INTRODUCTION

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Amphiregulin (AR)¹ is one of six mammalian ligands that bind the epidermal growth factor receptor (EGFR/HER-1). AR, which was originally isolated from conditioned medium of a PMA-treated human breast carcinoma cell line, is a cell type-dependent bifunctional modulator of cell growth (1). AR protein is synthesized as a 252-amino acid transmembrane glycoprotein, and two major soluble forms of 78 and 84 amino acids, which migrate between 20 and 25 kDa on SDS-PAGE, have been characterized (1-3). In addition, soluble AR forms of 9.5-10, 35, and 55-60 kDa have been identified in several other cell types (4-8). The variety of soluble AR forms implies intricacies in AR processing that have not yet been elucidated.

AR mRNA has been isolated from normal human tissues including ovary, placenta, testis, pancreas, spleen, kidney, lung, breast, and colon (3). For normal colonic mucosa, AR protein expression has been localized to more differentiated cells in the non-proliferative compartment (9). AR mRNA and protein have been detected in many colon cancer cell lines, primary colorectal tumors, and metastatic colorectal tumors (9-12). Although AR is expressed in normal colonic mucosa, it is uniformly overexpressed in colon cancer (11). In addition, AR mRNA and protein have been detected in normal human mammary epithelial cell and normal human keratinocyte cultures (6, 13, 14). AR has been shown to act as an autocrine factor for human colon carcinoma cell lines, normal human mammary epithelial cell lines, and normal human keratinocytes (9, 13-18). Recent studies indicate that AR may be involved in up-regulation of matrix metalloproteases, invasion by tumor cells, as well as hyperproliferation and inflammation observed with psoriasis (19-21). These findings suggest that AR is involved in both normal and aberrant cellular processes.

AR elicits biological effects by binding directly to and activating EGFR/HER-1 (22). Heterodimerization of EGFR/HER-1 with other HER family members enables AR to activate other HER family members, such as HER-2 (23, 24). Whereas initial receptor binding assays suggested that AR has a lower affinity for EGFR/HER-1 than EGF (2), recent studies indicate that the affinities of EGF and AR for EGFR/HER-1 may be more similar than previously observed (13, 27). Studies of recombinant AR forms with extensions C-terminal to mature AR have indicated that these forms have increased biological activity (25-27), suggesting that differential processing of the C terminus of mature AR may be important in regulating the different biological responses elicited by AR.

To date, most of the biochemical characterization of AR has been performed under steady state conditions (1, 3, 5-8). The present studies provide a detailed analysis of the biosynthesis and processing of AR in epithelial cells, both endogenous AR in human colorectal and mammary cancer cells, as well as various human pro-AR forms stably expressed in MDCK cells. We show that a predominant 50-kDa pro-AR form can be detected at the cell surface and is preferentially cleaved at the distal site within the ectodomain to release a major 43-kDa AR form into the medium. Sequential ectodomain cleavage of 50-kDa cell surface pro-AR, however, can lead to the appearance of other cell surface pro-AR forms and to the release of several soluble AR forms. The distal cleavage event can be stimulated by PMA and inhibited by the metalloprotease inhibitor, batimastat.

	EXPERIMENTAL PROCEDURES

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Reagents and Antibodies-- Cell culture reagents were purchased from Life Technologies, Inc. Chemicals were purchased from Sigma, unless otherwise stated. Tran³⁵S-label was purchased from ICN Biomedicals (Costa Mesa, CA). Sulfo-NHS-LC-biotin and protein A-agarose were purchased from Pierce. N-Glycosidase F, endoglycosidase H (endo H), O-glycosidase, and neuraminidase were purchased from Boehringer Mannheim. All electrophoresis reagents were purchased from Bio-Rad. Rainbow markers and ECL kit were purchased from Amersham Pharmacia Biotech. Long form of recombinant human AR was purchased from R & D Systems Inc. (Minneapolis, MN). The matrix metalloprotease inhibitor batimastat (BB94) (28) was kindly provided by Dr. Peter Brown (British Biotech, Oxford, UK).

Cells and Cell Culture-- MDCK strain II and Caco-2 cells were obtained from Dr. Enrique Rodriguez-Boulan (Cornell University Medical College, NY). HCA-7 (clone 29) and MCF-7 cell lines were obtained from Dr. Susan Kirkland (ICRF, London, UK) and Dr. Lynn Matrisian (Vanderbilt University, Nashville, TN), respectively. Chinese hamster ovary (CHO) cell lines CHO-K1, CHO-745, and CHO-677 were obtained from Dr. Jeffrey Esko (University of Alabama, Birmingham, AL). The CHO-677 mutant lacks N-acetylglucosaminyl- and glucuronosyltransferases and cannot make heparan sulfate glycosaminoglycans (GAGs) (29). The CHO-745 mutant lacks xylosyltransferase and cannot make heparan or chondroitin sulfate GAGs (30). For each cell line, all experiments were performed within 10 passages. MDCK-II, Caco-2, HCA-7, and MCF-7 were cultured in DMEM supplemented with 10% fetal bovine serum (Intergen, Purchase, NY), as described previously (31). The CHO cell lines were grown in Ham's F-12 supplemented with 10% fetal bovine serum as described previously (30). For culture on Transwell filters (0.4-µm pore size, Costar, Cambridge, MA), cells were seeded at 1 × 10⁵ and 5 × 10⁵ cells on 12- and 24-mm Transwell filters, respectively. For cells grown on Transwell filters, medium was changed daily, except for Caco-2 and HCA-7 which were changed every other day.

AR Constructs, Transfection, and Selection of Expressing Clones-- Two human AR cDNAs encoding the wild type AR precursor were obtained from Dr. Gary Shipley (Oregon Health Sciences University, Portland, OR) (13) and Dr. Greg Plowman (Sugen, Redwood City, CA) (3) and were subcloned into the pCB6 CMV expression vector. Both human AR cDNAs were used in these studies and gave equivalent results. A secreted AR construct (ARsec184) which ends in  ... ERCGEK¹⁸⁴ was generated by excising the EcoRI-NotI fragment (885 base pairs) from the full-length pro-AR cDNA, digesting with HphI to isolate a 576-base pair fragment, and ligating the HphI fragment with HphI/BamHI annealed adapter oligonucleotides (5'-GGGAAAAGTGATAAG-3' and 3'-CCCCTTTTCACTATTCCTAGG-5') into Bluescript II (SK). Subsequently, an XbaI-EcoRI fragment was excised and subcloned into pCB6. A secreted AR construct (ARsec190) which ends in  ... SMKTHS¹⁹⁰ was generated in the same manner as ARsec184 using annealed adapter oligonucleotides (5'-GGGAAAAGTCCATGAAAACTCACAGCTGATAAG-3' and 3'-CCCCTTTTCAGGTACTTTTGAGTGTCGACTATTCCTAGG-5'). The C-terminal MYC-tagged AR construct (AR/c-MYC) was generated by isolating an EcoRI-RsaI fragment and ligating the RsaI fragment with RsaI/BamHI-annealed oligonucleotides (5'-ACATGCTATAGCAGAGCAGAAGCTGATCTCCGAGGAGGACCTTTAATGAG-3' and 3'-TGTACGATATCGTCTCGTCTTCGACTAGAGGCTCCTCCTGGAAATTACTCCTAG-5'). All constructs were confirmed by DNA sequencing.

Metabolic Labeling-- All cells were grown on 24-mm Transwell filters in the appropriate medium for 4-6 days. To increase AR expression in transfected cell lines, cells were treated with 5 mM sodium butyrate in the appropriate medium for 16-18 h before labeling. For pulse and 2-h labeling, cells were rinsed 2 times with serum-free, L-cysteine/L-methionine-free DMEM (DME) and incubated in DME for 30 min at 37 °C. Cells were labeled with 1-2 mCi/ml Tran³⁵S-label as described previously (31). For pulse-chase experiments, cells were labeled at 37 °C for the indicated amount of time, rinsed once with 18 °C chase medium (DMEM containing 10 times excess L-cysteine and L-methionine), and incubated at 37 °C with chase medium for the specified times.

Cell Surface Biotinylation-- Cells were grown on 24-mm Transwell filters as described above. All steps were performed on ice or at 4 °C, unless otherwise stated. Prior to biotinylation, cells were rinsed 3 times with PBS containing 0.1 mM CaCl₂ and 1.0 mM MgCl₂ (PBS⁺). For polarizing cells, 1.5 mg/ml sulfo-NHS-LC-biotin in PBS⁺ was added to the basal compartment, and PBS⁺ was added to the apical compartment, and cells were incubated for 30 min. Non-polarizing cells were incubated with 1.5 mg/ml sulfo-NHS-LC-biotin in both the apical and basal compartments. Following biotinylation, cells were rinsed and washed 2 times with ice-cold 100 mM glycine in PBS⁺ and were rinsed 3 times with PBS⁺ containing 0.2% BSA (PBS/BSA).

AR Immunoprecipitation-- All immunoprecipitation protocols were performed at 4 °C or on ice, unless otherwise stated. After final washes, filters were cut out, and cell lysates were prepared as described previously (31). Cell lysates were incubated overnight with AR mAb (0.2 µg/ml). Specificity of AR mAb for the different forms of AR was determined by preincubating the antibody with an excess (10-100 ng/sample) of recombinant human AR before immunoprecipitation. In order to precipitate antibody-AR complexes, affinity purified rabbit anti-mouse IgG antibody was added for 1 h followed by addition of a 50% slurry of protein A-agarose for 2 h. For immunoprecipitation from conditioned medium, phenylmethylsulfonyl fluoride (Boehringer Mannheim) was added to 2 mM, and the medium was precleared, filtered through a 0.2-µm filter (Gelman Sciences, Ann Arbor, MI), and incubated overnight with AR mAb (0.05 µg/ml). Protein A-agarose was pelleted and complexes were stringently washed. Immunoprecipitates were analyzed under reducing conditions on 12.5% SDS-PAGE, unless otherwise specified. For metabolically labeled samples, gels were fixed, treated with Amplify (Amersham Pharmacia Biotech) for 30 min, and dried, and fluorography was performed with BioMax MR film (Eastman Kodak Co.).

AR Western Blotting-- Biotinylated samples were separated by SDS-PAGE under reducing conditions, and non-labeled samples were separated under non-reducing conditions to eliminate confounding detection of heavy and light immunoglobulin chains. After separation by SDS-PAGE, immunoprecipitates for biotinylated or non-labeled samples were electrophoretically transferred overnight at 30 V to nitrocellulose (0.2 µm, Bio-Rad). All steps were performed at room temperature. Membranes were rinsed twice in TBS-T (1× TBS, Tween 20) and blocked for 30 min in TBS-T containing 3% BSA (TBS-T/BSA). Once blocked, membranes were incubated for 1 h with primary antibody in TBS-T/BSA (AR mAb at 5 µg/ml or MYC mAb at 10 µg/ml) and then rinsed and washed with TBS-T. After washing, membranes were incubated with secondary antibody in TBS-T/BSA (SA-HRP at 1:20,000 or donkey anti-mouse IgG-HRP at 1:5,000) for 30 min, washed extensively with TBS-T, and rinsed with TBS. Membranes incubated in SA-HRP received no primary antibody. The ECL Western blotting kit was used per manufacturer's directions, and fluorography was performed using BioMax MR film.

PMA and Batimastat Treatment-- Cells seeded on 24-mm Transwell filters were biotinylated and washed for chase experiments as described above. For the first 5 min of chase, cells were incubated at 37 °C in either serum-free DMEM containing Me₂SO (control) or 5 µM batimastat. After 5 min, the chase medium was changed to serum-free DMEM containing either Me₂SO, 5 µM batimastat, 1 µM PMA, or a combination of batimastat and PMA, and incubated at 37 °C for the indicated time. Cells and conditioned medium were collected, and AR immunoprecipitation combined with detection of biotinylated AR by Western blotting was performed as described previously.

	RESULTS

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Multiple Endogenous Soluble and Cellular Forms of AR Are Detected for Epithelial Cells-- To identify the forms of AR produced in epithelial cells under basal conditions, we examined newly synthesized AR in two human colon cancer cell lines, HCA-7 and Caco-2, as well as a human breast cancer cell line, MCF-7, that produce AR. Cell lysates and conditioned medium from cells metabolically labeled with Tran³⁵S-label for 2 h were immunoprecipitated with AR mAb, and immunoprecipitates were analyzed by SDS-PAGE and fluorography. A predominant diffuse 43-kDa AR form, a less intense 19- and 21-kDa doublet, and weak immunoreactive form of <14.3 kDa running at the dye front were detected in conditioned medium (Fig. 1A). In separate experiments on higher percentage SDS-PAGE, the weak immunoreactive form was resolved as a 9-kDa band (data not shown). In addition, clonal MDCK cell lines stably transfected with a cDNA for wild type human pro-AR (MDCK-AR) released similar AR forms (Fig. 1A).

View larger version (51K): [in this window] [in a new window]   Fig. 1.   Multiple cellular and soluble AR forms are detected in epithelial cells. A, HCA-7, Caco-2, MCF-7, as well as MDCK cells expressing wild type human pro-AR (MDCK-AR) grown on Transwell filters were metabolically labeled with Tran35S-label for 2 h and chased for 2 h. Conditioned medium was collected and immunoprecipitated with AR mAb. B, HCA-7 or MDCK-AR cells grown on Transwell filters were metabolically labeled with Tran35S-label for 2 h. After labeling, total cell lysates were prepared, and AR immunoprecipitation was performed. All immunoprecipitates were analyzed by SDS-PAGE and fluorography. Arrowheads indicate the various AR forms, and molecular masses are indicated in kilodaltons.

Biosynthesis and Processing of Wild Type Pro-AR in MDCK Cells-- To delineate potential relationships among cellular and soluble AR forms, we performed pulse-chase experiments using MDCK-AR cells. Cells were metabolically labeled with Tran³⁵S-label for 20 min and chased for various times, and total cell lysates were analyzed. At 0 min of chase, a narrow band of 45 kDa and a diffuse band of 50 kDa were detected (Fig. 2A). In independent pulse-chase experiments examining total cell lysates, only the 45-kDa band was detected by 0 min of chase after 5 min of metabolic labeling, indicating it was a more immature pro-AR form (data not shown). By 20 min of chase, the 45-kDa band had diminished, which coincided with an increase in intensity of the 50-kDa AR. Intensity of the 50-kDa AR peaked at 20 min of chase and decreased with a half-life of ~20 min. At 20 min of chase, a 26- and 28-kDa doublet and a 16-kDa AR form were also detected; however, intensities of these forms were significantly less than the 50-kDa form. Based on an independent pulse-chase experiment involving a 5-min metabolic labeling with Tran³⁵S-label, increasing intensities of the 26- and 28-kDa doublet and the 16-kDa band were concurrent with loss of the 50-kDa AR through 40 min of chase, suggesting that the 26- and 28-kDa doublet may be derived from the 50-kDa AR (data not shown). By 4 h chase, no cellular AR forms were detectable.

View larger version (41K): [in this window] [in a new window]   Fig. 2.   Biosynthesis of wild type pro-AR in polarized MDCK cells. MDCK-AR cells grown on Transwell filters were metabolically labeled for 20 min and chased for various amounts of time. After chase, cell surface biotinylation was performed. AR immunoprecipitation was performed on total cell lysates (A) and conditioned medium (C). For cell surface AR (B), AR immunoprecipitates from total cell lysates were eluted and re-immunoprecipitated with streptavidin-agarose. All immunoprecipitates were analyzed by SDS-PAGE and fluorography. Arrowheads indicate the various AR forms, and molecular masses are indicated in kilodaltons.

N-Glycosylation, but Not O-Glycosylation or GAG Incorporation, Contributes to the Size of the Predominant Cellular and Soluble AR Forms-- Previous studies suggest that the major species of soluble AR are low molecular mass forms of 18-35 kDa (1, 6). One possibility to account for the high molecular mass AR forms detected in these studies is the presence of posttranslational modifications. Potential N- and O-linked glycosylation sites have been identified for pro-AR (3), and N-glycosylation of AR has been demonstrated in PMA-treated cells (1, 5, 8). To determine the extent of glycosylation of cellular and soluble AR forms, cell lysates and conditioned medium from MDCK-AR cells metabolically labeled with Tran³⁵S-label for 2 h were immunoprecipitated with AR mAb and digested with different glycosidases. We first examined which forms were endo H-sensitive. As shown in Fig. 3A, only the 45-kDa cellular AR is endo H-sensitive, confirming that it represents an immature pro-AR form that has not been modified in the Golgi. In addition, tunicamycin treatment resulted in the appearance of an ~39-kDa AR form (data not shown). These data also suggest that unmodified pro-AR has an electrophoretic mobility of ~39-kDa, which is much slower than the predicted electrophoretic mobility of ~26-kDa for the full-length, unmodified AR precursor minus the signal peptide (3). A similar electrophoretic mobility for pro-AR has been proposed based on in vitro transcription and translation of the full-length AR cDNA (8).

View larger version (33K): [in this window] [in a new window]   Fig. 3.   The AR precursor is modified by N-glycosylation. MDCK-AR cells grown on Transwell filters were metabolically labeled with Tran35S-label for 2 h and chased for 2 h. Total cell lysates and conditioned medium were prepared and immunoprecipitated with AR mAb. Immunoprecipitates were washed in appropriate buffers and digested with either endo H (A) or various combinations of N-glycosidase F, neuraminidase, and O-glycosidase (B) as described under "Experimental Procedures." All digests were analyzed by SDS-PAGE and fluorography. Arrowheads indicate the various AR forms; dashes indicate deglycosylated AR forms, and molecular masses are indicated in kilodaltons. * represents a minor AR precursor form with minimal glycosylation.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   Glycosaminoglycan attachment does not contribute to the molecular mass of AR. MDCK, CHO-K1, CHO-745, and CHO-677 cells transfected with wild type human pro-AR and grown on Transwell filters were metabolically labeled with Tran35S-label for 2 h and chased for 2 h. Total cell lysates (A) and conditioned medium (B) were prepared and immunoprecipitated with AR mAb. C, AR immunoprecipitates from total cell lysates of MDCK-AR and CHO-K1-AR cells metabolically labeled with Tran35S-label for 2 h were washed in appropriate buffers and digested with heparatinase as described under "Experimental Procedures." All immunoprecipitates were analyzed by SDS-PAGE and fluorography. Arrowheads indicate the various AR forms, and molecular masses are indicated in kilodaltons. * represents alternate glycosylated AR forms found in CHO cells.

Analysis of AR Secretory Mutants Suggests Soluble 43-kDa AR Contains N-terminal Pro-region-- Our results indicate that posttranslational modifications, such as glycosylation and GAG attachment, did not fully account for the increased molecular mass of 43-kDa soluble AR. Using an antibody to the AR pro-region, previous studies in transfected human mammary epithelial cells indicated that a 42-kDa cellular AR form contains the full-length N-terminal pro-region (6). To address the possibility that 43-kDa soluble AR may retain the N-terminal pro-region, we stably transfected two different secreted AR constructs containing the N-terminal pro-region (Fig. 5A) into MDCK cells. One construct (ARsec184) contains the first 184 amino acids of pro-AR terminating at the C-terminal end of the predicted mature AR (3) and therefore does not contain the six C-terminal residues of the EGF-like domain. The other construct (ARsec190) contains the first 184 amino acids of pro-AR plus the 6 amino acid C-terminal extension, and therefore contains the full-length EGF-like domain. Wild type and mutant soluble AR were immunoprecipitated from conditioned medium, and the immunoprecipitates were analyzed on SDS-PAGE. As shown previously, wild type 43-kDa, 19- and 21-kDa doublet, and 9-kDa soluble AR forms were detected. ARsec190 and ARsec184 are represented with one form each of 41 and 39 kDa, respectively (Fig. 5B). N-Glycosidase F digestion of wild type and mutant AR resulted in the same pattern of electrophoretic mobilities, which confirmed that N-glycosylation did not completely account for the increased molecular mass of 43-kDa soluble AR (Fig. 5C). The observation that the electrophoretic mobility of high molecular mass 43-kDa wild type AR was similar to both secreted AR mutants suggests that it retains the N-terminal pro-region. In addition, previous studies using PMA-treated MCF-7 cells had demonstrated that the distal cleavage site for the 78 and 84 amino acid AR forms is within the EGF-like domain (3). In the present study, this cleavage is represented by the ARsec184 mutant. Our results show that wild type 43-kDa soluble AR migrates significantly slower than either secreted AR mutant, and particularly ARsec190, raising the possibility of a cleavage site more distal to the EGF-like domain.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   High molecular mass soluble AR may contain the entire N-terminal domain of the AR precursor. A, a schematic comparing wild type human pro-AR with the ARsec190 and ARsec184 mutants. ARsec190 and ARsec184 comprise the first 190 and 184 amino acids, respectively, of full-length pro-AR. MDCK cells transfected with wild type or mutant pro-AR and grown on Transwell filters were metabolically labeled with Tran35S-label for 2 h and chased for 2 h. Untreated conditioned medium (B) or conditioned medium digested with N-glycosidase F (C) was immunoprecipitated with AR mAb. Immunoprecipitates were analyzed by SDS-PAGE and fluorography. Arrowheads indicate the various AR forms produced by MDCK-AR cells; dashes indicate the mutant AR forms, and molecular masses are indicated in kilodaltons.

Cell Surface AR Forms Are Membrane-anchored-- These results suggested that the various soluble AR forms were derived by sequential processing of different cell surface forms; however, the nature of AR cell surface forms remained unclear. Because the only antibodies available are those raised against portions of the AR ectodomain, it has not been possible to distinguish whether cell surface AR forms are membrane-anchored or cell-associated. To address these two possibilities, we stably expressed wild type pro-AR containing a C-terminal c-MYC epitope tag in MDCK cells (MDCK-AR/c-MYC) (Fig. 6A). Cell surface AR forms which contain the MYC epitope tag would be predicted to possess both transmembrane and cytoplasmic domains, whereas those that did not would be predicted to have resulted from ectodomain cleavage. Cell surface biotinylation combined with AR or MYC mAb immunoprecipitation and SA-HRP Western blotting was used to specifically examine cell surface AR forms. Total cell lysates from biotinylated MDCK-AR or MDCK-AR/c-MYC cells were immunoprecipitated with AR mAb, separated by SDS-PAGE, transferred to nitrocellulose, and probed with SA-HRP. As shown in Fig. 6B, we confirmed the 50-kDa, 26- and 28-kDa doublet, and 16-kDa cell surface pro-AR forms were present in both cell lines and electrophoretic mobilities of all AR cell surface forms were reduced by addition of the C-terminal c-MYC tag. To confirm the presence of MYC-tagged pro-AR at the cell surface, cell lysates from cell surface biotinylated MDCK-AR/c-MYC cells were immunoprecipitated with either AR or MYC mAb, separated by SDS-PAGE, transferred to nitrocellulose, and probed with SA-HRP. With the exception of the 16-kDa cell surface AR form, similar AR forms were detected in MDCK-AR/c-MYC cells with both antibodies (Fig. 6C). Although the reason for lack of detection of the 16-kDa cell surface AR is unknown, the consistency of its detection after metabolic labeling (Figs. 1-4) suggests that there may be some variability in the sensitivity of detection of 16-kDa AR by cold cell surface biotinylation (see also Fig. 7). We predict that the 16-kDa cell surface form has lost much of the heparin-binding domain, and therefore the sensitivity of detection of this form by cold cell surface biotinylation may be decreased due to loss of many of the lysines that serve as potential sites for biotinylation. These results demonstrated that all cell surface AR forms contain the transmembrane and cytoplasmic domains of pro-AR.

View larger version (39K): [in this window] [in a new window]   Fig. 6.   Cell surface AR forms contain transmembrane and cytoplasmic domains. A, a schematic illustrating wild type human pro-AR and wild type human pro-AR with a C-terminal c-MYC epitope tag (AR/c-MYC). MDCK-AR or MDCK-AR/c-MYC cells grown on Transwell filters were cell surface-biotinylated and chased for 1 h. B, total cell lysates from MDCK-AR and MDCK-AR/c-MYC cells were immunoprecipitated with AR mAb. Total cell lysates (C) and conditioned medium (D) from MDCK-AR/c-MYC cells were immunoprecipitated with either AR or MYC mAb. All immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with SA-HRP. Immunoreactivity was detected by using the ECL Western blotting detection kit in combination with fluorography. Arrowheads indicate the various AR forms, and molecular masses are indicated in kilodaltons.

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Cleavage of the AR ectodomain is stimulated by PMA and inhibited by the metalloprotease inhibitor, batimastat. MDCK-AR cells grown on Transwell filters were cell surface-biotinylated and chased for various lengths of time in the presence of Me2SO (control), PMA, batimastat, or PMA and batimastat. Total cell lysates (A) and conditioned medium (B) were immunoprecipitated with AR mAb. Immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose, and probed with SA-HRP. Immunoreactivity was detected by using the ECL Western blotting kit in combination with fluorography. Arrowheads indicate the various AR forms, and molecular masses are indicated in kilodaltons.

Soluble Forms Are Derived from Ectodomain Cleavage of Cell Surface Pro-AR-- We next used MYC-tagged pro-AR in a similar strategy to determine whether soluble AR forms were derived directly by ectodomain cleavage of membrane-bound cell surface forms. MDCK-AR/c-MYC cells were cell surface biotinylated and chased for 2 h to release biotinylated AR forms into the conditioned medium. Conditioned medium was immunoprecipitated with either AR or MYC mAb, separated by SDS-PAGE, transferred to nitrocellulose, and probed with SA-HRP. Unlike cell surface AR forms which can be immunoprecipitated with both AR and MYC mAbs (Fig. 6C), soluble AR forms were only immunoprecipitated by AR mAb (Fig. 6D), suggesting that soluble AR forms do not contain the transmembrane and cytoplasmic domains. It is also of interest to note that the 19- and 21-kDa soluble AR doublet detected in conditioned medium of MDCK-AR cells was not detected in conditioned medium of MDCK cells expressing secreted AR mutants (Fig. 5B). Absence of the 19- and 21-kDa AR doublet in conditioned medium of AR mutants further supports pulse-chase data (Fig. 2) suggesting these forms derive from cell surface forms of AR and not from processing of 43-kDa soluble AR. Taken together, these data support the hypothesis that soluble AR forms are derived directly from ectodomain cleavage of membrane-bound cell surface forms.

AR Ectodomain Cleavage Is Stimulated by PMA and Inhibited by Batimastat-- To characterize further the release of AR from the cell surface, we performed cold biotinylation chase experiments. MDCK-AR cells grown on Transwell filters were biotinylated for 30 min and chased up to 60 min. Total cell lysates were prepared, separated by SDS-PAGE, transferred to nitrocellulose, and probed with SA-HRP. A serum-free chase was performed to determine the basal rate of cell surface pro-AR cleavage. At 0 min chase, the 50-kDa, 26- and 28-kDa doublet, and less apparent 16-kDa forms of pro-AR were detected at the cell surface (Fig. 7A). By 30 min chase, the intensity of the 50-kDa and 26- and 28-kDa doublet forms of pro-AR had begun to diminish, and at 60 min chase, the 50-kDa form was significantly reduced. The intensity of the 16-kDa pro-AR peaked at 30 min and was diminished by 60 min chase. Concomitantly, the 43-kDa, 19- and 21-kDa doublet, and 9-kDa soluble AR forms are detected in the conditioned medium at 30 min chase (Fig. 7B) and continued to accumulate up to 60 min chase confirming that soluble AR forms represent forms released from the cell surface. The difference in rates of cleavage of the 50-kDa pro-AR form and the 26- and 28-kDa pro-AR doublet combined with differences in rates of appearance of the soluble 43-kDa and soluble 19- and 21-kDa AR doublet suggest that cleavage of the doublet is inefficient.

	DISCUSSION

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Amphiregulin was originally isolated and characterized from serum-free conditioned medium of phorbol ester-treated MCF-7 cells (1). Amino acid sequencing of mature AR indicated it exists in two forms, a 78 amino acid truncated form and an 84 amino acid form which has a 6 amino acid N-terminal extension (1, 2). It is predicted that these forms arise from differential processing at two distinct N-terminal, or proximal, cleavage sites within the pro-AR ectodomain, which is different from the single proximal site observed with pro-TGF processing (33, 40). Another difference between processing of pro-AR and other EGF-like ligand precursors is that the predicted C-terminal, or distal, cleavage site for mature AR (3) does not encompass the entire EGF-like domain. Both mature AR forms are truncated at the C terminus by six amino acids and therefore lack conserved amino acids required for high affinity binding to EGFR/HER-1 (41-43). More recent studies have demonstrated that C-terminal extension of mature AR increases its biological activity (25, 27) suggesting that different forms of AR may perform distinct biological functions.

Since the initial characterization of AR, several groups have identified other cellular and soluble AR forms from a variety of cell types (1, 3, 5-8). In general, these studies have analyzed AR expression under steady state conditions, and most have focused on AR isolated from MCF-7 cells after prolonged PMA treatment. Only limited biochemical characterization of these novel AR forms has been performed. In the current study, we sought to define the biosynthesis and processing of pro-AR by epithelial cells under basal conditions.

High Molecular Mass Cellular and Soluble AR Forms Expressed in Epithelial Cells-- In non-transformed MDCK cells transfected with pro-AR as well as in two transformed colon cell lines, HCA-7 and Caco-2, which express endogenous pro-AR (data not shown), we show that newly synthesized AR is modified during transit through the secretory pathway and is rapidly delivered to the plasma membrane as an N-glycosylated 50-kDa pro-AR. Once at the cell surface, pro-AR is rapidly cleaved (t1/2 = 20 min) to release soluble AR forms. Interestingly, the only other report on the biosynthesis and processing of pro-AR in a transformed mammary epithelial cell line (184A1N4-TH) indicated that cellular pro-AR persisted for at least 4 h and was only partially processed into a soluble form (6). The reasons for the differences in pro-AR processing observed between the present study and this previous report are not known, although similar findings have been described for pro-TGF processing in different cell types (44).

View larger version (16K): [in this window] [in a new window]   Fig. 8.   A proposed model for the origins of cellular and soluble AR forms. The domain structure of 50-kDa cell surface pro-AR is indicated as follows: N-terminal pro-region (a), heparin-binding (b), EGF-like (c), transmembrane (d), and cytoplasmic (e). A, primary proteolytic cleavage of the ectodomain of 50-kDa pro-AR at a distal site releases a high molecular mass 43-kDa soluble AR, which contains extensions of both N and C termini, into conditioned medium (#1). Alternatively, in the absence of primary proteolytic cleavage at the distal site, secondary cleavage at proximal sites removes the N-terminal domain resulting in 26- and 28-kDa cell surface pro-AR forms, which appear as a doublet in our experiments (#2). B, the 26- and 28-kDa pro-AR doublet is subject to cleavage at a distal site to release a 19- and 21-kDa soluble AR doublet from the cell surface (#3). This soluble doublet probably represents the originally described mature AR forms (1). The basal rate of cleavage of the pro-AR doublet appears to be slower than for the 50-kDa AR form, thereby increasing the cell surface half-life of the doublet. In the absence of cleavage at the distal site, further proteolytic processing at a proximal site may result in a 16-kDa pro-AR cell surface form, which has lost much of the heparin-binding domain and is not glycosylated (#4). C, the 16-kDa cell surface pro-AR may be cleaved at a distal site to release a 9-kDa soluble AR, which has lost much of the heparin-binding domain, into the conditioned medium (#5).

Nuclear Localization of AR-- Another interesting point raised by rapid delivery and processing of newly synthesized pro-AR at the cell surface concerns the mechanism for nuclear localization of AR. Several studies have immunolocalized AR to the nucleus in a variety of cell types both in vitro (23, 46) and in vivo (9). Two stretches of basic residues within the pro-AR heparin-binding domain are predicted not only to confer heparin binding capacity but also to act as a bipartite nuclear localization signal (2, 3). Earlier studies with Schwannoma-derived growth factor (47), the rat homolog of AR, suggested that these nuclear targeting signals are required for transport of Schwannoma-derived growth factor into the nucleus and for induction of its mitogenic activity in mouse NIH 3T3 cells (48). Although we have not directly addressed nuclear localization of AR in these studies, our pulse-chase data suggests that most, if not all, newly synthesized 50-kDa pro-AR is delivered directly to the cell surface where it is rapidly cleaved to release a major 43-kDa soluble AR form into the medium. This result indicates that a nuclear localization signal does not function in the secretory pathway. Interestingly, Martinez-Lacaci et al. (8) have suggested that lower molecular mass AR forms are differentially expressed in the nucleus of MCF-7 cells after PMA and 17-estradiol treatment. This observation raises the possibility that nuclear transport of AR occurs only after initial cell surface cleavage of 50-kDa pro-AR and involves lower molecular mass cell surface and soluble AR forms derived from secondary and tertiary cleavage events. The mechanism(s) by which different low molecular mass cellular and soluble AR species are internalized from the plasma membrane and subsequently gain access to the nucleus are not known.

All Cell Surface AR Forms Are Membrane-anchored-- A distinguishing feature of several EGF-like ligands, including high molecular mass EGF, HB-EGF, AR, and betacellulin, is that they can bind heparin or heparan sulfate proteoglycans (13, 49-51). In the case of HB-EGF, there is a heparin-binding region in the extracellular domain of the precursor (52). HB-EGF may be presented at the cell surface as either a membrane-bound precursor or as a soluble form which is cell-associated through interactions with cell surface heparan sulfate proteoglycans (37). Similarly, pro-AR contains a heparin-binding region within its ectodomain (3) raising the possibility that cell surface AR forms also may be membrane-anchored or cell-associated. We sought to distinguish between these two possibilities using a pro-AR construct that contains a C-terminal epitope tag. Cell surface biotinylation studies with MYC-tagged pro-AR indicate that all cell surface AR forms are membrane-anchored (Fig. 6C). This is a surprising finding considering numerous reports have demonstrated that soluble AR can bind to heparin (6, 8, 13, 22, 53), but it is in agreement with our pulse-chase data which show that cell surface pro-AR is rapidly released and that soluble AR accumulates in the conditioned medium (Fig. 2). Interestingly, the inability of 43-kDa soluble AR to bind to cell surface heparan sulfate proteoglycans may correlate with its significantly lower affinity for heparin (8).

Sequential Cell Surface Processing of AR-- Cell surface processing of pro-TGF has been studied extensively in CHO and NIH 3T3 cells and occurs by a sequential two-step process (54, 55). The first cleavage step, which removes the glycosylated N-terminal domain, occurs rapidly (t1/2 = 15 min) leaving a membrane-anchored form of pro-TGF that contains the sequence of mature TGF (32, 33, 56). The second cleavage step, which releases soluble mature TGF, occurs slowly (t1/2 = 120 min) but can be stimulated by a variety of factors, including PMA, serum, and Ca²⁺ (32, 34). The same sequential cleavage of pro-TGF occurs in polarized MDCK cells (31). In retrovirally transformed embryo fibroblasts and hepatocellular carcinoma cells, however, high molecular mass soluble TGF forms are found in the medium, suggesting that sequential cleavage of pro-TGF may be regulated differently in some transformed cell lines (44). Our analysis of the cell surface processing of pro-AR in transformed colon cell lines and in non-transformed MDCK cells indicates that the initial cleavage event for pro-AR is quite different to that for pro-TGF. The distal cleavage site of pro-AR is the preferred site, which causes release of 43-kDa soluble AR into the medium. In contrast to pro-TGF, several tyrosine sulfation sites are predicted to be within the pro-AR N-terminal domain (3) and would be juxtaposed to the proximal cleavage sites. Interestingly, tyrosine sulfation has been shown to regulate proteolytic processing of several proteins, including caerulein and gastrin (45). Although the protease(s) involved in cleavage at the proximal sites of pro-TGF and pro-AR have not been characterized, one possible explanation for lack of proximal pro-AR cleavage would be that tyrosine sulfation interferes with efficient cleavage at this site.

AR Ectodomain Cleavage Is Stimulated by PMA and Blocked by a Metalloprotease Inhibitor-- Cell shedding is a process in which membrane proteins undergo limited proteolysis to release ectodomains into the extracellular medium (57, 58). For membrane-anchored pro-TGF, pro-HB-EGF, and pro-EGF, ectodomain cleavage causes release of soluble ligands into the conditioned medium (49, 59, 60). In addition, cell shedding is a regulated process for many membrane proteins (55, 57). For pro-TGF (32-34) and pro-HB-EGF (35-37), but not pro-EGF (38), ectodomain cleavage can be stimulated by phorbol esters. In the present study, we demonstrate that ectodomain cleavage of membrane-anchored pro-AR can also be stimulated by the phorbol ester, PMA. Surprisingly, distal cleavage of the 26- and 28-kDa pro-AR doublet, which is inefficiently processed under basal conditions, is stimulated upon PMA activation to a level equivalent to that of the 50-kDa pro-AR form. At present, the mechanism(s) by which differential cleavage of the various membrane-anchored pro-AR forms occurs under basal conditions and after PMA activation are not known. One interesting possibility, however, is that under basal conditions the presence of the N-terminal domain in 50-kDa pro-AR allows a more favorable conformation of the stalk region (61) of the AR ectodomain for proteolytic activity and that this difference is circumvented upon PMA stimulation.