THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 5, Issue of February 15, pp. 2280-2284,1988 Printed in U.S.A.

Kinetics of Receptor-mediated Binding of Tropoelastin to Ligament Fibroblasts”

(Received for publication, June 16, 1987)

David S. Wrenn, Aleksander HinekS, and Robert P. Mecham4 From the Respiratory Disease and Critical Care Division, Department of Medicine, and Department of Cell Biology and Physiology, Jewish Hospital at Washington University Medical Center, St. Louis, Missouri 631 10

Soluble 129-labeled tropoelastin bound to confluent cultures of bovine ligamentum nuchae fibroblasts and to fibroblast plasma membrane preparations in a time- dependent, saturable, and reversible manner. Scat- chard analysis indicates that there are approximately 2 X 10’ binding sites/cell with a binding efficiency (Ka) of 8 X lo-’ M. Binding of tropoelastin to cells and membranes reached equilibrium by 90 min and was reversible with 50% of specifically bound material re- leased by 40 min. Specific binding o f tropoelastin to cells pre-treated with dilute trypsin solutions was re- duced significantly when compared with controls. Four polypeptides of estimated molecular masses of 67, 61, 55, and 43 kDa were obtained from detergent extracts of plasma membranes by elution affinity chromatog- raphy on elastin-Affi-Gel. Our findings establish that elastin-specific binding proteins displaying character- istics of a true receptor are present on the surface of elastin-producing cells.

Interactions between cells and macromolecules of the ex- tracellular matrix are critically important for the maintenance of normal cellular structure and function. Binding of matrix components to the cell membrane is mediated by specific receptor proteins or protein complexes (1) that promote cel- lular attachment, migration, growth, and differentiation (2- 4). Many receptors have been identified by their ability to mediate cellular attachment and spreading on matrix proteins bound to solid surfaces (5). Additional evidence for specific cell-matrix interaction is provided by observations that a variety of cell types display chemotaxis, a receptor-mediated process (6), to components Qf the extracellular matrix (7-11).

Studies with laminin and fibronectin have documented that cell surface receptors for these adhesive macromolecules rec- ognize specific domains or sequences on the polypeptide chain (1,5). In the case of fibronectin, this sequence can be as small as 3-4 amino acids (12). Our previous studies with elastin suggest that it, too, has the ability to interact specifically with cells. Fibroblasts and monocytes demonstrated directed mi- gration to tropoelastin and to peptides derived from insoluble

* This work was supported by National Institutes of Health Grants HL-26499, HL-29594, and Training Grant HL-07317. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $On leave from the Pomeranian Medical Academy, Szczecin,

Poland. 8 To whom reprint request should be addressed Respiratory Dis-

ease and Critical Care Div., Dept. of Medicine, The Jewish Hospital of St. Louis, 216 S. Kingshighway Blvd., P. 0. Box 14109, St. Louis, MO 63178.

elastin (8), and at least one site of chemotactic activity on elastin was found to be the repeating hexapeptide sequence Val-Gly-Val-Ala-Pro-Gly (8,13). Additional evidence for elas- tin binding proteins on cells has been provided recently by Hornebeck et al. (14) who demonstrated that purified elastic fibers bind strongly to fibroblasts and smooth muscle cells.

To provide additional, more direct evidence for an elastin receptor, we have undertaken binding studies with intact fibroblasts and with fibroblast plasma membrane fractions. Utilizing radiolabeled tropoelastin as ligand, we demonstrate high affinity binding sites that provide for specific interaction between elastin and receptors on the cell membrane.

MATERIALS AND METHODS

Cell Culture and Plasma Membrane Preparation-Fetal bovine ligamentum nuchae fibroblasts (FCL) were obtained and cultured as described previously (15). All experiments described in this report were with first or second passage cells. Plasma membranes were prepared using a modification of the procedure of Wileman et al. (16). Confluent cultures of fibroblasts were rinsed with phosphate-buffered saline and harvested with a rubber policeman into 10 ml of membrane isolation buffer consisting of 20 mM HEPES’ (pH 7.4), 250 mM sucrose, 2 mM benzamidine, 2 mM e-amino-caproic acid, 2 mM phenyl- methylsulfonyl fluoride, and 1 mM EDTA. Cells were transferred to a 50-ml conical centrifuge tube and collected by centrifugation at 500 X g for 10 min at 4 ‘C. The supernatant was discarded, the cells resuspended in 10 ml of fresh membrane isolation buffer with gentle trituration, and then lysed by N P cavitation following a 10-min pressurization at 250 p.s.i. The cell lysate was centrifuged as above to pellet nuclei, and the postnuclear supernatant, containing the crude membrane fraction, was transferred to polypropylene tubes and centrifuged at 33,000 X g for 45 min at 4 ‘C. The supernatant was discarded, the pellet resuspended in 0.5 ml of membrane isolation buffer, layered onto 30 ml of Percoll (1.045 g/ml), and centrifuged as above. A distinct band of flocculent membrane material approxi- mately 6 mm from the top of the gradient was removed with a flame- drawn Pasteur pipette and centrifuged at 110,000 X g for 45 min at 4 “C in a Beckman SW 50.1 rotor to pellet residual Percoll. Plasma membranes were removed from above the Percoll pellet, aliquoted into 0.5-ml Microfuge tubes using plastic pipette tips, and were stored at -70 “C until further use. Protein content of plasma membrane fractions was determined from amino acid analysis of an aliquot of known volume.

branes (280 pg total protein) from FCL-230 cells were suspended in Cell Surface Iodination und Immunoprecipitation-Plasma mem-

0.2 ml of 0.05 M Tris (pH 7.5) and radiolabeled with 0.5 mCi of NalZ5I using Iodobeads (Pierce Chemical Co.). Labeled membranes were pelleted by centrifugation at 10,000 X g and diaIyzed against 0.01 M Tris (pH 7.4) to remove unbound radioactivity. Membrane proteins were solubiiized with 1% (w/v) octyl-f3-glucoside, 50 mM HEPES (pH 7.3), 30 mM NaCl, 10 mM glucose, 1 mM EDTA and immunoprecipi- tated with anti-elastin IgG (17). Precipitated protein was analyzed on SDS-PAGE and fluorography (13).

Purification and Iodination of Tropoehtin-Tropoelastin was pre-

The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate-poly- acrylamide gel electrophoresis.

-

2280

Kinetics of Elastin Receptor Binding 2281

pared from fetal bovine ligamentum nuchae as described previously (13). Ten pg of tropoelastin in 20 p1 of 0.1 M potassium phosphate (pH 7.4), 0.02% Nonidet P-40 was labeled with 0.5 mCi of lZsII-Bolton- Hunter reagent (Amersham Corp., 2050 Ci/mmol) according to the manufacturer's instructions. Iodinated tropoelastin was separated from unbound radioactivity by chromatography on a 5-ml P-2 column (Bio-Rad) previously equilibrated with iodination buffer containing 0.1% ovalbumin. Greater than 95% of recovered peak material was precipitable with 10% trichloroacetic acid and was labeled to a specific activity of 1 X lo6 dpm/pg. The integrity of labeled tropoelastin was confirmed by SDS-PAGE followed by autoradiography. Aliquots of '2sI-tropoelastin were stored at -70 "C and thawed immediately before use. Binding Studies-Binding of tropoelastin to fetal calf ligament

fibroblasts was assessed with monolayer cultures. Cells were grown to confluence in 24-well cluster dishes in Dulbecco's minimal essential medium supplemented with nonessential amino acids and 10% fetal calf serum. Because tropoelastin and large elastin peptides undergo a phase separation (coacervation) a t temperatures in excess of 25 "C (18), binding studies were performed at 22 "C to minimize the risk of coacervation of labeled ligand. Binding studies were performed in triplicate in 1.0 ml of Dulbecco's minimal essential medium contain- ing 20 mM HEPES and 0.5% ovalbumin. Monolayers were washed three times (10 min each) with binding buffer to remove serum components and then incubated with 1 ml of binding buffer for 30 min at 22 "C with gentle agitation provided by an orbital shaker. The cells then received 1 ml of fresh binding buffer containing radiolabeled tropoelastin with or without excess unlabeled tropoelastin and were incubated as described in the figure legends. After various times of incubation, the binding buffer was rapidly aspirated and the mono- layers rinsed three times with ice-cold binding buffer to remove unbound material. Cells were solubilized in 0.5 ml of 0.5 N NaOH for 30 min and cell-associated radioactivity determined (Beckman LS 8000 y spectrometer). Specific binding was defined as the total radioactivity bound minus the amount bound in the presence of excess unlabeled material.

Binding of 12sI-tropoelastin to isolated plasma membranes was assessed by pipetting plasma membranes (20 pg total protein) in membrane isolation buffer onto 4-mm squares of nitrocellulose. After standing at room temperature for 2 h, the filters were transferred to 96-well polyvinylchloride microtiter dishes and incubated for 1 h a t 22 "C in 200 pl of binding buffer containing 1% bovine serum albumin or ovalbumin to block nonspecific binding sites. After extensive washing with binding buffer, the filters were incubated with 150 pl of binding buffer containing iodinated tropoelastin with or without excess unlabeled tropoelastin. Binding of iodinated tropoelastin to membrane coated filters was terminated by aspiration of binding medium followed by washing the filters three times for 5 min in ice- cold binding buffer. Specifically bound radioactivity was then deter- mined as described for cells. Binding of radiolabeled tropoelastin to nitrocellulose filters coated with bovine serum albumin or ovalbumin alone averaged about 200 cpm compared to 6000-8000 cpm observed for specific binding of tropoelastin to plasma membranes.

To determine whether fibronectin would compete with tropoelastin for receptor binding, plasma membranes were incubated for 100 min with 2 X IO5 cpm of iodinated tropoelastin in the presence or absence of unlabeled plasma fibronectin. Cell-associated radioactivity was then determined after 60 min.

Partitioning of bound tropoelastin between the cell surface and cytoplasm was assessed by incubating cellular monolayers with iodi- nated tropoelastin followed by a 10-min incubation at 22 "C with 0.025% trypsin. Radioactivity remaining with the cell pellet and in the supernatant following centrifugation at 500 X g was then deter- mined. Radioactivity released to the supernatant was taken as tro- poelastin accessible on the cell surface, while that associated with the cell pellet was assumed to be protected from proteolysis and, hence, internalized.

To determine whether elastin binding was protein-mediated, fibro- blast monolayers were incubated for 5 min at 22 "C with 0.0125% trypsin in Earle's balanced splt solution. The monolayers were washed three times with binding bufi'er containing 1% bovine serum albumin, 1% soybean trypsin inhibitcjr and binding assays performed as de- scribed above. Adherent cell:: were not observed to detach from the culture dish during this brief incubation.

Affiniw Isolation of Elustin Receptor-Elastin binding proteins were isolated from detergent extracts of purified plasma membranes usingaffinitychromatography. All procedures were performed at 4 "C. To prepare the affinity resins, approximately 20 mg of bovine serum

albumin (Sigma) or porcine pancreatic elastase-derived peptides of bovine insoluble elastin (19) in 0.1 M sodium bicarbonate (pH 8.0) were mixed overnight by end-over-end rotation with 3.0 ml of Affi- Gel 10 resin (Bio-Rad) in a siliconized glass tube. Unreacted resin binding sites were blocked by the addition of 0.1 M ethanolamine (pH 8.01, and the incubation was continued for 1 h. The resin with bound protein was transferred to a small siliconized glass column and washed with membrane extraction buffer until the Am of the effluent reached background. Porcine pancreatic elastase-derived peptides were used to prepare the affinity resin because bovine tropoelastin is difficult to purify in the required quantity.

Purified plasma membrane aliquots (0.5 ml) were quickly thawed and extracted with 1.5 ml of 3 M guanidine, 0.5% octyl-@-glucoside, 0.1 M dithiothreitol in the presence of proteinase inhibitors (2 mM benzamidine, 2 mM phenylmethylsulfonyi fluoride, 2 mM c-aminoca- proic acid) a t 4 "C overnight. Insoluble material was pelleted by centrifugation at 10,000 X g, and the supernatant was dialyzed against phosphate-buffered saline containing protease inhibitors. Aliquots of membrane extract (0.5 ml) were loaded on the columns and allowed to penetrate fully into the gel. Buffer flow was stopped, and the incubation was allowed to proceed for 2-4 h. Unbound material was eluted by washing the columns with buffer until a stable Am base line was obtained. Elution of bound material was achieved with 3 M guanidine, 0.5% octyl-8-glucoside in 0.1 M bicarbonate buffer (pH 8). Fractions were monitored for Am and peak fractions were pooled, dialyzed exhaustively against water, and lyophilized. Lyophilized samples were resuspended in sample buffer and subjected to SDS- PAGE as described by Matsudaira and Burgess (20).

RESULTS

Because fibroblasts from ligamentum nuchae synthesize elastin in culture, it was important to determine whether tropoelastin was associated with the cell surface and, hence, possibly bound to receptors. Purified plasma membranes were radioiodinated, and receptor-ligand complexes were disrupted in a buffer containing octyl-/3-glucoside. Immunoprecipitation with an antibody specific for elastin revealed iodinated tro- poelastin in the supernatant of the solubilized membranes (Fig. l), providing suggestive evidence that receptor sites on the membrane are occupied with tropoelastin synthesized by

Tropoelastin

FIG. 1. Identification of tropoelastin bound to the surface of ligament fibroblasts. Iodinated plasma membranes from con- fluent cultures of FCL-270 cells were solubilized using octyl-@-gluco- side and precipitated with a monoclonal antibody to elastin. SDS- PAGE of the precipitated proteins followed by fluorography demon- strated the characteristic tropoelastin trimer (33) indicative of tro- poelastin bound to the surface of ligament cells.

2282 Kinetics of Elastin Receptor Binding

the cell. Thus, the following binding experiments must be interpreted in the context of a receptor that is occupied initially with unlabeled ligand. Clearly, this unlabeled tropoe- lastin must be displaced before binding of labeled ligand can occur. The implications of this finding are discussed below.

The successful identification and study of receptor-ligand interactions is critically dependent upon the nature and qual- ity of radiolabeled probe. Iodination of bovine elastin peptides or of bovine tropoelastin using either chloramine T or lacto- peroxidase was not successful most likely due to a paucity of tyrosine residues in the bovine molecule. High specific activity

04 1

Tropoelastin 50 100 150

Bound (ng)

2o 1 Plasma Membranes

15 -

0 50 100 150

Time (minutes) FIG. 2. Time course of binding of '251-tropoelastin at 22 OC

to confluent cultures of FCL-270 fibroblasts (top) or plasma membranes isolated from FCL-260 cells spotted onto nitro- cellulose (bottom). Cells and membranes were incubated for the indicated times with 2-3 X lo5 cpm "'I-Bolton Hunter-labeled tro- poelastin with or without 100 pg/ml unlabeled tropoelastin. Results are mean of triplicate determinations for total, nonspecific, and specific binding.

6 l 5 -1

0 4 8 12 16 20 Bound

0 200 400 600 800

prnoleg added

FIG. 3. Saturability of binding of tropoelastin to FCL-270 fibroblasts at 22 "C. Incretsing amounts of unlabeled tropoelastin (0-50 mg) and a fixed amount of '"I-tropoelastin (200 ng) were added to confluent cultures of FCL-.<70 fibroblasts. Bound radioactivity was determined after 120 min and moles of ligand (labeled and unlabeled) added and specifically bound were calculated. Results are means of triplicate determinations. Scatchard analysis of the binding data is shown in the inset. R/F, bound/free.

3 0 m

10 I 0 20 40 60 80 100

Time (minutes) FIG. 4. Rate of dissociation at 22 "C of I2%tropoelastin

bound by FCL fibroblasts or by purified fibroblast plasma membranes. Confluent cultures or plasma membranes on nitrocel- lulose were incubated with lZsII-tropoelastin (2 X lo5 cpm) for 120 min, rinsed three times with binding buffer, and then overlaid with fresh binding buffer. A t the indicated times, cell- or membrane- associated radioactivity was determined. Results are means of tripli- cate determinations.

Ttypsin Sensitrve Trypsm InsensIIIve

30 60 90 120 Time (minutes)

FIG. 5. Internalization of '251-tropoelastin bound by FCL fibroblasts. Confluent cultures were incubated with 200 ng '*'I- tropoelastin for 120 min to ensure equilibrium conditions. Binding was terminated by washing cells three times with ice-cold binding buffer. The cells were incubated with 1.0 ml of Earle's balanced salt solution containing 0.025% trypsin for 10 min at 22 "C. Cells were pelleted in a Microfuge and radioactivity in the supernatant and associated with the cell pellet was determined.

c -61 - 67

I- -43 I

FIG. 6. SDS-PAGE of elastin binding proteins from liga- ment fibroblast plasma membranes. The gel on the left is total membrane extract and the gel on the right shows proteins that bound to the elastin-affinity column. Proteins were visualized by silver stain. Molecular masses are XlO-:'.

Kinetics of Elastin Receptor Binding 2283

probes (a-elastin, porcine pancreatic elastase-derived elastin peptides, or the synthetic hexapeptide Val-Gly-Val-Ala-Pro- Gly) could be made using the iodination method of Bolton and Hunter (21), but they did not bind to fibroblast mono- layers or plasma membrane preparations. Loss of biological activity following radiolabeling was corroborated in chemo- taxis assays; conjugation with Bolton-Hunter reagent (either the iodinated or unlabeled form) abolished chemotactic activ- ity (data not shown). Tropoelastin, however, remained chem- otactically active following iodination with Bolton-Hunter reagent and was used as ligand in subsequent binding exper- iments. The integrity and biological activity of radiolabeled tropoelastin was confirmed by demonstrating that binding of radiolabeled tropoelastin was displaced from cells at the same rate in the presence or absence of unlabeled protein.

Membrane-associated ‘251-tropoelastin in monolayer fibro- blast cultures and in fibroblast plasma membranes as a func- tion of incubation time is presented in Fig. 2. Binding was saturable and time-dependent. Equilibrium was generally reached by 90 min and was stable to 180 min, the longest time point examined. Nonspecific binding in both assay systems ranged from 20 to 40% of total binding. Binding of tropoelas- tin to plasma membranes was not altered by the presence of plasma fibronectin. Specific binding of tropoelastin to cells which had been preincubated with dilute solutions of trypsin was reduced by greater than 90% when compared with un- treated cells, suggesting that association of radiolabeled tro- poelastin with fibroblasts is mediated by protease-sensitive moieties on the cells’ surface and not by pinocytosis.

Fig. 3 shows that binding of ‘251-tropoelastin to fibroblasts was saturable, with half-maximal binding occurring at 2.5 x lo”* M. Scatchard analysis indicated approximately 2 x lo6 sites/cell, with an equilibrium dissociation constant of 8 X lo-’. High affinity binding was reproducible over a number of experiments using different batches of cells, but the con- tribution of low affinity binding was less consistent. Given the sensitivity of Scatchard analysis to various sources of error at high levels of bound ligand, this variability may result from imprecise measurement of nonspecific binding or could result from a second population of elastin binding sites with lower affinity constants. It should be noted, however, that binding studies were done on substrate-attached cells, so it is possible that our determination of receptor number may be an underestimate because of receptors on the basal surface of the cell that might not be accessible for ligand binding.

Reversibility of binding was assessed after preincubation of fibroblasts or fibroblast plasma membranes with ‘251-tropo- elastin in the presence or absence of excess unlabelled tropo- elastin. As shown in Fig. 4, the dissociation curves demon- strated first order kinetics with approximately 50% of specif- ically bound ligand released from cells by 40-50 min and from plasma membranes by 55-65 min.

The degree of ligand internalization is shown in Fig. 5. By 30 min, approximately 30% of specifically bound tropoelastin was trypsin-insensitive and presumably internalized. The amount of trypsin-insensitive radioactivity reached a steady state within one h at levels averaging between 30 and 40%. Similar results were obtained using pepsin to distinguish cell surface and internalized ligand (data not shown). Approxi- mately 5-10% of “‘I-associated counts at the 90-min time point were not precipitable with trichloroacetic acid, suggest- ing that internalized tropoelastin is being degraded.

To isolate the tropoelastin receptor, detergent extracts of fibroblast plasma meqbranes were subjected to affinity chro- matography as described under “Materials and Methods.” Complete elution of material bound to the elastin-affinity

column was obtained with 3 M guanidine, 0.5% octyl-p-glu- coside elution buffer. When proteins in the retained fraction were analyzed by SDS-PAGE, four major bands were resolved with estimated molecular masses of 67, 61, 55, and 43 kDa (Fig. 6). A minor component at 160 kDa was also detected, although infrequently. No specific protein bands could be obtained by the same elution procedure when albumin-Affi- Gel was used instead of the elastin affinity column.

DISCUSSION

There are a number of difficulties inherent in attempting to demonstrate binding of a radiolabeled ligand to cells which produce significant amounts of that same ligand, especially if the protein is bound by a receptor of high affinity. fn such a system, one would predict that binding of labeled probe might be low due to competition with endogenous ligand and that the time course to binding equilibrium would be lengthy due to a slow rate of dissociation of endogenous ligand-receptor complexes. Our demonstration of native tropoelastin bound to the ligament cell plasma membrane (presumably to recep- tors) may account for the relatively long period required for radiolabeled tropoelastin to reach binding equilibrium with ligament cell receptors (90 min; Fig. 2). Even though “total” membrane elastin receptor concentration is constant in these experiments, the concentration of receptor assessable to bind radiolabeled probe is increasing in a time-dependent fashion related inversely to the rate of dissociation of the native tropoelastin. Based on this assumption, saturation with ra- diolabeled ligand cannot be reached until dissociation of all unlabeled tropoelastin from the receptor pool is complete, which, as estimated from the results in Fig. 4, should require approximately 80-90 min. This estimate is in excellent agree- ment with our experimentally obtained finding that equilib- rium binding of radiolabeled tropoelastin occurs only after approximately 90 min of incubation.

The kinetic parameters determined in this study for elastin- receptor binding agree favorably with receptor binding of other matrix proteins. For type I collagen, Goldberg (22) has reported a high affinity receptor with a Kd of lo-” M on 3T3 cells, and Chiang and Kang (23) described the affinity isola- tion of a type I collagen receptor from detergent extracts of platelets which displays a Kd of 5 X M. Receptors have been isolated from murine fibrosarcoma cells (24) and human MCF-7 breast carcinoma cells (25) that bind laminin with a Kd = 2 X lo-’ M. Perhaps the best characterized receptor is the 140 kDa fibronectin binding receptor complex (26-28) that mediates the adhesion of cells to fibronectin as well as other proteins such as laminin (29). Akiyama and Yamada (27) have reported that baby hamster kidney cells have 5 x lo5 such receptors/cell with a Kd of 8 X M.

At present, the functional significance of the elastin recep- tor is unknown. We do not know whether the elastin receptor initiates biological activity within the cell in response to elastin binding or functions as a shuttle protein to transport tropoelastin to the cell surface and to anchor and align tro- poelastin monomers preparatory to cross-linking and fiber formation. It is interesting to note that studies by Jacob et al. (30) have shown that extracellular elastin peptides alter ion fluxes in mononuclear cells, fibroblasts, and smooth muscle cells to produce effects that have not yet been defined. In addition to the possible alteration of cellular metabolism related to elastin binding, the receptor may providing impor- tant chemotactic signals that direct the movement of cells to sites of tissue damage (19, 31).

It is yet to be firmly established whether there are in fact multiple tropoelastin binding proteins or a single receptor

2284 Kinetics of Elastin Receptor Binding

complex as has been demonstrated for fibronectin (26). Our identification by affinity chromatography of four elastin bind- ing proteins is consistent with findings of Hornebeck et al. (14), who described elastin binding proteins of 120, 67, 60, and 45 kDa. The 120-kDa protein, designated as “elastonec- tin,” was proposed by Hornebeck et al. (14) to be directly responsible for the adhesion of mesenchymal cells to elastic fibers, although the adhesive properties of this protein have not been established conclusively. We could not detect a 120- kDa protein in our preparations from ligament fibroblasts, although it should be noted that we used different extraction and purification methods than those of Hornebeck. We do not yet know the functional relationship between the four proteins identified in this study, although our preliminary results indicate strongly that the 67-kDa component is re- sponsible for the high affinity binding of tropoelastin. Inter- estingly, comparative immunochemical studies suggest that this 67-kDa elastin binding protein is similar if not identical to the 67-kDa laminin receptor purified from MCF-7 cells (32). Further work is in progress to characterize rigorously the elastin binding proteins and the molecular domains they recognize.

Acknowledgments-We thank Loren Whitehouse and Gertrude Crump for technical assistance and Gail Griffin and Robert Senior for performing the chemotaxis assays. We also wish to acknowledge Terese Hall for valuable secretarial help.

Note Added in Proof-A similar elastin-binding receptor complex has been found on Lewis lung carcinoma cells (C. H. Blood and B. R. Zetter, personal communication).

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