Prostaglandin E and F2α receptors in bovine corpus luteum plasma membranes are two different macromolecular entities

Vol. 85, No. 3, 1978

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

December 14, 1978

Pages

PROSTAGLANDIN E AND F,,

1054-1060

RECEPTORS IN BOVINE CORPUS LUTEUM PLASMA MEMBRANES

ARE TWO DIFFERENT MACROMOLECULAR ENTITIES Ch. V. Rao and Candace Departments

of Obstetrics

University

- Gynecology

of Louisville Louisville,

Received

October

20,

W. Harker and Biochemistry

School

of Medicine

Kentucky

40232

1978 SUMMARY

Greater numbers of prostaglandin (PG) F,u as compared to PGE receptors were solubilized from bovine corpus luteum plasma membranes by sodium deoxycholate (SDC) concentrations from 0.01% to 0.1%. However, at 0.5% SDC concentration, virtually 100% of both PGs receptors were solubilized. When these solubilized PGs receptors were chromatographed on a calibrated Sepharose 6B they exhibited a small but reproducible difference in their distribucolumn, tion coefficients. The above results suggest that PGE and PGF,, receptors represent two different macromolecular entities with a relatively small size difference between them. INTRODUCTION Recent F,,

studies

receptors

subcellular

in intact

probe

that

PGE and PGF,o

receptors

more peripheral

(10).

The present

not

from

(1)

receptors

were with

study

PGE and PGFao receptors

association intrinsic

respect

corpora

and plasma

detergents

membrane

two different

with

and other The specificity

membranes

(3,5)support

respect

to spe-

and dimethylsulfoxide receptors

proteins

to solubilize

(PG) E and

(Z-6) lutea.

are discrete

to PGE receptors

MATERIALS Unlabeled PGs were The following Company. sources: [3~1~~~1 (89.5

membranes

of these

was undertaken are

of prostaglandin

bovine

The use of various

of membrane

were

plasma

in cells

PGE and PGF,,

presence

(l),

prepared

receptors

and affinity. the nature

the

cells

(6-9)

of these

conclusion

cificity

viable

fractions

and affinity the

have demonstrated

in

revealed but

and to examine

macromolecular

that

the PGF,,

the membrane

to

receptors

structure whetheror

entities.

AND METHODS

generously supplied by Dr. John Pike of the Upjohn items were purchased from the indicated commercial from New c'/1 mmole) and [3HlPGF~o (178 Ci/mmole)

0006-291x/78/0853-1054$01.00/0 Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

1054

both

BIOCHEMICAL

Vol. 85, No. 3, 1978

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

England Nuclear Corp.; catalase (M.W.-232,000), ferritin (M.W.-400,000), thyroglobulin (M.W.-669,000) and Sepharose 6B from Sigma Chemical Co.; sodium deoxycholate (SDC) from Nutritional Biochemical Corp.; RPI Scintillator No. 111023 from Research Products International Corp.; Gelman Metricel filters (0.45 urn pore size) from Scientific Products and blue dextran 2000 from Pharmacia Fine Chemicals. Human hemoglobin (M.W.-64,500) was a generous gift from Dr. Robert Gray of our institution. Both [3~1~~~ were checked for purity and if it was less than 95%, they were further purified as described earlier (10). Aliquots of r3H]PGs stocks were diluted with redistilled ethanol and stored under nitrogen at -20' between uses. The procedures for the collection of bovine corpora lutea and isolation of purified plasma membrane fractions have already been described (6,ll). These membranes exhibited about 32 fold enrichment of 5'-nucleotidase activity Plasma membrane fractions were stored in as compared to the homogenate (6). aliquots at -20' until used. Aliquots of plasma membrane protein (3.2 to 14.0 mg in different experiments) were preincubated with either 30 nM r3H]PGEr (38O, 1 hr) or 10 nM [3H] in 0.5 ml of 10 mM Tris-HCl, pH 7.3 containing 250 mM suPGFz, (22', 2 hrs), crose and 1 mM Ca*+. Then 0.5 ml of 1% SDC in 10 mM Tris-HCl, pH 7.0 was added and incubation continued for another 1 hr at 4'. Following this incubation, the tubes were centrifuged at 48,000 x g for 30 min at 4' and supernates were careAliquots of the supernates were counted and fully decanted into clean tubes. the remainder was layered on a Sepharose 6B column (1.5 x 53 cm) and eluted with 10 mM Tris-HCl, pH 7.0 buffer containing 0.25% SDC (receptor aggregation occurred in SDC less than 0.25%) at 4" in fractions of 1.6 ml each. Absorbance at 280 nm and radioactivity in 0.5 ml aliquots of the fractions were measured. The radioactivity was measured (-20.0% counting efficiency) in a Packard liquid scintillation counter with 10 ml of scintillation fluid which consisted of toluene, Triton X-100, and RPI's scintillator No. 111023 in the ratio of 2750: 900:150 ml respectively. Protein was determined in selected column fractions (as well as in plasma membranes) according to Lowry et al (12) using bovine serum albumin as the standard. For column fractions, a blank containing detergent was employed. The Sepharose 6B column was calibrated with hemoglobin, catalase, ferritin and thyroglobulin. These proteins were chromatographed using the same buffer as the solubilized PG receptors. The void volume (V,) was determined as the elution volume for blue dextran 2000. The trailing end of the elution curve of r3HlPGs was used to determine the total volume (V,). K,,, (13) were calculated for the receptor bound r3H]PGs and marker proteins. All the experiments were repeated at least three times. ether pertinent details are piven in figure and table legends. RESULTS Table

I shows

centrations both

pared

It

should

a maximal

with

be noted, were difference

of plasma

in a progressively

receptors

to PGE receptors

0.1% with

pretreatment

of SDC resulted

PGE and PGFz,

0.5% SDC.

that

virtually

with

increasing

that

a'greater

number

at SDC concentrations

at 0.05%

1055

SDC.

increasing

solubilization

100% solubilization

however,

solubilized

membranes

occurring

conof at

of PGF2, as comfrom 0.01%

to

8lOCHEMlCAL

Vol. 85, No. 3, 1978

AND BIOPHYSICAL

TABLE

The

Effect

of

Concentration (v/v),

Pretreatment on Subsequent

of

I

of Plasma

Membranes

[3~1~~~ Specific [3H]PGEl

SDC

RESEARCH COMMUNICATIONS

with Binding

Bound Percent

%

of

I

Sodium

Deoxycholate

[3H]PGF,,

Bound

control

0.01

97.0

f 1.0

88.0

t 2.0

0.05

68.0

_+ 2.0

25.0

f 1.0

0.1

14.0 + 1.5

4.0

0.5

0.0

i

1.7

0.0

Membrane protein aliquots were preincubated for 1 hr at 4' with increasing concentrations of SDC, centrifuged (6,000 x g, 15 min) and the pellets were washed once with 10 mM Tris-HCl, pH 7.0. The washed pellet;+were resuspended in 10 mM Tris-HCl pH 7.0 containing 250 mM sucrose, 1 mM Ca , 1 mM dithiothreitol and 0.1% gelatin, aliquots of3200-600 ug g rotein were incubated with 12 nM [3H]PGE, (38'. 1 hr) and 6 nM [ H]PGFzcl (22 , 2 hrs). The rest of the details on this binding assay are the same as those described previously [9]. The membranes for controls were handled in a manner identical to treatment tubes except that SDC was not present during preincubation. The binding in control tubes, 69.0 and 143.1 fmoles/mg membrane protein for [3H]PGE, and [3H]PGF,a

respectively,

tors

was taken as 100%.

When SDC solubilized

prelabeled

were

on a Sepharose

chromatographed The first

two peaks. protein

came off

peak corresponds

radioactive

The rechromatography sulted

in

free

bound

['HlPGs.

hibition

of

binding

(Fig.

presence

of

(Fig. lar

[3~1~~~1

1B) in their

1B) but not

of excess (Fig. respective

unlabeled

of

[3~1~~~1

PGF

the

volume

peak

second

1A) but

it

(Fig.

peaks.

10.56

following

while

in

the second the first

for

r3H]PGFza.

lypholization peak

little peaks.

re-

represents peak

PGEI resulted had very

1B) recep-

was eluted

* 0.000

radioactive

macromolecular

(Fig.

The Kav for

radioactive

unlabeled

2cL resulted

binding

the void

and 0.289

The first while

binding

protein

peak.

[3H]PGE1

radioactive

profile.

presence

The

with

the

radioactive for

first

radioactivity,

,

excess

first

1A) and PGFzo

6B column,

column

+ 0.014

of the

the same elution

romolecular

the

to the

peak was 0.272

PGE (Fig.

mac-

represents

in a complete effect

on [3H]PGFlg

Similarly,

in a complete

inhibition

1A) in their

respective

in-

the of

[3H]PGF~o

macromolecu-

BIOCHEMICAL

Vol. 85, No. 3, 1978

PH]

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

PGE,

VI J.

VO 24-

4

te-

120.5

0.3 z ::

,I

3 : 0.1 a D z 0 1

[Vi] PGF,a

1

0.5

6 0.3

0.1

IO

20 Fraction

40 50 30 Number. lI.6ml/FracIion)

60

70

Figs. 1A & 1B - Gel filtration of free and [3H1PGs-receptor complexes solubilized from bovine corpus luteum plasma membranes with SDC. Plasma membranes were preincubated with t3~Ip~s alone (closed triangles), or with t3HIPGs and 5.6 x 10m6M unlabeled PGEl (open circles) or 5.6 x 10m6M unlabeled PGFzo (closed circles) prior to solubilization. The average total recovery of the radioactivity from the column was 85.9% of which 26.8% was recovered in the macromolecular peak. Variation in fraction sizes (open and closed circles in 1B) required us to normalize the data before graphic display. The flow rate in this figure and in figure 2 was 1.6 ml/5 min.

1057

BIOCHEMICAL

Vol. 85, No. 3, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.5

2

0.3

0.1

I

I

I

I

5.0

LOG

Fig. 2 - Estimation of molecular complexes by gel filtration on a patterns for the marker proteins 405 nm (hemoglobin, ferritin and elution patterns for [3H]PG-receptor measurements. The K avs presented

In order Sepharose globular

to obtain

6B column proteins

whose

The molecular

weights

tor

respectively

complexes

log molecular

weights

molecular

were

MW

estimate

in the above

of 346,736

I

6.0

weights of solubilized PGE and PGF,o receptor calibrated Sepharose 6B column. The elution were followed by measurement of absorbances at thyroglobulin) and at 280 nm (catalase). The complexes were followed by radioactivity are the mean of 3 experiments.

a preliminary

used

I

of their

studies

weights

was calibrated

ranged

and 323,953 obtained

of the marker

molecular

from

for

from

using

64,500

[3~1~~~1

the

weights, four

marker

to 669,000

and

relationship

a

(Fig.

[3HlPGFzo-recepbetween

Kav vs

proteins.

DISCUSSION The SDC solubilized at g forces size truly

up to 322,000,

and not

adsorbed

solubilized

tracts of which

PGE and PGFno-receptor

by charcoal

PGs-receptor

on a Sepharose corresponded

not

6B column,

retained

by Metricel

(data complexes. the

complexes

not

shown).

This

Following

gel

radioactivity

to the macromolecular

1058

filters

appeared

were

non-sedimentable

of 0.45 indicated filtration

Urn pore that

SDC

of SDC ex-

in two peaks,

peak and the other

to free

one

[3HIPG~.

2).

BIOCHEMICAL

Vol. 85, No. 3, 1978

The macromolecular not

radioactive

t3H]PGs-detergent

ited plasma

plexes

a calibrated were

weights

represent

two different

assigned

this

with

but

peak exhib-

cells

molecular

to the PGE-receptor of SDC.

macromolecular

from membranes

and

then

should

have been

of detergent

is

only

If

these

receptor

identical.

com-

difference

7.5%

the two receptors

these

of

and PGFzo-receptor

Despite

entities.

weights

appear

in to

two receptors

losses

But clearly

were

due to solubilizathey were

not

(see

I).

ations long

as its

absence

as detergent

(14,15)

is

filtration

teins

present,

problems

tion.

This

leaves

one possible

receptor

corrected

for

receptor

complexes

in

Even in this

detergent sucrose

procedure,

above discussion, be considered

certain

the molecular as preliminary

variety

of other

in the presence

of detergents

There

was one study

a gel

filtration

solubilized

from bovine

gel avenue

for

binding. density

This

gradients

assumptions weight

which

pattern

have

corpus

plasma

11159

are

simple

pro-

more

gel

filtra-

weight

of the of

H,O and D,O (18).

presented for

In view in this

which

of the

paper

comparison

weights

should

with

were

a

determirmd

19).

used an uncalibrated

luteum

that

to be made.

as a basis

(0.35)

of marker

centrifugation

containing

in ref.

and a K,,

(13,16,17)

a molecular

molecular

(reviewed

comparison

involves

estimates

receptors

SDC binding

than

obtaining

as

anomalous

to that

electrophoresis)

determin-

Therefore,

even in techniques

and can serve

membrane

in the

PGs receptors faced

weight

aggregation.

involved

are

polyacrylamide

molecular

such as possible

of solubilized weights

for

receptor

and limitations

of known molecular (i.e.

essential

to inevitable

behavior

sophisticated

quite

leads

to PG receptors

of the

tain

in

studies

approximate

of PGE and PGFzo complexes,

The presence

wide

complexes

the binding from

6B column,

on the same macromolecule,

Table

because

receptors

in the presence

molecular

tion

Sepharose

respectively,

present

of these

[3H]PGs-receptor

(l-5).

and 323,593

346,736

represent

complexes

expected

membranes Using

peaks

micelle

a specificity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Sepharose for

PGFzo-receptor

membranes

(20).

6B column

to ob-

complexes The difference

BIOCHEMICAL

Vol. 85, No. 3, 1978

in Kav between SDC concentrations

our

study used

and theirs in

the elution

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

(20)

be explained

may perhaps

by different

buffer.

ACKNOWLEDGEMENTS The work was supported Mr. Jan Hai Lin, the help of and helpful discussions and Robert Benson are gratefully

by NIH grant HD09577. The technical assistance of Dr. S. Mitra in the preparation of plasma membranes critique on the manuscript by Drs. Robert Burton and acknowledged. REFERENCES

1)

2) 3) 4) 5)

6) 7) 8) 9) 10) 11)

12) 13) 14) 15) 16) 17) 18) 19) 20)

Lin, M. T. and Rao, Ch. V. (1977) Biochem. Biophys. Res. Comm., 78, 510-516 Rao, Ch. V. (1974) J. Biol. Chem., 249, 7203-7209 Rao, Ch. V. (1975) Biochem. Biophys. Res. Comm., 64, 416-424 Powell, W. S., HammarstrGm, S. and Samuelsson, B. (1975) Eur. J. Biochem., 56, 73-77 Kimball, F. A. and Lauderdale, J. W. (1975) Prostaglandins, 5, 313-331 Rao, Ch. V. and Mitra, S. (1977) Biochem. Biophys. Res. Comm., 7&,636-643 Mitra, S. and Rao, Ch. V. (1978) Arch. Biochem. Biophys., 185, 126-133 S. and Rao, Ch. V. (1978) Arch. Biochem. Biophys., In Press. Mitra, Rao, Ch. V., and Mitra, S. (1978) Submitted for publication. Rao, Ch. V. (1977) Life Sci., 0, 2013-2022 Gospodarowicz, D. (1973) J. Biol. Chem., 248, 5050-5056 Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275 Laurent, T. C. and Killander, J. (1964) J. Chromatogr., l4, 317-330 Helenius, A. and Simon, K. (1972) J. Biol. Chem., 247, 3656-3661 Makino, S., Reynolds, J. A. and Tanford, C. (1973) J. Biol. Chem., 248, 49264932 Siegel, L. and Monty, K. J. (1966) Biochim. Biophys. Acta, 112, 346-362 18, 1-51 Andrews, P. (1970) Methods in Biochem. Anal., Smigel, M. and Fleischer, S. (1977) J. Biol. Chem., 252, 3689-3696 Cuatrecasas, P. (1974) Ann Rev. Biochem., (Snell,E.E.,Boyer,P. D.,Meister,A. and Richardson, C.C., eds)Vol.s,pp.169-214 Annual Reviews,Inc.,Palo Alto,Ca. Hammarstrb'm, S., Klyden, U., Powell, W. S. and Samuelsson, B. (1975) F.E.B.S. Lee. , -,50 306-310

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