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[15] I d e n t i f i c a t i o n a n d A s s a y o f F i b r o b l a s t G r o w t h Factor Receptors
By MIKIO KAN, ER-GANG SHI, and WALLACE L. MCKEEHAN Introduction Heparin-binding (fibroblast) growth factors (HBGF) constitute a structurally defined family of polypeptides that are widely distributed in tissues. Although originally described as fibroblast growth factors FGF, the polypeptides exhibit a wide spectrum of biological activities on both mesenchymal and epithelial cells that includes stimulation and inhibition of cell growth and effects on gene expression. Currently the HBGF family consists of seven cloned genes with overall amino acid sequence similarity of 19% (see Ref. 1). At least five members of the family exhibit an affinity for the glycosaminoglycan heparin or similar molecules which affect the structure, activity, and protease sensitivity of the polypeptides. An important advance in understanding the mechanism of action of the HBGF family has been the identification of specific cell membrane receptors for the polypeptides and dissociation of the receptors from the abundant cell-associated extracellular matrix heparin-like binding sites. This chapter describes methods for iodination and recovery of high specific activity ~25I-labeled HBGF and for assay of HBGF receptors. Methods underlying advances in characterization of HBGF receptors are also reviewed. Iodination of HBGF-1
Preparation of HBGF-1 for lodination HBGF-1 is a very sticky polypeptide, especially at neutral pH. Although bovine serum albumin (BSA), heparin, and reducing agents such as dithiothreitol (DTT) prevent HBGF-1 from being sticky (Table I), these additives and low pH interfere with the iodination reaction. Therefore, freshly isolated and reduced HBGF-1 must be prepared for the iodination to carry out the reaction under nonsticky conditions. Partially purified HBGF-1 is prepared from bovine brain as described previously. 2 One milligram of the bovine brain heparin-Sepharose (0.65 to I W. H. B u r g e s s a n d T. M a c i a g , Annu. Rev. Biochem. 58, 575 (1989). 2 j . W. C r a b b a n d W. L . M c K e e h a n , Anal. Biochem. 164, 563 (1987).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE I ADSORPTION OF HBGF-1 TO PLASTIC TUBES a Conditions PBS Acid PBS PBS PBS
Adsorption (%)
(pH 7.2) (pH 2.0) + B S A (1 mg/ml) + heparin (25/~g/ml) + D T T (10 m M )
63 2.8 6.8 13.0 18.6
A p p r o x i m a t e l y 500 pg of 125I-labeled HBGF-1 (specific activity 2.1 x 105 cpm/ng) was added in polypropylene tubes containing I ml of each indicated solution. After incubation for 15 hr at r o o m temperature, soluble and adsorbed (tube) radioactivity was determined in a y-scintillation counter.
2 M NaCI) fraction is reduced by adding 10 mM (final) DTT at neutral pH for 1 hr at 37 °. This reduction is very important to get good recovery by preventing adsorption of H B G F to reaction tubes (Table II). The solution is immediately acidified by adding trifluoroacetic acid and acetonitrile at final concentrations of 0.1% and 10% (v/v), respectively. After clarification by centrifugation, the solution is injected into C 4 reversed-phase HPLC column (25 cm x 4.6 mm Phenomenex, Palos Verdes, CA). HBGF-1 is eluted by linear gradient from 36 to 40% of acetonitrile as described previously. 2 The concentration of purified HBGF-1 is around 20-40 tzg/ml. The purified HBGF-I is immediately concentrated to 100/zg/ml
T A B L E II EFFECT OF REDUCTION ON RECOVERY OF HBGF-1 UNDER DIFFERENT IODINATION CONDITIONS a Reduction of starting material
Reduction after chloramine-T treatment
-
-
-
+
+ +
+
Recovery (%) 2.6
4.9 4.5 23.2
a Partially purified H B G F - I was purified by C 4 reversed-phase H P L C after t r e a t m e n t with or without D T T (10 m M ) . After chloramine-T treatment, the reaction was stopped with 10 m M D T T or e x c e s s KI and tyrosine.
160
FIBROBLAST GROWTH FACTOR
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and used for iodination. The remaining HBGF-1 is stored in the presence of 10 rnM (final) DTT at - 2 0 ° for use as a competitor.
Iodination Reaction The reaction is carried out at room temperature in an iodination hood in a 2-ml conical siliconized tube. Reagents are added in the following order: 1. 2. 3. 4. 5.
50/zl of HBGF-1 ( - 5 / z g ) 100/xl of 0.25 M phosphate buffer (pH 7.0) 20/.d of Na125I ( - 2 mCi, Amersham, Arlington Heights, IL) 50/zl of rinse of the NalZSI vessel with phosphate buffer 30/zl of chloramine-T (400/zg/ml in phosphate buffer, freshly made)
The total reaction volume is 250/xl. After each addition, the reactants are shaken gently every 30 sec. The reaction time is 90 sec. At the end of the reaction time, 250/xl of 10 mM DTT in phosphate buffer (pH 7.0, freshly made) is added to stop the reaction and reduce the iodinated HBGF-1 (total volume is 500/.d). This reduction is also very important to get good recovery (Table II). After a further 10-min incubation, a 5-tzl aliquot is removed from the iodination mixture and added to 1 ml of phosphate buffer containing 1 mg/ml BSA for a determination of trichloroacetic acid (TCA) precipitation. Incorporation of ~zsI into HBGF-1 is determined by mixing equal volumes of cold 20% (w/v) TCA with the solution and precipitating JzSI-labeled HBGF-1. TCA-precipitable radioactivity is usually over 50%, and specific activity is around 2.5-5 × 105 counts/min (cpm)/ng.
Separation of 125I-Labeled HBGF-1 from Free Na125I To remove unincorporated ~zsI, the reaction mixture is applied to a heparin-Sepharose column. Heparin-Sepharose beads (400/.d; Pharmacia, Piscataway, NJ) equilibrated by phosphate buffer containing 0.5 M NaC1 is poured into a disposable plastic column (2.0 ml, Bio-Rad, Richmond, CA). The column is washed first by 1.5 M NaCl and 1 mg/ml BSA (10-20 ml) and then equilibrated by 0.5 M NaCl in phosphate buffer (10-15 ml). The iodinated mixture (-500 ~l) is loaded on the column at a flow rate of about 0.1 ml/1.5 min. After washing the column with 15 ml of 0.5 M NaCl to remove free ~25I,the JESI-labeled HBGF-1 is eluted by 1.5 M NaCl at a flow rate of about 0.05 ml/min into conical tubes containing l0/~l of 5 mg/ml BSA. Each collection volume is approximately 0.5 ml, and the total collection volume is 2 ml (four tubes). Five microliters of each of the aliquots is removed to measure recovery. The lzSI-labeled HBGF-I is stored at - 70 ° until use. The recovery is around 20-50%. The ~25I-labeled
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FIBROBLAST GROWTH FACTOR RECEPTORS
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HBGF-1 is diluted 10-fold with NaCl-free phosphate buffer containing 1 mg/ml BSA prior to use. Binding Assay of HBGF-1 Cell Surface Receptors
Preparation of Cells HBGF-1 shows a variety of activities for many types of cells including epithelial types of cells. ~HepG2 is a well-differentiated human hepatoblastoma cell line 3 in which cell growth is stimulated by HBGF-1 at low concentrations (< 1 ng/ml), but inhibited at high concentrations (> 1 ng/ml) of HBGF-1. 4 To test whether the effects correlated with specific cell surface receptors, we tried to develop an assay system for HBGF-1 cell surface binding. HepG2 cells (2 × 104/well) are placed in medium WAJC 1015 containing 1% fetal bovine serum (FBS) into 24-well (2 cm z) collagen-coated tissue culture plates. The medium is replaced with MCDB107 serum-free medium 6 after overnight incubation. Incubation is continued for 2 to 3 days until the cell number reaches 5-8 x 10 4 cells/well. The cells are washed once with 500/xl of binding buffer [phosphate-buffered saline (PBS) or MCDB107, pH 7.2, containing 1 mg/ml BSA and 0.1 mM DTT], and then 250/zl of binding buffer is added to each of the wells.
Binding Assay of HBGF-1 125I-Labeled HBGF-1 and various concentrations of unlabeled HBGF-1 are incubated at 4 ° (on ice) with HepG2 cells. Unusually high amounts of unlabeled HBGF-1 are required to compete with detergent-extractable (1% Triton X-100) 125I-labeled HBGF-1 (Fig. 1). Although unlabeled HBGF-1 decreases cell-associated 125I-labeled HBGF-1, a 100-fold excess of unlabeled HBGF-1 only reduces the binding to 50%. Furthermore, at high concentrations of 125I-labeled HBGF-1, nonspecific binding (in the presence of 100-fold excess of unlabeled HBGF-1) dramatically increases and finally overcomes the total binding (Fig. 2). We reasoned that the affinity of HBGF-1 for pericellular heparin-like sites might distort the competition binding kinetics of HBGF-1 to specific cell membrane receptor sites. Labeled and unlabeled HBGF-1 may also aggregate and bind to the 3 B. B. Knowles, C. C. Howe, and D. P. Aden, Science 209, 497 (1980). 4 M. Kan, D. DiSorbo, J. Hou, H. Hoshi, P.-E. Mansson, and W. L. McKeehan, J. Biol. Chem. 263, 11306 (1988). 5 H. Hoshi and W. L. McKeehan, In Vitro Cell. Dev. Biol. 21, 125 (1985). 6 H. Hoshi and M. Kan, J. Tissue Cult. Methods 10, 83 (1987).
162
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FIG. 1. Competition of unlabeled HBGF-I with bound t251-labeledHBGF-I. The indicated amounts of unlabeled and 125I-labeled HBGF (260 pM) were incubated with HepG2 cells at 4° for 4 hr. Assay wells were washed 3 times with binding buffer and then treated with binding buffer alone (0) or binding buffer containing 250/xg/ml heparin (©), 0.5 M NaC1 (&), or 1.5 M NaCl (A) for l min at 4°. Cells were then extracted with 1% Trition X-100 and the radioactivity of the extract determined. (Inset) Various amounts (60, 180, and 600 pM) of I25I-labeled HBGF-1 were incubated with HepG2 cells in the presence of the indicated ratios of heparin to both labeled and unlabeled HBGF-l (w/w) at 4° for 4 hr. Specific HBGF-l binding was determined as described in text.
heparin-like sites because of the poor solubility of HBGF-1 at high concentrations and pH values above the isoelectric point. 2 Because HBGF-1 heparin binding is dissociated by 1.0-1.5 M NaC1, we treat the cells with 0.5-1.5 M NaC1 after binding to remove heparin-like site binding. Extraction of cells with high salt improves the competition curves (Fig. 1). "However, the salt extraction affected specific receptor-bound J25Ilabeled HBGF-1 (see Fig. 7). Extraction of cells with excess heparin (250/xg/ml) just after binding assay reveals a fraction of detergent-extractable 125I-labeled HBGF-1 that
[15]
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125I-HBGF-IAddod(nM) FIG. 2. Concentration dependence of 125I-labeled HBGF-1 binding to HepG2 cells in the absence of heparin. The indicated concentrations of 125I-labeled HBGF-l were incubated with cells at room temperature for 3 hr in the presence (e) or absence ((3) of 100-fold excess unlabeled HBGF-1. At the end of the incubation, the cells were washed with binding buffer (A) or heparin (250/.~g/ml) (B), and the J25I-labeled HBGF-1 bound to the cells was extracted with detergent (1% Triton X-100).
is reduced by 30-45% in the presence of an equal amount of unlabeled HBGF-1 (Fig. 1). A 100-fold excess of unlabeled HBGF-1 reduces detergent-extractable JzsI-labeled HBGF-1 by about 85%. However, at high concentrations of ligand, nonspecific binding (in the presence of 100-fold excess unlabeled ligand) still shows a relatively high ratio (Fig. 2). To further reduce the interference of heparin-like binding sites during equilibrium binding of HBGF-1 to specific receptor sites and to more closely correlate HBGF binding assays to cell growth assays containing heparin, a constant ratio (1000 : 1, w/w) of heparin to both lzSI-labeled and unlabeled HBGF-1 is included during the competition binding assays (Fig. l, inset). When added in equimolar amounts, unlabeled HBGF-1 reduces detergentextractable 125I-labeled HBGF-1 binding to the expected 50% levels (Fig. 3), and a 100-fold excess of unlabeled reduces ~25I-labeledHBGF-1 binding by 90-98% (Fig. 3, Table III). Table III compares the heparin- and detergent-extractable fractions of
164
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Unlabeled Competitor / 1251-HBGF-1 (w/w) FIG. 3. S p e c i f i c i t y o f 125I-labeled H B G F - I b i n d i n g to d e t e r g e n t - e x t r a c t a b l e sites. H e p G 2 c e l l s w e r e i n c u b a t e d for 3 hr a t r o o m t e m p e r a t u r e w i t h 150 p M o f 125I-labeled H B G F - 1 a n d t h e i n d i c a t e d c o n c e n t r a t i o n s o f u n l a b e l e d H B G F - 1 a n d H B G F - 2 . H e p a r i n w a s a d d e d at 1000fold ( w / w ) e x c e s s for H B G F - 1 (©) a n d 100-fold ( w / w ) e x c e s s for H B G F - 2 (O). T r a n s f e r r i n (TF), i n s u l i n (IN), a n d e p i d e r m a l g r o w t h f a c t o r ( E G F ) w e r e a d d e d at 2 5 0 / l g / m l (F1). C e l l s w e r e w a s h e d w i t h h e p a r i n (250 # g / m l ) , a n d t h e n d e t e r g e n t - e x t r a c t a b l e (1% T r i t o n X-100) ~25I-labeled H B G F - 1 w a s d e t e r m i n e d .
TABLE III HEPARIN- AND DETERGENT-EXTRACTABLE 125I-LABELED H B G F - 1 or~ H e p G 2 CELLS a '25I-Labeled HBGF-I extracted (IaM) ~2~I-Labeled HBGF-I added (pM)
Heparin Heparin in assay
50 50 560 560
+ +
Triton
Heparin + Triton
Total ~25I-labeled
+ Unlabeled HBGF-I
Total 125I-labeled
+ Unlabeled HBGF-1
1.0 0.8 7.8 4.4
1.3 0.05 22.2 0.16
5.5 4.7 11.3 10.4
0.34 0.11 1.80 0.51
-+ 0.05 .+ 0.13 --- 1.39 .+ 1.00
± 0.02 --- 0.01 "4- 0.44 -+ 0.03
-+ 0.10 --- 0.08 .+ 0.18 -+ 0.07
--+ 0.13 -+ 0.06 -+ 0.01 -+ 0.04
Total t25-I labeled
+ Unlabeled HBGF-I
6.5 5.5 19.1 14.8
1.6 0.16 24.0 0.67
a J251_Labeled HBGF-I (50 or 560 pM) was incubated with HepG2 cells at 22°. Cells were sequentially extracted with heparin and Triton X-100. Where indicated, binding assays contained a 100-fold excess of unlabeled to ~2~Ilabeled HBGF-I and heparin at a ratio of 1000:1 (w/w) to both t25I-labeled and unlabeled HBGF-1.
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FIBROBLAST GROWTH FACTOR RECEPTORS
165
cell-associated lZSI-labeled HBGF-I at two levels of 125I-labeled HBGF-1 in the absence and presence of soluble heparin and 100-fold excess unlabeled HBGF-1. At 50 pM JESI-labeled HBGF-1, heparin-extractable nSI-labeled HBGF-1 constitutes 15 and 81% of cell-associated nSI-HBGF-1 in the absence and presence of unlabeled HBGF-1, respectively. Inclusion of heparin in the binding reaction has a small effect on total heparin-extractable I25I-labeled HBGF-1 but reduces heparin-extractable nSI-labeled HBGF-1 binding in the presence of unlabeled HBGF-1 by 96%. At 560 pM ~zSI-labeled HBGF-1, the heparin-extractable binding in the absence of heparin during binding assays completely masks a reduction of total cellassociated J25I-labeled HBGF-1 due to addition of unlabeled HBGF-1. Heparin-extractable ~zSI-labeled HBGF-1 constitutes 41 and 93% of cellassociated JzSI-labeled HBGF-1 in the absence and presence of unlabeled HBGF-1, respectively. Heparin in the binding reaction reduces the total heparin-extractable ~zSI-labeled HBGF-1 by 44% and the residual heparinextractable ~251-labeled HBGF-1 in the presence of unlabeled HBGF-1 by 99%. Heparin in the binding assays reduces total and specific detergentextractable nSI-labeled HBGF-1 binding by less than 15% at both concentrations of ligand, but it reduces nonspecific detergent-extractable binding by 60-70%. In sum, inclusion of heparin at a constant 1000 : 1 (w/w) ratio to HBGF-1 in the binding reaction and the postreaction extraction of cells with excess heparin reveals the fraction of nSI-labeled HBGF- 1 specifically bound to detergent-extractable membrane receptor sites with minimal interference by nonreceptor, heparin-like sites. ~25I-Labeled HBGF-1 binding to detergent-extractable binding sites on HepG2 cells is unaffected by transferrin, insulin, and epidermal growth factor (EGF). HBGF-2, the homolog of HBGF-1, competes with 125Ilabeled HBGF-1 binding equally for HBGF-1 when 100-fold (w/w) heparin is added with HBGF-2 (Fig. 3). Heparin in the binding reaction mixture reduces the effectiveness of HBGF-2 at higher concentrations.
Kinetics of HBGF-1 Binding to Specific HepG2 Cell Surface Receptors Association of detergent-extractable l:5I-labeled HBGF-1 with HepG2 cells is complete after 3 hr of incubation at 4 ° (Fig. 4). In contrast, n51labeled HBGF-1 binding to heparin-extractable sites is complete after 1 hr at 4° (Fig. 4). Analysis of specific detergent-extractable nSI-labeled HBGF-1 binding at 4 ° as a function of nSI-labeled HBGF-1 concentration and following treatment of the data according to Scatchard 7 indicates primarily a class of 146,000 -+ 30,000 specific receptor sites per cell with 7 G. Scatchard, Ann. N. Y. Acad. Sci. 51, 660 (1949).
166
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FIG. 4. Kinetics of association of HBGF-1 with HepG2 cells, showing the time course of 125I-labeled HBGF-1 association with HepG2 cells at 4° (A) and 22° (B). HepG2 cells were incubated for the indicated times with 300 pM I25I-labeled HBGF-1. Heparin-extractable (A, A) and detergent-extractable (0, O) I25I-labeled HBGF-I was sequentially determined. Total (0, A) and nonspecific (O, A) 125I-labeled HBGF-1 binding was determined.
an apparent Kd of 2.5 --- 1.8 nM (Fig. 5). Data points at low concentrations of ]25I-labeled HBGF-1 indicate the possible presence of high affinity binding sites at 4 ° on HepG2 cells (Fig. 5). Performing the assays at room temperature (22°) reveals a significant increase in specific detergentextractable ~25I-labeled HBGF-1 binding (Fig. 4) without a significant increase in the extent of heparin-extractable or nonspecific detergentextractable 125I-labeled HBGF-1 binding (Fig. 4). 125I-Labeled HBGF-1 binding to detergent-extractable sites as a function of HBGF-1 concentrations at 22° is clearly biphasic (Fig. 5). Scatchard plots of the binding data at 22° can be resolved into high and low affinity phases of specific 125I-labeled HBGF binding. From the two linear extremes of the Scatchard plot at 22°, an apparent Kd of 9.2 - 0.9 pM is estimated for about 15,000 --- 900 high affinity receptor sites per cell, and an apparent Kd of 2 - 0.4 nM can be estimated for about 180,000 -+ 18,000 low affinity receptor sites per cell. Association of ~25I-labeled HBGF- 1 with heparin-extractable sites is also saturable, reversible, but temperatureindependent. From a linear Scatchard plot of the heparin-extractable fraction of uSI-labeled HBGF-1 associated with HepG2 cells, an apparent Ko of about 650 --- 60 pM for this type of nonreceptor binding site can be estimated (Fig. 6, inset). The temperature dependence of the curvilinear binding of 125I-labeled
[15]
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HBGF-1 to detergent-extractable binding sites on HepG2 cells is in contrast to other cell types which exhibit simple linear Scatchard curves (Fig. 6). For example, Scatchard analysis of HBGF-1 binding data obtained at 22° for the human hepatocellular carcinoma cell line, HUH-7, 8 indicates a single class of about 5600 --- 340 detergent-extractable receptors per cell with an apparent Kd of about 112 - 13 pM (Fig. 6). Kinetic parameters for the association of ~25I-labeled HBGF-1 to heparin-extractable binding sites on HUH-7 cells were similar to those for HepG2 cells (Fig. 6, inset). We interpret the data to indicate two discrete classes of specific HepG2 membrane receptor sites for HBGF-1 defined by both affinity and function. The association of HBGF-1 with high and low affinity classes of receptors correlates with opposite effects on cell growth, and only the low affinity receptor class correlates with stimulation of secretion of It~TI-related antigens. 4 Moreover, cells that exhibit a simple, positive mitogenic response to HBGF-1 exhibit linear Scatchard plots for HBGF-1 with no evidence of a ligand-dependent decrease in receptor affinity. 8 H. Nakabayashi, K. Taketa, K. Miyano, T. Yamane, and J. D. Sat•, CancerRes. 42, 3858 (1982).
168
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FIG. 6. Scatchard analysis of HBGF-I binding to Hcp3B, HUH-7, and PLC/PRF/5 cells. Cell densities were 77,600 Hep3B, 110,000 HUH-7, and 171,000 PLC/PRF/5 cells/well (2 cm2). (Inset, top) Scatchard plot of specific heparin-extractable 125I-labeledHBGF-1 binding to HepG3 and HUH-7 cells. (Inset, bottom) Affinitycross-linking of ~zSI-labeledHBGF-I to HUH-7 and Hep3B cells was performed as described in text. Lanes 2 and 4 contained a 100fold excess unlabeled HBGF-1.
Cross-Linking of 12SI-Labeled H B G F - 1 - R e c e p t o r Complex by Disuccinimidyl Suberate
Covalent Cross-Linking of ~25I-Labeled HBGF-1 to HepG2 Cells After ligand binding, the cells are washed 2 times with chilled PBS and 1 time with PBS containing 250/xg/ml heparin (Sigma, St. Louis, MO, bovine intestine or lung) followed by washing twice with PBS. The purpose of the heparin wash is to r e m o v e HBGF-1 bound to heparin-like sites on the cell surface or on the extracellular matrix. The duration of the heparin
[15]
FIBROBLAST GROWTH FACTOR RECEPTORS
169
wash should not be too long (
FIBROBLAST GROWTH FACTOR
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[15] I d e n t i f i c a t i o n a n d A s s a y o f F i b r o b l a s t G r o w t h Factor Receptors
By MIKIO KAN, ER-GANG SHI, and WALLACE L. MCKEEHAN Introduction Heparin-binding (fibroblast) growth factors (HBGF) constitute a structurally defined family of polypeptides that are widely distributed in tissues. Although originally described as fibroblast growth factors FGF, the polypeptides exhibit a wide spectrum of biological activities on both mesenchymal and epithelial cells that includes stimulation and inhibition of cell growth and effects on gene expression. Currently the HBGF family consists of seven cloned genes with overall amino acid sequence similarity of 19% (see Ref. 1). At least five members of the family exhibit an affinity for the glycosaminoglycan heparin or similar molecules which affect the structure, activity, and protease sensitivity of the polypeptides. An important advance in understanding the mechanism of action of the HBGF family has been the identification of specific cell membrane receptors for the polypeptides and dissociation of the receptors from the abundant cell-associated extracellular matrix heparin-like binding sites. This chapter describes methods for iodination and recovery of high specific activity ~25I-labeled HBGF and for assay of HBGF receptors. Methods underlying advances in characterization of HBGF receptors are also reviewed. Iodination of HBGF-1
Preparation of HBGF-1 for lodination HBGF-1 is a very sticky polypeptide, especially at neutral pH. Although bovine serum albumin (BSA), heparin, and reducing agents such as dithiothreitol (DTT) prevent HBGF-1 from being sticky (Table I), these additives and low pH interfere with the iodination reaction. Therefore, freshly isolated and reduced HBGF-1 must be prepared for the iodination to carry out the reaction under nonsticky conditions. Partially purified HBGF-1 is prepared from bovine brain as described previously. 2 One milligram of the bovine brain heparin-Sepharose (0.65 to I W. H. B u r g e s s a n d T. M a c i a g , Annu. Rev. Biochem. 58, 575 (1989). 2 j . W. C r a b b a n d W. L . M c K e e h a n , Anal. Biochem. 164, 563 (1987).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[15]
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TABLE I ADSORPTION OF HBGF-1 TO PLASTIC TUBES a Conditions PBS Acid PBS PBS PBS
Adsorption (%)
(pH 7.2) (pH 2.0) + B S A (1 mg/ml) + heparin (25/~g/ml) + D T T (10 m M )
63 2.8 6.8 13.0 18.6
A p p r o x i m a t e l y 500 pg of 125I-labeled HBGF-1 (specific activity 2.1 x 105 cpm/ng) was added in polypropylene tubes containing I ml of each indicated solution. After incubation for 15 hr at r o o m temperature, soluble and adsorbed (tube) radioactivity was determined in a y-scintillation counter.
2 M NaCI) fraction is reduced by adding 10 mM (final) DTT at neutral pH for 1 hr at 37 °. This reduction is very important to get good recovery by preventing adsorption of H B G F to reaction tubes (Table II). The solution is immediately acidified by adding trifluoroacetic acid and acetonitrile at final concentrations of 0.1% and 10% (v/v), respectively. After clarification by centrifugation, the solution is injected into C 4 reversed-phase HPLC column (25 cm x 4.6 mm Phenomenex, Palos Verdes, CA). HBGF-1 is eluted by linear gradient from 36 to 40% of acetonitrile as described previously. 2 The concentration of purified HBGF-1 is around 20-40 tzg/ml. The purified HBGF-I is immediately concentrated to 100/zg/ml
T A B L E II EFFECT OF REDUCTION ON RECOVERY OF HBGF-1 UNDER DIFFERENT IODINATION CONDITIONS a Reduction of starting material
Reduction after chloramine-T treatment
-
-
-
+
+ +
+
Recovery (%) 2.6
4.9 4.5 23.2
a Partially purified H B G F - I was purified by C 4 reversed-phase H P L C after t r e a t m e n t with or without D T T (10 m M ) . After chloramine-T treatment, the reaction was stopped with 10 m M D T T or e x c e s s KI and tyrosine.
160
FIBROBLAST GROWTH FACTOR
[15]
and used for iodination. The remaining HBGF-1 is stored in the presence of 10 rnM (final) DTT at - 2 0 ° for use as a competitor.
Iodination Reaction The reaction is carried out at room temperature in an iodination hood in a 2-ml conical siliconized tube. Reagents are added in the following order: 1. 2. 3. 4. 5.
50/zl of HBGF-1 ( - 5 / z g ) 100/xl of 0.25 M phosphate buffer (pH 7.0) 20/.d of Na125I ( - 2 mCi, Amersham, Arlington Heights, IL) 50/zl of rinse of the NalZSI vessel with phosphate buffer 30/zl of chloramine-T (400/zg/ml in phosphate buffer, freshly made)
The total reaction volume is 250/xl. After each addition, the reactants are shaken gently every 30 sec. The reaction time is 90 sec. At the end of the reaction time, 250/xl of 10 mM DTT in phosphate buffer (pH 7.0, freshly made) is added to stop the reaction and reduce the iodinated HBGF-1 (total volume is 500/.d). This reduction is also very important to get good recovery (Table II). After a further 10-min incubation, a 5-tzl aliquot is removed from the iodination mixture and added to 1 ml of phosphate buffer containing 1 mg/ml BSA for a determination of trichloroacetic acid (TCA) precipitation. Incorporation of ~zsI into HBGF-1 is determined by mixing equal volumes of cold 20% (w/v) TCA with the solution and precipitating JzSI-labeled HBGF-1. TCA-precipitable radioactivity is usually over 50%, and specific activity is around 2.5-5 × 105 counts/min (cpm)/ng.
Separation of 125I-Labeled HBGF-1 from Free Na125I To remove unincorporated ~zsI, the reaction mixture is applied to a heparin-Sepharose column. Heparin-Sepharose beads (400/.d; Pharmacia, Piscataway, NJ) equilibrated by phosphate buffer containing 0.5 M NaC1 is poured into a disposable plastic column (2.0 ml, Bio-Rad, Richmond, CA). The column is washed first by 1.5 M NaCl and 1 mg/ml BSA (10-20 ml) and then equilibrated by 0.5 M NaCl in phosphate buffer (10-15 ml). The iodinated mixture (-500 ~l) is loaded on the column at a flow rate of about 0.1 ml/1.5 min. After washing the column with 15 ml of 0.5 M NaCl to remove free ~25I,the JESI-labeled HBGF-1 is eluted by 1.5 M NaCl at a flow rate of about 0.05 ml/min into conical tubes containing l0/~l of 5 mg/ml BSA. Each collection volume is approximately 0.5 ml, and the total collection volume is 2 ml (four tubes). Five microliters of each of the aliquots is removed to measure recovery. The lzSI-labeled HBGF-I is stored at - 70 ° until use. The recovery is around 20-50%. The ~25I-labeled
[15]
FIBROBLAST GROWTH FACTOR RECEPTORS
161
HBGF-1 is diluted 10-fold with NaCl-free phosphate buffer containing 1 mg/ml BSA prior to use. Binding Assay of HBGF-1 Cell Surface Receptors
Preparation of Cells HBGF-1 shows a variety of activities for many types of cells including epithelial types of cells. ~HepG2 is a well-differentiated human hepatoblastoma cell line 3 in which cell growth is stimulated by HBGF-1 at low concentrations (< 1 ng/ml), but inhibited at high concentrations (> 1 ng/ml) of HBGF-1. 4 To test whether the effects correlated with specific cell surface receptors, we tried to develop an assay system for HBGF-1 cell surface binding. HepG2 cells (2 × 104/well) are placed in medium WAJC 1015 containing 1% fetal bovine serum (FBS) into 24-well (2 cm z) collagen-coated tissue culture plates. The medium is replaced with MCDB107 serum-free medium 6 after overnight incubation. Incubation is continued for 2 to 3 days until the cell number reaches 5-8 x 10 4 cells/well. The cells are washed once with 500/xl of binding buffer [phosphate-buffered saline (PBS) or MCDB107, pH 7.2, containing 1 mg/ml BSA and 0.1 mM DTT], and then 250/zl of binding buffer is added to each of the wells.
Binding Assay of HBGF-1 125I-Labeled HBGF-1 and various concentrations of unlabeled HBGF-1 are incubated at 4 ° (on ice) with HepG2 cells. Unusually high amounts of unlabeled HBGF-1 are required to compete with detergent-extractable (1% Triton X-100) 125I-labeled HBGF-1 (Fig. 1). Although unlabeled HBGF-1 decreases cell-associated 125I-labeled HBGF-1, a 100-fold excess of unlabeled HBGF-1 only reduces the binding to 50%. Furthermore, at high concentrations of 125I-labeled HBGF-1, nonspecific binding (in the presence of 100-fold excess of unlabeled HBGF-1) dramatically increases and finally overcomes the total binding (Fig. 2). We reasoned that the affinity of HBGF-1 for pericellular heparin-like sites might distort the competition binding kinetics of HBGF-1 to specific cell membrane receptor sites. Labeled and unlabeled HBGF-1 may also aggregate and bind to the 3 B. B. Knowles, C. C. Howe, and D. P. Aden, Science 209, 497 (1980). 4 M. Kan, D. DiSorbo, J. Hou, H. Hoshi, P.-E. Mansson, and W. L. McKeehan, J. Biol. Chem. 263, 11306 (1988). 5 H. Hoshi and W. L. McKeehan, In Vitro Cell. Dev. Biol. 21, 125 (1985). 6 H. Hoshi and M. Kan, J. Tissue Cult. Methods 10, 83 (1987).
162
FIBROBLAST GROWTH FACTOR
I
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HBGF-1/1251-HBGF-1
FIG. 1. Competition of unlabeled HBGF-I with bound t251-labeledHBGF-I. The indicated amounts of unlabeled and 125I-labeled HBGF (260 pM) were incubated with HepG2 cells at 4° for 4 hr. Assay wells were washed 3 times with binding buffer and then treated with binding buffer alone (0) or binding buffer containing 250/xg/ml heparin (©), 0.5 M NaC1 (&), or 1.5 M NaCl (A) for l min at 4°. Cells were then extracted with 1% Trition X-100 and the radioactivity of the extract determined. (Inset) Various amounts (60, 180, and 600 pM) of I25I-labeled HBGF-1 were incubated with HepG2 cells in the presence of the indicated ratios of heparin to both labeled and unlabeled HBGF-l (w/w) at 4° for 4 hr. Specific HBGF-l binding was determined as described in text.
heparin-like sites because of the poor solubility of HBGF-1 at high concentrations and pH values above the isoelectric point. 2 Because HBGF-1 heparin binding is dissociated by 1.0-1.5 M NaC1, we treat the cells with 0.5-1.5 M NaC1 after binding to remove heparin-like site binding. Extraction of cells with high salt improves the competition curves (Fig. 1). "However, the salt extraction affected specific receptor-bound J25Ilabeled HBGF-1 (see Fig. 7). Extraction of cells with excess heparin (250/xg/ml) just after binding assay reveals a fraction of detergent-extractable 125I-labeled HBGF-1 that
[15]
I63
FIBROBLAST GROWTH FACTOR RECEPTORS i
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125I-HBGF-IAddod(nM) FIG. 2. Concentration dependence of 125I-labeled HBGF-1 binding to HepG2 cells in the absence of heparin. The indicated concentrations of 125I-labeled HBGF-l were incubated with cells at room temperature for 3 hr in the presence (e) or absence ((3) of 100-fold excess unlabeled HBGF-1. At the end of the incubation, the cells were washed with binding buffer (A) or heparin (250/.~g/ml) (B), and the J25I-labeled HBGF-1 bound to the cells was extracted with detergent (1% Triton X-100).
is reduced by 30-45% in the presence of an equal amount of unlabeled HBGF-1 (Fig. 1). A 100-fold excess of unlabeled HBGF-1 reduces detergent-extractable JzsI-labeled HBGF-1 by about 85%. However, at high concentrations of ligand, nonspecific binding (in the presence of 100-fold excess unlabeled ligand) still shows a relatively high ratio (Fig. 2). To further reduce the interference of heparin-like binding sites during equilibrium binding of HBGF-1 to specific receptor sites and to more closely correlate HBGF binding assays to cell growth assays containing heparin, a constant ratio (1000 : 1, w/w) of heparin to both lzSI-labeled and unlabeled HBGF-1 is included during the competition binding assays (Fig. l, inset). When added in equimolar amounts, unlabeled HBGF-1 reduces detergentextractable 125I-labeled HBGF-1 binding to the expected 50% levels (Fig. 3), and a 100-fold excess of unlabeled reduces ~25I-labeledHBGF-1 binding by 90-98% (Fig. 3, Table III). Table III compares the heparin- and detergent-extractable fractions of
164
FIBROBLAST GROWTH FACTOR I
I
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I
I
I
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75
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25
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1
3
10
30
100
Unlabeled Competitor / 1251-HBGF-1 (w/w) FIG. 3. S p e c i f i c i t y o f 125I-labeled H B G F - I b i n d i n g to d e t e r g e n t - e x t r a c t a b l e sites. H e p G 2 c e l l s w e r e i n c u b a t e d for 3 hr a t r o o m t e m p e r a t u r e w i t h 150 p M o f 125I-labeled H B G F - 1 a n d t h e i n d i c a t e d c o n c e n t r a t i o n s o f u n l a b e l e d H B G F - 1 a n d H B G F - 2 . H e p a r i n w a s a d d e d at 1000fold ( w / w ) e x c e s s for H B G F - 1 (©) a n d 100-fold ( w / w ) e x c e s s for H B G F - 2 (O). T r a n s f e r r i n (TF), i n s u l i n (IN), a n d e p i d e r m a l g r o w t h f a c t o r ( E G F ) w e r e a d d e d at 2 5 0 / l g / m l (F1). C e l l s w e r e w a s h e d w i t h h e p a r i n (250 # g / m l ) , a n d t h e n d e t e r g e n t - e x t r a c t a b l e (1% T r i t o n X-100) ~25I-labeled H B G F - 1 w a s d e t e r m i n e d .
TABLE III HEPARIN- AND DETERGENT-EXTRACTABLE 125I-LABELED H B G F - 1 or~ H e p G 2 CELLS a '25I-Labeled HBGF-I extracted (IaM) ~2~I-Labeled HBGF-I added (pM)
Heparin Heparin in assay
50 50 560 560
+ +
Triton
Heparin + Triton
Total ~25I-labeled
+ Unlabeled HBGF-I
Total 125I-labeled
+ Unlabeled HBGF-1
1.0 0.8 7.8 4.4
1.3 0.05 22.2 0.16
5.5 4.7 11.3 10.4
0.34 0.11 1.80 0.51
-+ 0.05 .+ 0.13 --- 1.39 .+ 1.00
± 0.02 --- 0.01 "4- 0.44 -+ 0.03
-+ 0.10 --- 0.08 .+ 0.18 -+ 0.07
--+ 0.13 -+ 0.06 -+ 0.01 -+ 0.04
Total t25-I labeled
+ Unlabeled HBGF-I
6.5 5.5 19.1 14.8
1.6 0.16 24.0 0.67
a J251_Labeled HBGF-I (50 or 560 pM) was incubated with HepG2 cells at 22°. Cells were sequentially extracted with heparin and Triton X-100. Where indicated, binding assays contained a 100-fold excess of unlabeled to ~2~Ilabeled HBGF-I and heparin at a ratio of 1000:1 (w/w) to both t25I-labeled and unlabeled HBGF-1.
[15]
FIBROBLAST GROWTH FACTOR RECEPTORS
165
cell-associated lZSI-labeled HBGF-I at two levels of 125I-labeled HBGF-1 in the absence and presence of soluble heparin and 100-fold excess unlabeled HBGF-1. At 50 pM JESI-labeled HBGF-1, heparin-extractable nSI-labeled HBGF-1 constitutes 15 and 81% of cell-associated nSI-HBGF-1 in the absence and presence of unlabeled HBGF-1, respectively. Inclusion of heparin in the binding reaction has a small effect on total heparin-extractable I25I-labeled HBGF-1 but reduces heparin-extractable nSI-labeled HBGF-1 binding in the presence of unlabeled HBGF-1 by 96%. At 560 pM ~zSI-labeled HBGF-1, the heparin-extractable binding in the absence of heparin during binding assays completely masks a reduction of total cellassociated J25I-labeled HBGF-1 due to addition of unlabeled HBGF-1. Heparin-extractable ~zSI-labeled HBGF-1 constitutes 41 and 93% of cellassociated JzSI-labeled HBGF-1 in the absence and presence of unlabeled HBGF-1, respectively. Heparin in the binding reaction reduces the total heparin-extractable ~zSI-labeled HBGF-1 by 44% and the residual heparinextractable ~251-labeled HBGF-1 in the presence of unlabeled HBGF-1 by 99%. Heparin in the binding assays reduces total and specific detergentextractable nSI-labeled HBGF-1 binding by less than 15% at both concentrations of ligand, but it reduces nonspecific detergent-extractable binding by 60-70%. In sum, inclusion of heparin at a constant 1000 : 1 (w/w) ratio to HBGF-1 in the binding reaction and the postreaction extraction of cells with excess heparin reveals the fraction of nSI-labeled HBGF- 1 specifically bound to detergent-extractable membrane receptor sites with minimal interference by nonreceptor, heparin-like sites. ~25I-Labeled HBGF-1 binding to detergent-extractable binding sites on HepG2 cells is unaffected by transferrin, insulin, and epidermal growth factor (EGF). HBGF-2, the homolog of HBGF-1, competes with 125Ilabeled HBGF-1 binding equally for HBGF-1 when 100-fold (w/w) heparin is added with HBGF-2 (Fig. 3). Heparin in the binding reaction mixture reduces the effectiveness of HBGF-2 at higher concentrations.
Kinetics of HBGF-1 Binding to Specific HepG2 Cell Surface Receptors Association of detergent-extractable l:5I-labeled HBGF-1 with HepG2 cells is complete after 3 hr of incubation at 4 ° (Fig. 4). In contrast, n51labeled HBGF-1 binding to heparin-extractable sites is complete after 1 hr at 4° (Fig. 4). Analysis of specific detergent-extractable nSI-labeled HBGF-1 binding at 4 ° as a function of nSI-labeled HBGF-1 concentration and following treatment of the data according to Scatchard 7 indicates primarily a class of 146,000 -+ 30,000 specific receptor sites per cell with 7 G. Scatchard, Ann. N. Y. Acad. Sci. 51, 660 (1949).
166
[15]
FIBROBLAST GROWTH FACTOR
A
30
i
i
i 4oc
I
I
I
I
I
B
I
|
8
10
22oc
25 =on20
--
10 in
'-
./
"0~
O-- •
~--
A--&
5 ~0____.-------0 ~ 0
2
4
6
8
2 10 Time (hr)
4
6
I 12
14
FIG. 4. Kinetics of association of HBGF-1 with HepG2 cells, showing the time course of 125I-labeled HBGF-1 association with HepG2 cells at 4° (A) and 22° (B). HepG2 cells were incubated for the indicated times with 300 pM I25I-labeled HBGF-1. Heparin-extractable (A, A) and detergent-extractable (0, O) I25I-labeled HBGF-I was sequentially determined. Total (0, A) and nonspecific (O, A) 125I-labeled HBGF-1 binding was determined.
an apparent Kd of 2.5 --- 1.8 nM (Fig. 5). Data points at low concentrations of ]25I-labeled HBGF-1 indicate the possible presence of high affinity binding sites at 4 ° on HepG2 cells (Fig. 5). Performing the assays at room temperature (22°) reveals a significant increase in specific detergentextractable ~25I-labeled HBGF-1 binding (Fig. 4) without a significant increase in the extent of heparin-extractable or nonspecific detergentextractable 125I-labeled HBGF-1 binding (Fig. 4). 125I-Labeled HBGF-1 binding to detergent-extractable sites as a function of HBGF-1 concentrations at 22° is clearly biphasic (Fig. 5). Scatchard plots of the binding data at 22° can be resolved into high and low affinity phases of specific 125I-labeled HBGF binding. From the two linear extremes of the Scatchard plot at 22°, an apparent Kd of 9.2 - 0.9 pM is estimated for about 15,000 --- 900 high affinity receptor sites per cell, and an apparent Kd of 2 - 0.4 nM can be estimated for about 180,000 -+ 18,000 low affinity receptor sites per cell. Association of ~25I-labeled HBGF- 1 with heparin-extractable sites is also saturable, reversible, but temperatureindependent. From a linear Scatchard plot of the heparin-extractable fraction of uSI-labeled HBGF-1 associated with HepG2 cells, an apparent Ko of about 650 --- 60 pM for this type of nonreceptor binding site can be estimated (Fig. 6, inset). The temperature dependence of the curvilinear binding of 125I-labeled
[15]
167
FIBROBLAST GROWTH FACTOR RECEPTORS I
14_
I
I
I
1
A
l
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I
1
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i
i
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i
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6
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I
I
I
I
6
8
10
12
22°C 4
6
8
10
2
HBGF-I B o u n d (pM) F[o. 5. Scatchard analysis of ]25I-labeled HBGF-I binding to HepG2 cells. Specific detergent-extractable HBGF-1 binding was determined at 4 ° (A) and at 22 ° (B). B, Bound ligand; F, free ligand. (Inset a) Expanded plot of binding data from 1 to 7 pM from (A). (Inset b) E x p a n d e d plot of the data from 6 to 12 pM from (B).
HBGF-1 to detergent-extractable binding sites on HepG2 cells is in contrast to other cell types which exhibit simple linear Scatchard curves (Fig. 6). For example, Scatchard analysis of HBGF-1 binding data obtained at 22° for the human hepatocellular carcinoma cell line, HUH-7, 8 indicates a single class of about 5600 --- 340 detergent-extractable receptors per cell with an apparent Kd of about 112 - 13 pM (Fig. 6). Kinetic parameters for the association of ~25I-labeled HBGF-1 to heparin-extractable binding sites on HUH-7 cells were similar to those for HepG2 cells (Fig. 6, inset). We interpret the data to indicate two discrete classes of specific HepG2 membrane receptor sites for HBGF-1 defined by both affinity and function. The association of HBGF-1 with high and low affinity classes of receptors correlates with opposite effects on cell growth, and only the low affinity receptor class correlates with stimulation of secretion of It~TI-related antigens. 4 Moreover, cells that exhibit a simple, positive mitogenic response to HBGF-1 exhibit linear Scatchard plots for HBGF-1 with no evidence of a ligand-dependent decrease in receptor affinity. 8 H. Nakabayashi, K. Taketa, K. Miyano, T. Yamane, and J. D. Sat•, CancerRes. 42, 3858 (1982).
168
FIBROBLAST GROWTH FACTOR
,0!
I
I
I
r
[15]
I
I I
I
i I
I
2 l_Heparin.extractablel ~o ~1 ~ Sites
35 ®
,| 0 2 4 6 HBGF-I Bound (pM)
Hep3B
30
/
Top-~
"~
O4 O
200 1
M.
,.~PLC/PRF~K~~I 11:-1 1 27 3ep;B
10L 5L
o "A\
z~
r, ~ t
" o
I
0
10
20
30
40
I
I
50
60
HBGF-IBound(pg/ 105cells)
N~l
70
FIG. 6. Scatchard analysis of HBGF-I binding to Hcp3B, HUH-7, and PLC/PRF/5 cells. Cell densities were 77,600 Hep3B, 110,000 HUH-7, and 171,000 PLC/PRF/5 cells/well (2 cm2). (Inset, top) Scatchard plot of specific heparin-extractable 125I-labeledHBGF-1 binding to HepG3 and HUH-7 cells. (Inset, bottom) Affinitycross-linking of ~zSI-labeledHBGF-I to HUH-7 and Hep3B cells was performed as described in text. Lanes 2 and 4 contained a 100fold excess unlabeled HBGF-1.
Cross-Linking of 12SI-Labeled H B G F - 1 - R e c e p t o r Complex by Disuccinimidyl Suberate
Covalent Cross-Linking of ~25I-Labeled HBGF-1 to HepG2 Cells After ligand binding, the cells are washed 2 times with chilled PBS and 1 time with PBS containing 250/xg/ml heparin (Sigma, St. Louis, MO, bovine intestine or lung) followed by washing twice with PBS. The purpose of the heparin wash is to r e m o v e HBGF-1 bound to heparin-like sites on the cell surface or on the extracellular matrix. The duration of the heparin
[15]
FIBROBLAST GROWTH FACTOR RECEPTORS
169
wash should not be too long (
