asialoglycoprotein (or galactosyl; Gal) receptors are hetero-oligomeric. In the present study more direct evidence was obtained using specific antibodies. The Gal receptor contains three different subunits; RHL 1, RHL 2 and RHL 3. Polyclonal antisera that specifically recognize either RHL 1 or RHL 2/3 subunits (Halberg et al., J. Biol. Chem. 262, 9828, 1987) were tested for their abi 1 ity rat to interfere with the specific binding of asialo-orosomucoid to intact hepatocytes. The different antisera used all completely inhibited spec fit These results indicate that functional Gal ligand binding to the receptor. In receptors on the cell surface are composed of multiple types of subunits. addition, no evidence was found to suggest that the two previously descr bed d by functionally distinct receptor populations in hepatocytes can be explaine these receptor populations containing different RHL subunits. We conclude that all receptors on the cell surface are composed of multiple subunits. eJ 1990 Academic

Press,

The designated

Inc.

affinity

purified

rat

hepatic

RHL 1, RHL 2 and RHL 3 (1).

two different

genes

and differ

(1-4).

in their of cells

expression

of functional

ligands

such

gives

a receptor

. . Abbrev1atlons

;

capable

subunits

are

cDNAs for

both

Gal

receptors

However, of

from the

post-translational gene

products

subunits of only

same polypeptide

glycosylation

products

are

required

(4). for

the

can endocytose

naturally

occurring

transfection

with

cDNA for

RHL 1 alone

internalizing

Inc. reserved.

the

three

that

ASOR, asialo-orosomucoid;

$1.50 0 1990 6y Academic Press, of reproducfion in any form

These

of

a highly

BSA, bovine

galactosyl. 0006-291x/90 Copyright All rights

contains

and types

with

as ASOR (5).

receptor

RHL 2 and RHl 3 are derived

extent

Transfection

Gal

1308

the

galactosylated

serum

albumin;

synthetic

Gal,

Vol.

170, No. 3, 1990

ligand

(6).

receptor

data

Antibodies

are

cells,

BIOCHEMICAL

able

that that

the

studies

have

reported

above

results

also

do not

upon detergent

some aspect

of receptor

not for

ligand

specific

antibodies

of receptor

only

receptor

is

that rule

Gal out

solubilization

stability

to bind

detergent

subunits

or function

(e.g.

of These

although (4).

The

subunits

may

are needed

for

recycling)

but

study we have used subunit-

whether

a natural

extracts

one subunit,

receptor

multiple

or human

(7-9).

homo-oligomeric that

In this

more directly

is needed

are

possibility

itself.

to address

of more than

or that

biosynthesis,

from

in the rat

a hetero-oligomer

receptors

the

RESEARCH COMMUNICATIONS

subunits

subunits

are comprised

internalization

subunits

one of the all

native

Gal receptors

other

rearrange

for

to immunoprecipitate

indicating indicate

specific

AND BIOPHYSICAL

a hetero-oligomeric

complex

ligand.

METHODS Materials. Orosomucoid obtained from Sigma was desialylated and ASOR was radiolabelled as described (10). BSA was from Armour Biochemicals (CRG-7). Collagenase (Type D) was from Boehringer Mannheim. Medium l/BSA contains modified Eagle's medium (Grand Island Biological Co.) supplemented with 2.4 g/L Hepes, pH 7.4, 0.22 g/L NaHCO, and 0.1% BSA. Buffer 1 contains 143 mM NaCl, 6.7 mM KCl, and 10 mM Hepes, pH 7.4. Cell DreDaration. Hepatocytes were prepared from male Sprague-Dawley rats Final cell pellets (250 g) by a collagenase liver perfusion procedure (11). Cells were routinely >85% single cells and were suspended in Medium l/BSA. Experiments were performed in the viable as judged by trypan blue exclusion. In some cases, cells were first incubated at 37°C for 1 hr to absence of serum. increase and stabilize cell surface receptor number (12). Antibodies. The polyclonal goat antibody against affinity purified rat Gal receptor has been previously characterized (9). Two different rabbit antisera specific for either RHL 1 or RHL 2/3 were the generous gift of Kurt Drickamer (4). Non-immune goat or rabbit sera were from Sigma. SDS-PAGE using with second antibody-alkaline 10% gels and immunoblotting and detection phosphatase conjugates were performed as recently described (13). Centrifugations of cell suspensions were at 800 rpm for 2 min General. Protein was determined by the in a Sorvall GLC-1 Model Tabletop Centrifuge. method of Bradford (14) using BSA as standard.

RESULTS AND DISCUSSION Western study

with

blot

purified

analysis Gal receptor

of

the

reactivity

verified

of the

the specificity 1309

antibodies

used

of the antibodies

in

this (Fig.

Vol.

170,

No.

3, 1990

BIOCHEMICALANDBIOPHYSICAL

RESEARCH

COMMUNICATIONS

Mr -kDa -92

-66 RHL

3 -

RHL

2 -

RHL

1 -

-45

1

2

3

4

Fiqure 1. Soecificity of antisera for RHL subunits of ourified rat Gal receptor. Affinity purified Gal receptor was run on SDS-PAGE, transferred to nitrocellulose and probed with either polyclonal chicken antiserum to whole receptor (lane l), Appropriate rabbit anti-RHL 1 (lane 2), or rabbit anti-RHL 2/3 (lane 3). secondary antibodies conjugated to alkaline phosphatase were used for visualizaNo reactivity with RHL 1,2 or 3 is observed tion of antibody reactive bands. with any nonimmune or irrelevant primary antisera from goat (lane 4), rabbit or chicken and the appropriate secondary reagents. Polyclonal goat antireceptor antisera, which has already been described (18), shows the same reactivity to RHL 1,2 and 3 as in lane 1.

Polyclonal

1). purified

antisera

rat

receptor

antisera,

however,

against

RHL 1, for

2/3

and likewise

antisera

were

on isolated binding serum

were

had

allowed

of binding

anti used

hepatocytes.

the antiserum

blocks to bind

prior

to distinguish showed

essentially

to determine

(Fig.

against

the

was not effect

binding

observed.

to incubation

to

its

which all

However, with

1310

RHL 1.

are

specific

These

Gal receptor

blocks

antibody,

cells

RHL

to receptors

all

the

Non-immune three

receptors.

with

ASOR, then

with

internalization.

functional

if

against

binding

recognizes

to incubation

Antiserum

reactivity

reactive

active

affinity

The subunit-specific

on ligand

to intact

antiserum,

the

RHL 1 and RHL 2/3.

no cross

2) and prevents

ASOR prior is

(13)

between

RHL 2/3 antiserum

This ligand

or chicken

RHL 1, RHL 2 and RHL 3.

The antiserum

no effect.

or uptake

able

example,

the then

in goat

recognize

of ASOR to cells

effectively first

raised

subunits,

If then

incubated binding

cells

are

no inhibition at 4“C with of ligand

is

Vol.

170,

No.

3,199O

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

60

I 0

I

10

20

02

SERUM

30

40

CONCENTRATION

50

0

60

03

(pi/ml)

----_--_--_---____________________ ' 0 3 6 9 ANTISERUM ( pL)

Fisure 2, Effect of anti-Gal receptor antiserum on liqand bindino to heoatoa. Cell suspensions were incubated at 4'C with 8 rg/ml 3H-ASOR for 90 min, centrifuged, and resuspended in Medium 1 with the indicated final concentrations of goat immune (D) or nonimmune (0) sera against the whole receptor. Suspensions were incubated on ice for 1 hr at 4°C and then 3O'C for 12 min to allow internalization. Other cell suspensions were first incubated with nonimmune (0) or immune (e) sera on ice for 1 hr. 3H-ASOR was added to a concentration of 8 pg/ml, the cells were incubated for 1 hr at 4'C and then transferred to 37'C for 12 min. Internalized radioactivity was determined after washing cells twice by centrifugation at 4'C with Medium 1 containing 15 mM EDTA (10). Fiqure 3. Effect heoatocvtes exoressinq State 1 or State 1 and State 2 Gal receptors. Freshly isolated cells kept at 4'C (State 1 cells) or cells pretreated at 37'C for 1 hr (State 2 cells) and then chilled to 4'C were incubated with the indicated volume of anti-RHL 1 (0) or anti-RHL 2/3 (a) antiserum in Buffer 1 (0.24 ml final volume) for 90 min at 4'C. The cells were washed and resuspended in Buffer 1 containing 5 mM CaCl, and 1.5 fig/ml "'1-ASOR. After 60 min at 4'C the cells were washed and cell associated radioactivity was determined. Dashed lines represent nonspecific "'1-ASOR binding determined in the presence of a go-fold excess of nonradiolabeled ASOR.

inhibited the

by -90%

ability

Therefore,

is not

bound

receptor

the

The presence

2).

of previously

37OC.

The

(Fig.

bound

antibody

ligand

of antibody to

does

be internalized

has no nonspecific

not

interfere

by the

effects

with

receptor

on endocytosis

at if

to receptor.

hepatic subpopulations

Gal

receptor that

system internalize

operates and

1311

by two process

functionally

ligand

by

distinct two

different

it

Vol. 170,

BlOCHEMlCALANDBlOPHYSlCAL

No. 3, 1990

intracellular

pathways

two

populations

receptor

isolated

hepatocytes

37°C express cell

both

surface.

isolated

cells

receptor

receptor

State

activity

including

monensin,

chloroquine

the

2 Gal

transfection

To test

these

the

2 or

only

1 and anti-RHL of

cells

(i.e.

to

finding

with

numbers

on the

only

The State

surface

a variety

depolymerizing

Freshly

1-2 hr express

cell

in at

2 Gal receptors.

surface.

on the

when

drugs,

low

one 2 Gal

cells

of perturbing

differences

the

are agents

temperature,

identical

affinity

would

be

exist.

receptor

is

hepatocytes

1 Gal

activity

1251-ASOR.

present,

then

inhibited

1251:ASOR binding.

on ligand competed If

subunit-specific However,

studies However,

binding effectively

functional antiserum no difference 1312

It

is

several

State

possible one type

results (4)

based

on

indicating other

that studies

(5,7-g).

expressing were

both

of only

with

hetero-oligomeric

possibilities, receptor

ASOR.

is comprised

consistent

of RHL subunits

in receptors

For example, for

population

the two receptor

difference

and pathways.

crosslinking

Gal

between

structural

and chemical

2/3 antiserum

either

of ligand

show

antisera

bind

lost)

cell

defined

equilibrated

equal

at 24'C for

on the

2 Gal receptor

(6)

State

subunit-specific

incubated

populations

receptors

species that

1 and State

of functional

This

concluded

Cells

these

(12,17).

1 or State

homo-oligomeric

(16).

and

in approximately

receptors,

allow

be differentiated

is as yet no identified

studies

characteristics

in suspension

microtubule

receptor

RHL subunit.

to

as State

absence

azide,

two

the State

State

the

there

1 and State

have

these

characterization

subpopulations,

different

subpopulations

1 Gal

or vanadate

Despite

or

is modulated

at 37OC in

nine

pathways

at 4'C or cells

incubated

of

and in culture

kept the

to explain

At least

We designate

population,

that

(15).

RESEARCH COMMUNJCATIONS

either

examined

for

at 4'C (Fig.

3).

and completely homo-oligomeric should

State the

effect

Both for

was observed

of

anti-RHL

the ability

receptors have

1 and

only in the

were

partially ability

Vol.

170,

No. 3, 1990

of either

to inhibit

We conclude

from

in

State

both

Gal

the

receptors

These

BIOCHEMICAL

ligand this

result

support

are

State

to either both

the

receptor

evidence

studies

1 or State

State

(7-9)

2 Gal receptors.

subunits

populations.

obtained

from

RHL 2/3

the

by us and others

are All

RHL 1 and

and contain

indirect

RESEARCH COMMUNICATIONS

RHL 1 and RHL Z/3

2 Gal

hetero-oligomeric

and co-immunoprecipitation receptors

that

1 and the

are

results

binding

AND BIOPHYSICAL

that

present

functional subunits.

transfection

(5)

functional

Gal

and Glenda

Romero

hetero-oligomeric.

ACKNOWLEDGMENTS We thank for

help

of Health,

with

Deborah

Baudy for

the manuscript.

Grant

the preparation

This

research

of hepatocytes was supported

by National

Institutes

GM 30218.

REFERENCES 1.

Drickamer,

2.

259, 770-778. Leung, J.O., Holland,

3. 4.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

K.,

Mamon, J. F.,

Binns,

G. and Leung,

J .O. (1984) J. Biol.

Chem.

K. (1985) J. Biol. Chem. 260, E.C. and Drickamer, 12,523-12,527. McPhaul, M. and Berg, P. (1987) Mol. Cell. Biol. L, 1841-1847. Halberg, D.F., Wager, R.E., Farrell, D.C., Hildreth IV, J., Quesenberry, M.S., Loeb, J.A., Holland, E.C. and Drickamer, K. (1987) J. Biol. Chem. 262, 9828-9838. McPhaul, M. and Berg, P. (1986) Proc. Natl. Acad. Sci. USA 83, 8863-8867. Braiterman, L.T., Chance, S.C., Porter, W.R., Lee, Y.C., Townsend, R.R. and Hubbard A.L. (1989) J. Biol. Chem. 264, 1682-1688. Sawyer, J.T., Sanford, J .P. and Doyle, D. (1988) J. Biol. Chem. 263, 10,534-10,538. M.A. and Lodish, H.F. (1988) J. Cell Bischoff, J., Libresco, S., Shia, Biol. 106, 1067-1074. Herzig, M.C.S. and Weigel, P.H. (1989) Biochemistry 28, 600-610. Weigel, P.H. and Oka, J.A. (1982) J. Biol. Chem. 257, 1201-1207. Oka, J.A. and Weigel, P.H. (1987) J. Cell. Physiol. 133, 243-252. Weigel, P.H. and Oka, J.A. (1983) J. Biol. Chem. 258, 5089-5094. Her-zig, M.C.S. and Weigel, P.H. (1990) Biochemistry 29, (in press). Bradford, M. (1976) Anal. Biochem. 72, 248-254. Weigel, P-H., Clarke, B.L. and Oka, J.A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50. Weigel, P.H. (1987) in Vertebrate Lectins (K. Olden and J.B. Parent, eds) Van Nostrand Reinhold, NY p. 65-91. McAbee, D.D., Clarke, B.L., Oka, J.A. and Weigel, P.H. (1990) J. Biol. Chem. 265, 629-635. McAbee, D.D. and Weigel, P.H. (1988) Biochemistry 27, 2061-2069.

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