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[24] I s o l a t i o n o f C e l l M e m b r a n e for E p i d e r m a l G r o w t h Factor Receptor Studies

By

PETER

H.

LIN,

RICHARD

SELINFREUND,

and

WALKER

WHARTON

Introduction We have developed a rapid procedure for the purification of cell membrane from cultured cells. Two versions of this purification method are included in this chapter. The large-scale version provides a convenient way to process either liter quantities of high-density spin cultures or several hundred large plates of cell cultures. ~ The small-scale version is routinely used to extract milligrams amounts of cell membrane from as few as four confluent plates (25 × 150 mm) using a 7 rain centrifugation protocol. 2 Traditional extraction of cell membranes from cultured systems relies heavily on lengthy ultracentrifugation cycles (i.e., 2 to 20 hr) to selectively sort out the plasmalemma from other organellar membranes. 3-5 The yield and lot-to-lot reproducibility is typically poor because a large number of physical parameters has to be carefully controlled. Small variations in key steps, such as the type and duration of cell homogenization, can significantly alter the size of different membrane fractions and decrease the purity of the final product. The new purification method developed here is substantially faster (i.e., < ! hr of centrifugation time) and has eliminated the need for density gradient ultracentrifugation. Deviations during cell homogenization is also less critical since the process is taken to completion with all membranous components sheared into small vesicles. The new method takes advantage of a unique property of calcium ion, which at millimolar concentrations preferentially induces aggregation of nonplasmalemma membranes. 6-8 The membrane aggregate is easily removed from the homogenate by a

t p. H. Lin, R. Selinfreund, E. Wakshull, and W. Wharton, Biochemistry 26, 731 (1987). z p. H. Lin, R. Selinfreund, E. Wakshull, and W. Wharton, Anal. Biochem. 168,300 (1988). 3 D. Thorn, A. Powell, C. W. Lloyd, and D. A. Rees, Biochem. J. 168, 187 (1977). 4 S. Cohen, this series, Vol. 99, p. 379. 5 T. D. Butlers and R. C. Hughes, Biochem. J. 140, 469 (1974). 6 j. B. Shenkman and D. L. Cinti, this series, Vol. 52, p. 83. 7 S. A. Kamath and E. Rubin, Biochem. Biophys. Res. Commun. 49, 52 (1972). 8 p. Malathi, H. Preiser, P. Fairclough, P. Mallett, and R. K. Crane, Biochim. Biophys. Acta 554, 259 (1979).

METHODS IN ENZYMOLOGY.VOL. 198

Copyright © 1991by Academic Press. Inc. All rights of reproduction in any forrn reserved.

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E G F RECEPTOR AND RELATED RECEPTORS

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simple centrifugation step. The plasma membrane is subsequently collected from the supernatant with a second centrifugation cycle. Cell Membrane Purification Procedure An outline of the cell membrane purification is shown in Fig. 1. All steps are performed at 4°. Reagents. All reagents are stored at 4°. Phosphate-buffered saline (PBS: 140 mM NaC1, 10 mM sodium phosphate, pH 7.4) Hypotonic lysis buffer (50 mM mannitol, 5 mM HEPES, pH 7.4) CaCI2, 1 M in deionized water

Cell Harvest and Wet Weight Determination Cells are grown in 5% CO2 at 37 °. The culture medium is changed every 4 days. On reaching confluency, cells are rinsed once with PBS and harvested from the plates with a wide blade rubber policeman. The wet weight of the cells is determined by pelleting the cells in preweighed centrifuge tubes (10 min, 2000 g). Approximately 0.1-0.2 g of cells can be obtained from a single confluent P150 (25 x 150 mm) culture plate. Highdensity spin cultured cells are directly harvested by centrifugation using preweighed 250-ml centrifugation bottles. Large-scale Procedure. The large-scale procedure should be used for cell harvests that are greater than 1 g per harvest. Thirty volumes (1 : 30, w/v) of hypotonic lysis buffer is added to the harvested cells. The mixture is immediately homogenized in a Waring blendor at maximum speed for 5 min. The 1 M calcium chloride solution is added to the homogenate with stirring to a final concentration of 10 mM. The mixture is stirred vigorously for I0 min more to ensure even distribution of the calcium ion. Calcium-induced membrane aggregates are sedimented by a 3000 g centrifugation (i.e., SS-34 rotor at 5000 rpm) for 15 min. The solid pellet (P1 fraction) containing nuclei, lysosomes, and endoplasmic reticulum is discarded. The slightly turbid supernatant is tranferred to a clean set of centrifuge tubes. The plasma membrane is pelleted with a 48,000 g spin (i.e., SS-34 rotor at 20,000 rpm in a RC-5C centrifuge) for 30 min. The whitish, translucent pellet (P2 fraction) is the purified cell membrane. Small-scale Procedure. Cells from four confluent P150 plates are harvested. Ten volumes (1 : 10, w/v) of the hypotonic buffer is added to the harvested cells. The mixture is homogenized by aspiration (6 times) through two 25-gauge needles using the vacuum homogenization apparatus (Fig. 2). Alternatively, when very small amounts of cells are used, homoge-

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ISOLATION OF CELL MEMBRANES

253

small-scale

large-scale Harvest cells, measure net wet weight

1

add mannitol buffer (1:30, w/v) Waring blender (5 min, full speed)

I

.

add mannitol buffer (1:10, w/v) vacuum homogenization (6 passes, 25-gauge needles) Add CaCI to 10 mM

_

I

I

1

SS-34 rotor (3,000 g, 15 rain) =-

Eppendorf microfuge (15,600 g, 1min) Discardpellet (P1)

SS-34 rotor (48,200 g, 30min) I I

~

Beckman TL-100 (430,000 g, 6 min) Discard supirnatani (S)

~

I~.R~i~SPo~ndnc~e/Itorme,mbirqaun ~ Pi~lle~(eP2.~

II FIG. 1. Outline of the membrane purification procedure. Distinct steps of the large-scale and small-scale purifications are bracketed on the left-hand and right-hand sides, respectively. Common steps for the two protocols are positioned in the middle.

254

E G F RECEPTOR AND RELATED RECEPTORS

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tO Vacuum (

b ,.°-

II

p

-7 m

C

..

A

13

FIG. 2. The vacuum homogenization apparatus is constructed from sterile, disposable plasticware. A 50-ml conical centrifuge tube (A) is stopped with a silastic rubber stopper (a). Vacuum is applied to the tube through a 1.5-mm cannula and 18-gauge needles (b, shown with Leur adaptors). Cells in hypotonic lysis buffer (B) are homogenized by aspiration through the two 25-gauge needles (c) and is collected in tube A. Multiple units can be constructed for parallel sample processing. Needles are replaced upon clogging.

nization can be done manually by aspiration through a l-ml syringe and a 25-gauge needle. Calcium chloride solution (1 M) is immediately added to the homogenate to a final concentration of 10 raM. The mixture is vortexed vigorously for 1 min. Aliquots of the homogenate are placed into 1.5-ml microfuge tubes. Calcium-induced membrane aggregates (i.e., the PI fraction) are sedimented by a l-min 15,600 g centrifugation (14,000 rpm in a Eppendorf microfuge). The supernatant is transferred to 1-ml polycarbonate tubes, and the cell membranes (i.e., the P2 fraction) are collected with a 6-min centrifugation in a Beckman TL-100 tabletop ultracentrifuge (TLA100.2 fixed-angle rotor at 430,000 g).

Resuspension and Storage of Purified Cell Membrane Resuspension of the purified membrane pellet (P2 fraction) from the 1-ml polycarbonate tubes is easily accomplished by aspiration through a 25-gauge needle using a 1-ml plastic syringe. For the large-scale preparation, membrane resuspension is done automatically using the vacuum homogenization unit. The membrane pellets are scraped away from the

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ISOLATION OF CELL MEMBRANES

255

centrifugation tubes and repeatly aspirated through 25-gauge needles as explained (Fig. 2). The purified cell membranes can be resuspended in different buffers by repeating the 430,000 g centrifugation cycle as needed. Excess calcium in the membrane is removed, if necessary, with one wash of EDTA (2 mM) buffer. The protein concentration is determined using the bincinchoninic acid assay 9 (available through Pierce, Rockford, IL). Aliquots of the cell membrane preparation are stored frozen in liquid nitrogen. Under such storage conditions, the biological activity of the membranes is preserved with no loss of activity for 6 months. Characterization of Purified Cell Membranes Plasma membranes from human epidermoid carcinoma A431 cells or KB cells are purified as described above. The purity of the cell membranes from both large-scale and small-scale methods is determined by membrane marker enzyme assays. In addition, membrane samples from the largescale purification are processed for transmission electron microscopy. The activity of the EGF receptor is determined by 125I-labeled EGF binding and receptor phosphorylation assays.

Relative Specific Activity Analysis The purified A431 membranes are enriched 7- to 12-fold over the starting homogenate, as indicated by the relative specific activity (RSA) analysis of alkaline phosphatase (Table I). The ratios are substantially better than the 1.8- to 3.1-fold purification of a 2-hr procedure 3 and are comparable to the purification obtained using a 20-hr gradient procedure. 5 Cell membranes purified from KB cells have a similar profile (RSA ratio of 9) and has been reported. ~ Consistent with the electron microscopy data, RSA analysis also demonstrates that contamination from endoplasmic reticulum or lysosomal membranes in the purified cell membrane preparation is minimal (Table I).

Transmission Electron Microscopy The purified A431 cell membrane fraction consists of a population of small vesicles with an average diameter of 100 A (Fig. 3). No contamination from nuclei, lysosomes, or endoplasmic reticulum is detected. Transmis9 p. K. Smith, R. I. Krohn, G. R. Hermanson, A. K. Mallia, F. H. Gartner, M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Anal. Biochem. 150, 76 (1985).

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E G F RECEPTOR AND RELATED RECEPTORS

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TABLE 1 RELATIVE SPECIFIC ACTIVITY ANALYSIS OF PURIFIED MEMBRANE PELLET FROM A431 CELLS" RSA ratio (P2/H) b

Enzyme Alkaline phosphatase 5'-Nucleotidase Giucose-6-phosphatase Acid phosphatase

Marker of

Largescale

Smallscale

Cell membrane Cell membrane Endoplasmic reticulum Lysosome

12.0 5.2 1.2

6.7 3.6 0.3

2.3

1.3

a P2 fraction. From Refs. 1 and 2. b RSA ratio equals the enzyme specific activity of purified cell membrane (P2) divided by the enzyme specific activity of homogenate (H).

sion electron microscopy of purified cell membranes from KB cells reveals a similar morphology.I Epidermal Growth Factor Receptor Profile of Purified Cell Membranes

Binding Profile ~25I-Labeled EGF binding to purified cell membranes is rapid, saturable, and reaches equilibrium within 30 min. L2 The binding specificity of ~25I-labeled EGF to purified cell membranes of both A431 and KB cells is greater than 90% (Table II). Scatchard analysis shows that the equilibrium binding constants for both cell lines are in the nanomolar range (Table II). In particular, the Kd of 1.2 nM for purified A431 cells membranes agrees with a previously reported value of 1.5 nM. ~°

EGF-Dependent Receptor Autophosphorylation Using Purified A431 Cell Membranes Using the purified cell membranes, distinct EGF-dependent receptor autophosphorylation can be demonstrated at 170 kDa without the need for prior immunoprecipitation (Fig. 4a). ~l'j2 The known divalent cation dependency of the EGF receptor kinasC 3 is illustrated in Fig. 4b where l0 j. A. Fernandez-Pol, J. Biol. Chem. 2611, 5003 (1985). II G. Carpenter, L. King, and S. Cohen, J. Biol. Chem. 254, 4884 (1979). t2 p. H. Lin, R. Selinfreund, and W. Wharton, Anal. Biochem. 167, 128 (1987). 13 E. M. Wakshull and W. Wharton, Proc. Natl. Acad. Sci. U.S.A. 82, 8513 (1985).

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FIG. 3. Transmission electron microscopy of purified cell membranes from A431 cells. The membrane sample was fixed in 3% glutaraldehyde, 1% OsO3 and stained with lead citrate and uranyl acetate as described. 1 Black Box: 100 A.

the addition of 3 mM EGTA inhibits the EGF-dependent receptor autophosphorylation. Western blotting of the purified cell membranes with ~25I-labeled EGF detects both forms of the EGF receptor at 150 and 170 kDa (Fig. 4c). The

258

E G F RECEPTOR AND RELATED RECEPTORS

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TABLE II 125I-LABELED EGF-BINDING TO PURIFIED CELL MEMBRANES FROM A431 AND KB CELL LINESa Cell type

Specificb binding (%)

Kd (nM)

Bmax (pm/mg protein)

A431 KB

93 92

1.2 0.14

5.3 0.1

" From Ref. 1. b Specific binding equals total 1251-labeled EGF bound minus background binding (in the presence of excessive unlabeled EGF).

170 kDa

~i~~i~ i~ ~ ~ ~

+

--

a

-I-

b

c

d

e

FIG. 4. EGF receptor detection by autophosphorylation and Western blot using purified A431 cell membranes. (a) A431 receptor autophosphorylation in the presence (+) and absence ( - ) of EGF. (b) EGF receptor autophosphorylation with ( + ) and without ( - ) EGF in the presence of 3 mM EGTA. (c) Western blot of EGF receptor using 1251-labeled EGF. (d) Immunoblot of EGF receptor using 125I-labeled anti-EGF receptor antibody (#528). (e) lmmunoblot of EGF receptor using 125I-labeled anti-EGF receptor antibody (#225). EGF receptor autophosphorylation using [y-32p]ATP was performed as described. HI Western blot and immunoblots using 125I-labeled proteins were processed as reported, r2

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259

150 kDa form of the receptor has been suggested to be a proteolytic product of the 170 kDa receptor. 14-16Last, immunoblot analysis using ~25Ilabeled anti-EGF receptor antibody (#528 and #225) to the purified cell membrane shows a strong reactive band at 170 kDa indicating the presence of EGF receptor (Fig. 4d,e). Summary The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron microscopy, membrane marker enzyme analysis, binding study, EGF-dependent receptor autophosphorylation, and Western blots all demonstrate the biological activity of the purified cell membranes. The membrane purification procedure has been adapted by others 17in assessing EGF kinase activity and has been used for the purification of cell membranes from other types of cultured cells. 18 Acknowledgments The authors wish to thank Dr. D. Urquhart for helpful comments. This work was supported in part by the United States Department of Energy under Contract W-7405-ENG36 with the University of California, and in part by the United States Army Medical Research and Development Command, Fort Detrick, Maryland.

14 S. t5 R. 16 p. 17 S. 18 R.

A. Burow, S. Cohen, and J. V. Staros, J. Biol. Chem. 257, 4019 (1982). Gates and L. E. King, Biochemistry 24, 5209 (1985). Ghosh-Dastidar and C. F. Fox, J. Biol. Chem. 259, 3864 (1984). E. Shoelson, M. F. White, and C. R. Kahn, J. Biol. Chem. 264, 7831 (1989). Hattori, K. K. Hamilton, R. P. McEver, and P. J. Sims, J. Biol. Chem. 264, 9053 (1989).

Isolation of cell membrane for epidermal growth factor receptor studies.

The cell membrane isolation procedure we developed here can be scaled up from four to several hundred plates of cultured cells. Transmission electron ...
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