(Received for publication, August 7, 1995; and in revised form, September 20, 1995)
From the
In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal growth factor (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of 20-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine.
The amino-terminal sequence of the purified
160-kDa protein,
HN-SAPQHXSXPEGTXA-, matched
residues 21-34 of the predicted sequence of human prepro-EGF and
established that the 160 kDa protein (pro-EGF) is a product of the
prepro-EGF gene. Characterization of the carboxyl terminus of the
purified protein by digestion with carboxypeptidase B and by
immunoblotting with antisera against synthetic carboxyl-terminal and
juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl
terminus has been truncated at an arginine residue that corresponds,
most likely, to the carboxyl-terminal arginine of the EGF moiety.
The intact 160-kDa pro-EGF is biologically active as evidenced by
its specific binding to the EGF receptor and activation of the EGF
receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF
competitively inhibited the binding of I-EGF to human
fibroblasts, and it stimulated the proliferation of these cells in
culture. When immobilized onto culture dishes, the heparin-binding
pro-EGF appeared to function both as an adhesion molecule and as a
growth factor for serum-free mouse embryo cells.
Epidermal growth factor (EGF) ()is a 53-amino acid
polypeptide originally discovered in the mouse submaxillary gland as an
agent that induced precocious eyelid opening and early incisor eruption
in the newborn mouse(1) . EGF has been subsequently shown to
elicit an array of biological responses (reviewed in (2, 3, 4) ) that are mediated by specific
binding to an EGF receptor/tyrosine kinase located on the cell surface
(reviewed in (5) and (6) ). EGF is structurally and
functionally identical to urogastrone, an inhibitor of gastric acid
secretion(7) , and is a member of a family of EGF-related
growth factors (reviewed in (3) and (8) and 9). The
soluble growth factors appear to originate from the extracellular
domain of their respective transmembrane glycoprotein precursor
(reviewed in (10) ).
Early evidence that EGF might be derived from a larger precursor arose from the observation of high molecular weight forms of EGF in urine (11) and from the observation that urogastrone might exist as a high molecular weight glycoprotein(12) . Indeed, cloning of the EGF gene from mouse (13, 14) and human (15) sources revealed cDNA that encodes precursors of 1217 and 1207 amino acids, respectively. Human prepro-EGF contains an EGF sequence that is flanked on its amino terminus by a 970-residue polypeptide and on its carboxyl terminus by a 184-residue segment. The large amino-terminal polypeptide contains 8 additional EGF-like regions, the biological significance of which is unknown, and the smaller carboxyl-terminal segment contains a 25-residue hydrophobic transmembrane domain that serves to anchor the protein to the cell membrane.
NIH 3T3 cells transfected with human prepro-EGF cDNA express the precursor as a 160-170-kDa glycoprotein that exists both as an intrinsic membrane-associated form and as a soluble form found in the conditioned medium(16, 17) . Partially purified EGF precursor binds to the EGF receptor on cultured fibroblasts, activates the EGF receptor tyrosine kinase, and supports the growth of EGF-dependent mouse keratinocytes(17) .
The mechanism whereby prepro-EGF is processed to mature EGF is not well understood. In the salivary gland, prepro-EGF appears to be processed intracellularly to mature 6-kDa EGF that is stored as a high molecular weight complex within secretory granules(1, 18, 19) . In contrast, other organs such as the kidney contain mostly unprocessed precursor with relatively low levels of mature EGF(20) . A 140-kDa EGF precursor has been isolated and partially characterized from mouse kidney membranes(21) . Since urine contains relatively large amounts of mature 6-kDa EGF(22) , which appears to arise from the kidney(23) , the renal/urinary system may be a useful model for investigating the mechanism of prepro-EGF processing. Higher molecular weight EGF-immunoreactive glycoproteins have been detected in the urine of rodents (24) and humans (25, 26, 27) and were reported to exhibit EGF-like activity.
In the present report, we describe a procedure for the isolation of a heparin-binding 160-kDa EGF-immunoreactive glycoprotein from 20-30-liter batches of fresh human urine. Characterization of the purified protein indicated that it is a pro-EGF and that it is derived from a membrane-anchored prepro-EGF by amino-terminal cleavage of a 20-residue signal peptide and by carboxyl-terminal truncation at an arginine residue between the EGF moiety and the transmembrane domain. The intact 160-kDa pro-EGF bound specifically to the EGF receptor and activated the EGF receptor tyrosine kinase in A-431 cell membranes, and it stimulated the proliferation of cultured human fibroblasts. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a growth factor for serum-free mouse embryo cells.
The supernatant fraction (80 ml) was subjected to batch chromatography on DEAE-Sephacel (10 ml packed volume) equilibrated in 0.1 M NaCl, 5 mM imidazole, pH 7.0. The suspension was mixed overnight, and the beads were then washed eight times with 30-ml aliquots of the same buffer. Adsorbed proteins were eluted by two washes (15 min each) with 10 ml of 0.5 M NaCl, 5 mM imidazole, pH 7.0. The eluates were combined and then desalted by 5-fold concentration in an Amicon stirred cell equipped with a YM-30 membrane, followed by reconstitution to 20 ml with 5 mM imidazole, pH 7.0.
The desalted DEAE-eluate (20 ml) was applied batchwise to heparin-agarose (4 ml packed volume) equilibrated in 90 mM NaCl, 5 mM imidazole, pH 7.0 (HA buffer). The suspension was incubated overnight and the beads were washed twice with 16 ml of HA buffer, three times with 16 ml of 20 mM TAPS buffer, pH 9.0, and then twice with 16 ml of HA buffer. Adsorbed proteins were eluted by two washes (15 min each) with 4 ml of 0.5 M NaCl, 5 mM imidazole, pH 7.0. The two HA eluates were pooled and stored in aliquots at -70 °C.
Figure 3:
Sephacryl S-300 chromatography of
heparin-agarose purified urinary EGF precursor.
Heparin-agarose-purified EGF precursor was denatured by boiling in the
presence of SDS and -mercaptoethanol and then chromatographed on
Sephacryl S-300 as described under ``Experimental
Procedures.'' A 5-µl aliquot of each column fraction was
subjected to SDS-PAGE (7% acrylamide) and stained with Coomassie
Blue.
Figure 1: Identification of EGF-immunoreactive proteins in human urine. A, fresh urine samples (10 ml) were incubated overnight at 4 °C with 100 µl of WGL-Sepharose 6MB. Adsorbed proteins were eluted by boiling in Laemmli sample buffer, resolved by SDS-PAGE (7%), electrotransferred to nitrocellulose, and immunoblotted with rabbit polyclonal antiserum against human EGF, 1:300 dilution. In lane 2, a 33-µl aliquot of antiserum was preincubated with 10 µg of recombinant human EGF overnight at 4 °C prior to immunoblotting. B, fresh human urine (50 ml) was dialyzed against water at 4 °C using 2-kDa molecular mass cutoff tubing, lyophilized, and reconstituted with 0.5 ml of water. A 40-µl aliquot was boiled for 3 min in Laemmli sample buffer, subjected to SDS-PAGE (6-12% gradient gel), electrotransferred, and blotted with human EGF antiserum, 1:500 dilution. DF, dye front.
Using human urine that was dialyzed and
concentrated by lyophilization, without isolation of the glycoprotein
fraction on WGL-Sepharose, immunoblotting detected both the 160- and
70-kDa EGF-immunoreactive proteins as well as an additional 55-kDa
protein (Fig. 1B). The 55- and 70-kDa proteins may
represent degradation products of the 160-kDa EGF-immunoreactive
glycoprotein. The amount of 160-kDa EGF-immunoreactive protein in
dialyzed urine was similar to that in an equivalent amount of urine
adsorbed onto WGL-Sepharose and suggests that nearly all of the urinary
160-kDa EGF-immunoreactive protein is glycosylated. Similar immunoblots
were obtained with a commercially available (Sigma) preparation of
dialyzed and lyophilized human urine, and with an acetone powder of
human pregnancy urine (22) that was utilized previously for the
isolation of mature 6-kDa EGF.
In contrast to the relatively large amounts of high molecular weight EGF-immunoreactive proteins in fresh urine or urine powders, only trace amounts of mature EGF were detected by immunoblotting. Similar findings were reported by Lakshmanan et al.(26) and were ascribed to the relative insensitivity of the immunoblotting procedure to detect mature EGF.
Comparison of fresh urine samples from 8 adult donors (7 male and 1 female) revealed the presence of the 160-kDa EGF-immunoreactive protein in each specimen with only minor variations in the total amount of immunoreactive material.
Ultracentrifugation of the dialyzed retentate
at 180,000 g for 4 h produced a loose gel-like pellet;
greater than 90% of the 160-kDa EGF-immunoreactive protein remained in
the supernatant fraction as determined by immunoblotting. Analysis of
the pellet fraction by SDS-PAGE and Coomassie Blue staining revealed
that it contained a relatively large amount of a 80-100-kDa
protein. This protein comigrated on SDS-PAGE with a purified sample of
Tamm-Horsefall glycoprotein (uromodulin), an abundant urinary protein
with EGF-like sequences that forms high molecular weight aggregates in
solution (34) .
After ultracentrifugation, the supernatant fraction was chromatographed on DEAE-Sephacel. Immunoblot analysis revealed that the 160-kDa EGF-immunoreactive protein was retained by the DEAE-Sephacel with little or no material in the flow-through fraction (Fig. 2A, lanes 1 and 2). The bulk of the immunoreactive material was eluted with 0.5 M NaCl (lane 3). On Coomassie Blue staining, a 160-kDa protein band was detected in the original retentate fraction (Fig. 2B, lane 1) and in the DEAE-eluate (lane 3), but not in the flow-through fraction (lane 2). Based on Coomassie Blue staining, the recovery of 160-kDa protein from DEAE-Sephacel was greater that 75%.
Figure 2: Chromatography of the 160-kDa urinary EGF precursor on DEAE-Sephacel and heparin-agarose. Chromatography was performed as described under ``Experimental Procedures.'' To detect the 160-kDa EGF precursor, aliquots of each fraction were subjected to SDS-PAGE (6-7% acrylamide), and either electrotransferred and blotted with human EGF antiserum (1:500 dilution) (A) or fixed and stained with Coomassie Blue (C.B.) dye (B). Gel lanes are as follows: 1, original retentate (200 µl); 2, DEAE flow-through (200 µl); 3, DEAE-eluate (50 µl); 4, heparin-agarose flow-through (50 µl); and 5, heparin-agarose eluate (50 µl). The data shown for DEAE-Sephacel (lanes 1, 2, and 3) and heparin-agarose (lanes 4 and 5) chromatography were from separate but representative urine preparations.
The 160-kDa protein
in the DEAE-eluate exhibited salt-dependent binding to heparin-agarose,
and this interaction was utilized for further purification. The
DEAE-eluate was desalted to 90 mM NaCl, adsorbed onto
heparin-agarose, and the 160-kDa protein eluted with 0.5 M NaCl. Immunoblot analysis of the heparin-agarose fractions
revealed the presence of the 160-kDa immunoreactive protein in the
eluate (Fig. 2A, lane 5) with little or no
immunoreactive material in the flow-through (lane 4).
Coomassie Blue staining of the heparin-agarose fractions indicated that
the 160-kDa protein bound selectively to heparin-agarose since the bulk
of the protein was present in the flow-through fraction (Fig. 2B, lanes 4). In the heparin-agarose
eluate (lane 5), the 160-kDa protein corresponded exactly to
the 160-kDa EGF-immunoreactive band detected on immunoblots and
represented 40-60% of the total protein. The overall recovery at
this stage, as estimated by Coomassie Blue staining, was 1 mg of
160-kDa protein from 25 liters of fresh urine.
To
purify further the 160-kDa protein, an aliquot of heparin-agarose
eluate was denatured by boiling in SDS (2%) and -mercaptoethanol
(5%) and then subjected to gel filtration chromatography on Sephacryl
S-300 in buffer that contained 0.1% SDS and 0.1% thioglycerol. Under
these conditions, the 160-kDa protein eluted with significant
resolution from lower molecular mass proteins as detected by SDS-PAGE
and Coomassie Blue staining of column fractions (Fig. 3).
Fractions 25 and 26 contained nearly homogeneous 160-kDa protein.
Comparison of the above amino-terminal sequence with the predicted amino acid sequence of human prepro-EGF (15) indicated a precise match, with the amino-terminal serine residue of the urinary EGF precursor corresponding to Ser-21 of prepro-EGF. The results indicate that the amino terminus of the urinary EGF precursor (pro-EGF) has been modified from that of prepro-EGF by cleavage of a 20-residue hydrophobic signal peptide.
To define the site of carboxyl-terminal modification, we have prepared a second polyclonal antiserum against a synthetic peptide (designated C2) that corresponds to a cytoplasmic region of prepro-EGF (residues 1058-1081) immediately adjacent to the transmembrane domain. In hEGF19 cells, where the expression of prepro-EGF is induced by sodium butyrate (16) , this antiserum blotted a 160-kDa protein in membranes isolated from butyrate-induced, but not uninduced, cells (Fig. 4, lanes 3 and 4); blotting of the 160-kDa protein was blocked by preincubation of the antiserum with excess C2 peptide, and preimmune serum was nonreactive (data not shown). In contrast, the antiserum exhibited no detectable immunoreactivity toward the heparin-agarose-purified EGF precursor (lane 2). These findings suggest that the 160-kDa urinary EGF precursor contains a truncated carboxyl terminus, with the processing site located between the EGF moiety and the juxtatransmembrane C2 region.
Figure 4: Immunoblot of human EGF precursors with prepro-EGF C2-peptide antiserum. Aliquots of heparin-agarose-purified urinary pro-EGF and membrane fractions from control and sodium butyrate-induced hEGF19 cells were subjected SDS-PAGE (6% acrylamide), electrotransferred, and immunoblotted with rabbit polyclonal antiserum against prepro-EGF C2 peptide (1:300 dilution) or antiserum against human EGF (1:500 dilution). Detection was by ECL using horseradish peroxidase-conjugated protein A (1:3000). Gel lanes are as follows: 1 and 2, heparin-agarose eluate (5 µl); 3, induced hEGF19 membranes (120 µg of protein); and 4, control hEGF19 membranes (120 µg of protein).
To establish more precisely the site of carboxyl-terminal
processing, we have subjected the urinary EGF precursor to
carboxypeptidase digestion in order to determine the carboxyl-terminal
residue. Since proteolysis of the precursor by a presumed arginine
esterase would result in a carboxyl-terminal arginine, digestion was
carried out with carboxypeptidase B, which preferentially cleaves
arginine or lysine residues. When Sephacryl S-300-purified 160-kDa EGF
precursor (125 pmol) was incubated with 0.43 µg of
carboxypeptidase B for 2 h at 37 °C, approximately 110 pmol of
arginine was recovered; little or no arginine was recovered from
reaction mixtures that lacked either enzyme or substrate. Also
recovered was a small amount of tyrosine, which is of uncertain
significance. The results suggest that arginine is the
carboxyl-terminal residue of the urinary 160-kDa pro-EGF.
Figure 5: Binding of intact pro-EGF to the EGF receptor in A-431 cell membranes. Urine retentate (0.25 ml) was incubated with A-431 membrane vesicles (60 µg of protein) for 2 h at 0 °C. Where indicated, A-431 membrane vesicles (60 µg) were first preincubated with recombinant human EGF (10 µg) for 1 h at 0 °C. Following the incubation period, the vesicle fraction was pelleted by centrifugation at 30,000 rpm (Beckman TL-100) for 20 min. The supernatants (concentrated to 50 µl by Centricon-30) and pellets were boiled for 3 min in Laemmli sample buffer and then subjected to SDS-PAGE (6% acrylamide), electrotransferred, and immunoblotted with human EGF antiserum at 1:500 dilution. DF, dye front.
To assess further the
interaction of purified urinary pro-EGF with the EGF receptor, its
ability to stimulate EGF receptor autophosphorylation was examined. In
a standard phosphorylation reaction mixture that contained A-431
membrane vesicles and P-labeled ATP, addition of 50 µl
(
0.5 µg) of purified urinary pro-EGF resulted in a 2- to
3-fold increase in
P incorporation into the 170-kDa EGF
receptor (data not shown). The degree of stimulation of EGF receptor
phosphorylation was similar to that obtained with 6 ng of pure
recombinant human EGF. Since the phosphorylation reaction was carried
out at 0 °C, a temperature where little or no processing of the EGF
precursor would be expected, the results again suggest that the intact
urinary pro-EGF binds to the EGF receptor and, in the presence of ATP,
stimulates receptor autophosphorylation.
The purified pro-EGF had a stimulatory effect on the growth of cultured human fibroblasts. When cells were incubated over a 10-day period with 100 µl of heparin-agarose-purified pro-EGF/ml of medium, there was a 1.6-fold increase in cell number as compared to control cultures (Table 2). The stimulation by pro-EGF was slightly less than the 1.9-fold stimulation observed with 10 ng/ml pure EGF.
Based on the
molar concentration of 160-kDa protein in the heparin-agarose eluate,
as estimated by SDS-PAGE and Coomassie Blue staining, the urinary
pro-EGF was calculated to possess 50% of the activity of mature
EGF as assayed by competitive inhibition of
I-EGF binding
to cultured fibroblasts and by stimulation of EGF receptor
autophosphorylation in A-431 cell membranes.
To examine the possibility that the heparin-binding pro-EGF could act both as a growth stimulant and as an adhesion molecule, we precoated a portion of a standard culture dish with heparin-agarose-purified pro-EGF prior to plating the dish with SFME cells (in medium without exogenous serum or EGF). This treatment resulted in cell attachment and growth only in the area coated with pro-EGF; a typical culture dish with hematoxylin-stained cells is shown (Fig. 6). We conclude that, when immobilized on a culture dish, the heparin-binding pro-EGF functions both as an adhesion molecule and as a growth stimulant for SFME cells.
Figure 6:
Adhesion and growth of SFME cells on
culture dishes precoated with purified urinary pro-EGF. An
1-cm
area was marked in the center of a standard 35-mm
culture dish, and the area then was coated with 20 µl of
heparin-agarose-purified pro-EGF. After 2 h at room temperature
(without drying), excess sample was removed by washing the coated
square 10 times with 0.25 ml of PBS and then the entire dish 4 times
with 2 ml of PBS. The dish was plated with 200,000 SFME cells in
serum-free medium (without supplemental EGF). The cultures were refed
daily, and, after 6 days, the cells were rinsed with PBS, fixed, and
stained with hematoxylin.
The production of peptide growth factors from the extracellular domain of a membrane-anchored precursor is a common theme in cell biology (reviewed in (10) and (35) ). While details of precursor processing have been elucidated for several growth factors, relatively little is known about the mechanism of processing of prepro-EGF to mature EGF. Since prepro-EGF and mature EGF are relatively abundant in kidney and urine, respectively, the renal/urinary system may serve as a useful model for understanding the mechanism of prepro-EGF processing.
In the present study, after confirming the presence of high molecular weight EGF-related glycoproteins in human urine(25, 26, 27) , the urinary 160-kDa EGF-immunoreactive protein was isolated and characterized. The results established that this protein is indeed a product of the human prepro-EGF gene, and that the amino-terminal serine residue of the 160-kDa protein corresponds to Ser-21 of the published sequence of human prepro-EGF(15) . The initial 20-residue hydrophobic sequence of prepro-EGF was presumably cleaved by the action of a signal peptidase.
Characterization of the carboxyl
terminus of the 160-kDa protein by carboxypeptidase digestion and by
immunoblotting with antisera against synthetic carboxyl-terminal and
juxtatransmembrane C2 peptides (prepro-EGF residues 1188-1207 and
1058-1081, respectively) indicated that the carboxyl-terminal
residue is an arginine and is located amino-terminal to the
juxtatransmembrane C2 region. The cDNA sequence of human prepro-EGF
predicts two arginine residues in a potential truncation region between
the EGF moiety and the C2 sequence; Arg-1023 corresponds to the
carboxyl terminus of the EGF moiety itself, and Arg-1058 corresponds to
the first arginine on the cytoplasmic side of the transmembrane domain.
The results suggest two possible molecular structures for the urinary
160-kDa pro-EGF (Fig. 7, A and B). Form B,
terminating at Arg-1023, is the more likely since the 160-kDa pro-EGF
in urine is a soluble protein and would be expected to lack the
transmembrane domain (residues 1033-1057). It is unlikely that
the truncation site would be at an arginine further upstream from
Arg-1023, e.g. at Arg-1011 or -1015 (see (15) ), since
this would truncate the EGF sequence and compromise biological
activity. A human EGF analog, hEGF, that lacks the
7 carboxyl-terminal residues of full-length hEGF, is reported to
possess less than 0.5% of the biological activity of intact
hEGF(36) .
Figure 7: Comparison of urinary pro-EGF to full-length human prepro-EGF. The predicted amino acid sequence of human prepro-EGF is from Bell et al.(15) . The SDS-PAGE gel is of an aliquot of heparin-agarose-purified 160-kDa pro-EGF from which the amino-terminal sequence was derived (see Table 1). Molecular schematics A and B represent proposed structures of urinary pro-EGF with potential carboxyl-terminal modifications. TM, transmembrane domain.
A specific proteolytic enzyme that cleaves the carboxyl terminus of the membrane-anchored EGF precursor, with release of soluble pro-EGF from the transmembrane domain, has not been described. However, there is some evidence to suggest the involvement of a membrane-associated endoprotease. Using hEGF19 membranes, autolysis for 1 h at 37 °C resulted in the formation of a soluble EGF precursor that was of similar molecular mass (on SDS-PAGE) to the EGF precursor extracted from the membranes with detergent(17) . Prolonged autolysis (24 h at 37 °C) of rat kidney membranes released two forms of soluble EGF-immunoreactive material, and this process was inhibited by aprotinin(37) .
Further processing of the 160-kDa pro-EGF to lower molecular mass species may occur within the lumen of the renal tubule and/or in the urine per se. Kallikreins and other endoproteases have been described in urine(38) , and some of these may be active toward urinary pro-EGF. Purified mouse submaxillary gland EGF binding protein(18) , also known as mouse glandular kallikrein-9, was shown to process mouse kidney EGF precursor to a 97-kDa species(21) , and rat submaxillary gland kallikreins K1, K7, and K10 were also shown to process a rat urinary 45-kDa EGF precursor to an approximately 6-kDa EGF species (39) . In the present study, it was of interest that the native pro-EGF appeared to be a component of a high molecular weight complex, as estimated by gel filtration chromatography of the heparin-agarose eluate. It is possible that association of pro-EGF with specific urinary proteins might serve to regulate its processing to mature EGF.
We have shown that the urinary 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor, activation of the EGF receptor tyrosine kinase, and stimulation of the proliferation of cultured human fibroblasts. Our data with A-431 cell membranes demonstrate that it is the intact 160-kDa pro-EGF that binds to the EGF receptor; binding did not require conversion of pro-EGF to a lower molecular mass species. High molecular weight EGF precursors extracted from hEGF19 cells (17) and mouse kidney membranes (21) have also been shown to possess biological activity. While several EGF-related growth factors appear to be active in their membrane-bound form(10, 40) , it remains to be established unequivocally that this is the case for the membrane-anchored EGF precursor with intact transmembrane and cytoplasmic domains.
The present data indicate that the urinary 160-kDa pro-EGF is a heparin-binding protein. It has been suggested that binding of growth factors to heparin-like molecules on the cell surface or extracellular matrix may serve to concentrate the soluble growth factor in the vicinity of its corresponding receptor (10) or to modulate growth factor activity(41) . Mature EGF has been reported not to require heparin for EGF receptor binding or activation(41) , but whether heparin affects the biological activity of urinary pro-EGF remains to be established.
Our experiments with SFME cells suggest an additional possible function for pro-EGF. When culture dishes were coated with urinary pro-EGF, the immobilized protein appeared to function as a cell adhesion molecule as well as a growth factor for SFME cells. The mechanism whereby pro-EGF promotes cell adhesion is not clear, but several possibilities exist. Immobilized heparin-binding pro-EGF may bind SFME cells via cell surface heparin-like molecules and/or EGF receptors, thereby promoting cell attachment. Alternatively, binding may occur due to other intrinsic adhesion properties of pro-EGF. It is of interest to note that the cell adhesion molecule laminin contains multiple EGF-like motifs(42) . Further characterization of the adhesion properties of pro-EGF may provide insight into possible physiological roles for the EGF precursor in cell-matrix or cell-cell interactions. It is possible that in the urinary tract pro-EGF might bind to urothelial glycosaminoglycans or to heparin-like molecules on damaged mucosa and thereby serve to enhance urothelial repair.