Cell, Vol. 16,265275, February 1979, Copyright 0 1979 by MIT

Binding of Soluble Type I Collagen Molecules to the Fibroblast Plasma Membrane

Burton Goldberg Department of Pathology New York University Medical Center New York, New York 10016

Summary

Soluble 1251-labeled type I collagen binds to cul- tured fibroblasts but not to cultured epithelia. The binding of the ligand to fibroblasts is reversible, saturable and highly specific for sequences con- tained within the helical portions of the ~yl and cu2 chains. The amount of ligand bound is dependent upon cell number and ligand concentration. Bind- ing is decreased but measurable at 4°C. The steady state binding is greater at 26” than at 37°C due to a more rapid dissociation of the ligand- acceptor complex at 37°C. The half-life of the complex is 46 min at 37°C and approximately 2.5 hr at 26°C. Scatchard plots of binding data indi- cate a single class of high affinity binding sites (K, = 1.2 x 10-l’ M) with each fibroblast binding approximately 500,000 molecules at saturation: Pretreatment of fibroblasts with bacterial colla- genase, chondroitinase ABC or testicular hyalu- ronidase does not affect the binding reaction, whereas pretreatment of the cells with phospho- lipase C increases the amount of ligand bound. Ligand binding is decreased but not abolished after fibroblasts are treated with trypsin concen- trations which remove surface fibronectin. Fibro- blast monolayers treated with antiserum against fibronectin bind the radiolabeled ligand normally. In contrast to collagen, addition of excess fibro- nectin does not accelerate the dissociation of bound ligand from fibroblasts. Possible functions for surface-bound collagen are discussed.

Introduction

Two aspects of collagen biosynthesis suggest re- quirements for the binding of collagenous mole- cules to the fibroblast surface. First, soluble pro- collagens secreted from fibroblasts are enzymati- tally converted to smaller native collagen mole- cules which assemble to form fibrils. Electron mi- croscopic studies indicate that fibrillogenesis is initiated at the fibroblast surface (Goldberg and Green, 1964; Trelstad, 1975); thus it is reasonable to suggest that the enzymatic processing of pro- collagens and fibrillar assembly could both occur while the collagenous molecules are bound to the plasma membrane of the fibroblast. Second, dur- ing normal morphogenesis and wound healing, collagen synthesis ceases when appropriate amounts of extracellular fibers are formed. This

suggests that feedback inhibition of collagen syn- thesis and secretion might be initiated by an inter- action between extracellular collagen and a fibro- blast surface receptor.

There is some experimental evidence for colla- gen binding to the fibroblast. Antibodies in combi- nation with fluorescence methods and cytotoxicity assays have demonstrated collagen on the surface of fibroblasts (Lustig, 1970; Duksin, Maoz and Fuchs, 1975; Faulk, Conochie and Temple, 1975; Lichtenstein et al., 1976). These studies, however, did not determine whether the collagen was in a soluble or fibrillar form; nor did they provide defin- itive proof of a direct collagen-plasma membrane interaction.

I have used soluble ‘251-labeled type I collagen as a binding ligand and have found that it binds specifically to monolayers or suspensions of fibro- blasts. The evidence indicates that the collagen binds to a structural element of the plasma mem- brane, and the quantitative binding data are com- patible with participation of the binding complex in one or more steps of collagen metabolism.

Results

The Binding Assay: Identity of Labeled and Unlabeled Ligands, and Evidence for Saturability Lathyritic type I collagen from rat skin (LRSC) was labeled with lZ51 by the chloramine T method to an average specific activity of 3 x lo6 cpm/pg. Figure 1 shows the polyacrylamide gel electrophoresis pattern of a representative preparation. Lathyritic collagen molecules lack covalent interchain cross- links; thus under the denaturing conditions of the gel, the al and a2 chains of collagen are sepa- rated. The observed (~l/cu2 ratio is less than the theoretical value of 2 because the a2 chain incor- porates relatively more radioiodine than does the (~1 chain. A small proportion of the collagen mole- cules in the preparation contained covalent inter- chain cross-links between all three chains or just two chains, and these are labeled in Figure 1 as y and p forms, respectively.

Binding experiments were performed with the radiolabeled collagen (1251-LRSC) and cultured fi- broblast lines and strains of mouse, human and avian origin. Evidence for binding was obtained with every type of cultured fibroblast. The contact- inhibited Swiss mouse fibroblast line 3T3/M was used for the majority of the quantitative binding, experiments of this report because these cells form an almost true monolayer at confluence (Todaro, Green and Goldberg, 1964). With such monolayers, artifacts due to hindered diffusion of the ligand and limited accessibility of the ligand to the total cell surface area are minimized.

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Figure 1. Sodium Dodecylsulfate (SDS)-Polyacrylamide Gel Elec- trophoresis of ‘251-Labeled Lathyritic Rat Skin Collagen

y and p forms of collagen contain three and two covalently cross- linked a chains, respectively.

Figure 2 shows the results of a representative experiment in which confluent cultures of 3T3 fibroblasts were incubated for different times with 1251-LRSC. A replicate set of control cultures re- ceived the radiolabeled collagen and a 3000 fold excess of unlabeled collagen. The radioactivity not displaced by the unlabeled collagen in the control cultures is considered to represent nonspecific binding and was subtracted to give specific bind- ing. After a 90 min incubation, 14% of the input radioactivity was specifically bound; in contrast, the nonspecific binding represented only 1% of the input radioactivity and 11% of the total bound radioactivity.

To examine the kinetics of the exchange of the unlabeled collagen with the radiolabeled ligand, the following experiment was performed. Two sets of confluent 3T3 cultures were incubated at 26°C with equal amounts of 1251-LRSC; 30 min after the start of the incubation, each plate of one set received 100 pg of unlabeled collagen. The radio- activity bound to the cells was measured at inter- vals thereafter and the results are shown in Figure 3. The control cultures which had not received unlabeled collagen showed continued net binding of the radioactive ligand over the 2.5 hr of the experiment. The addition of excess unlabeled col- lagen to the other cultures, however, decreased binding of lz51-LRSC by 70 and 92% after 20 min and 2 hr of incubation, respectively. The rapidity and efficiency of the exchange and displacement indicate that the labeled and unlabeled collagens behave identically in the binding reaction. In con- trast, noncollagen proteins in equally high concen- trations do not compete for binding sites or dis- place the bound radiolabeled ligand from the cells (see below).

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Figure 2. Rate of Binding of ‘251-LRSC to 3T3 Fibroblasts at 26°C

Each confluent culture plate (2 x lo6 cells) received 33 ng of 1251- LRSC (135,400 cpm) in the presence (O---O) and absence (O- --@) of 100 ,ug of unlabeled LRSC. Radioactivity corrected for nonspecific binding (0-O).

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MINUTES Figure 3. Exchange of V-LRSC and Unlabeled Collagen in the Binding Reaction

Confluent cultures of 3T3 (2 x IO6 cells) were incubated with 53 ng of ‘Z51-LRSC (143,400 cpm) at 26°C. At 30 min, each of six cultures (O-O) received 100 pg of unlabeled LRSC, and bound radioactivities were measured at intervals in these plates and in the controls (O-O).

To test whether the binding of collagen was a saturable process, confluent fibroblast cultures re- ceived a fixed amount of 1251-LRSC and increasing amounts of unlabeled collagen to a maximum of 100 pg. The cultures were incubated at 26°C for 2 hr to ensure that equilibrium conditions were achieved, and bound radioactivities were then measured. A plot of picomoles of ligand bound versus picomoles of ligand added is given in Figure 4, and it is evident that saturation of binding was achieved. Evidence for saturation after such a long (2 hr) incubation interval favors the interpretation

Collagen Binding to Fibroblasts

267

0 15 30 45 60 75 PICOMOLES ADDED

Figure 4. Saturability of Binding of Collagen to 3T3 Fibroblasts

Increasing amounts of unlabeled LRSC (O-100 pg) were premixed with a fixed amount of ‘Z51-LRSC (50 ng; 115,000 cpm) in 2 ml of binding buffer and added to confluent cultures (2 x lo6 cells). The cultures were incubated at 26°C for 2 hr. The bound radioac- tivity was measured, and the picomoles of collagen (radiolabeled and unlabeled) added and specifically bound to each culture were calculated.

that “binding” to a surface acceptor was being measured rather than “uptake” by a transport process (Cuatrecasas and Hollenberg, 1976).

Characterization of the Bound Radioactivity The gel pattern of Figure 1 shows that collagen accounted for 80% of the radioactive,species after the radioiodination of LRSC. To establish the fact that it was the radioactive collagen and not a minor contaminant of this preparation which bound to fibroblasts, the following experiment was per- formed. Confluent 3T3 cultures were incubated with the 1z51-LRSC for 90 min at 26”C, unbound radioactivity was removed by washing, and bound radioactivity was displaced by adding buffer which contained 150 pg/ml of unlabeled collagen. Figure 5 shows the gel pattern given by the radioactivity dissociated from the fibroblasts. The pattern is almost identical to that of the input 1251-LRSC (see Figure 1). The only discrepancy is that proportion- ately fewer cr2 chains were recovered in the gel of the bound radioactivity. This finding remains unex- plained at present, but the data allow the conclu- sions that it is only the radioactive collagen in the iodinated preparation which binds to the fibroblast, and that intact collagen molecules can be re- covered after binding and displacement reactions occur.

Specificity of Binding with Respect to Cell Type Cultured nonfibroblastic cell types were tested for

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mm FROM ORIGIN Figure 5. SDS-Polyacrylamide Gel Electrophoresis of Bound Ra- dioactivity Displaced from Fibroblasts

Confluent cultures of 3T3 fibroblasts were incubated with lZ51- LRSC for 90 min at 26°C. Unbound radioactivity was removed by washing. The cell layers were then overlaid with 2 ml of binding buffer containing 300 pg of unlabeled collagen. The plates were incubated for 60 min at 26”C, and the binding buffer was col- lected, dialyzed against water and lyophilized. The lyophilate was solubilized in electrophoresis buffer and an aliquot was applied to the polyacrylamide gel. 90% of the applied radioactivity was recovered in the gel.

their capacity to bind ‘251-LRSC. The assayed cloned strains or lines are judged to be epithelial because they express differentiated functions char- acteristic of the epithelia of the parent tissues. Table 1 shows that such cell types did not bind significant amounts of the ligand when compared to fibroblasts. The small amounts of radioactivity retained by the nonfibroblastic cells may well have represented nonspecific binding, since controls containing an excess of unlabeled collagen were not included in these assays. Thus within the limits of this small survey, I conclude that epithelial cell types do not possess specific binding sites for the radiolabeled type I collagen.

Specificity of Binding with Respect to the Molecular Form of the Ligand Several types of molecules were tested for their ability to compete with 1251-LRSC for binding sites on fibroblasts. In one experiment, unlabeled lath- yritic collagen was digested with pepsin under conditions which left the triple helix intact but removed the nonhelical telopeptides from the ends

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Table 1. Comparative Binding of ‘?-LRSC to Fibroblasts and Nonfibroblastic Cell Types

Cell Type cpm Bound per Relative % 1 OK Cells Bound

(1) Human fibroblast

(2) Rat hepatocyte

(3) Rat hepatoma

(4) Rat hepatoma

(5) Canine renal epithelium

(6) Porcine renal epithelium

19,638 100

681 3.4

277 1.4

63 0.3

192 0.9

195 0.9

Each confluent culture received 40 ng of ‘V-LRSC (129,000 cpm). After incubation at 26°C for 60 min, the bound radioactivi- ties were determined as described. Cell counts were made of replicate plates and binding values were normalized to lo6 cells per plate. The percentage of binding of each cell type relative to the fibroblast culture was then calculated.

(1) Human diploid skin fibroblast, American Type Culture Collection (ATCC), CRL 1121; (2) clonal strain 9 (Kaighn and Prince, 1971); (3) clonal line Fu5 (Schneider and Weiss, 1971); (4) clonal strain H4-12 (Wolf, Munkelt and Kaighn, 1974); (5) ATCC, CCL 34 (NBL-2) MDCK; (6) ATCC, CCL 33 PK(15). The epithelial cultures were supplied by W. Dolan (New York University Medical Center).

of the molecule. The pepsinized collagen proved to be equivalent to the nonpepsinized collagen as a competitive inhibitor in binding assays (data not shown). This result indicates that the binding se- quences are in the helical portion of the collagen molecule. Table 2 gives the results of competitive inhibition studies in which the added unlabeled proteins all contained collagen-type helical struc- ture. Unlabeled LRSC and cross-linked and individ- ual cy chains were about equally effective as com- petitors for the binding sites. Thus cooperative effects between chains do not seem to be impor- tant for binding specificity. Three cyanogen bro- mide peptides from helical portions of the ul chains differed in their effectiveness as inhibitors of binding. The al-CB7 peptide was the most effective competitor, and it was equivalent on a concentration basis to the intact collagen molecule and to the intact (Y chains. Clq, a complement component containing some collagen helical structure (Reid and Porter, 1976; Reid, 1977), did not displace significant amounts of ‘251-LRSC. Even at a 50 fold higher concentration (50 pg/ml), Clq inhibited binding by only 5%. Thus the binding specificity is sufficiently great that distinctions are made between molecules which share collagen helical structure.

For all the binding assays, the ‘251-LRSC stock solution was routinely incubated at 37°C for 30 min before dilution into the binding buffer. The prein- cubation was required for uniform and maximal

Table 2. Competitive Inhibition of Binding

Added Unlabeled Protein cpm Bound % Inhibition

0 12,785 0

(ffl )*a2 5,068 60

011 5,904 54

P12 6,116 52

LX1 5,683 56

a2 5,548 57

al -CB7 5,620 56

oil-CBB 8,536 33

al-CBS 11,757 a

Clq 12,515 2

Competing unlabeled proteins were dissolved in buffer [0.05 M Tris-HCI (pH 7.2), 0.15 M NaCI] and diluted to give 0.5 optical density units at 228 nm. Confluent cultures of 3T3 fibroblasts (2 x IO6 cells) received 0.01 ml (-2 wg) of the competing protein and 55 ng of ‘251-LRSC (126,000 cpm) in 2 ml of binding buffer. A control plate received only ‘251-LRSC. Both labeled and unlabeled protein solutions were incubated at 37°C for 30 min before they were mixed with the binding buffer. Plates were incubated for 90 min at 26°C and bound radioactivity was measured as described.

(al),a2 represents the unlabeled lathyritic rat skin collagen. p notation indicates a covalently cross-linked dimer of collagen a chains. CB notation identifies peptides derived from cyanogen bromide cleavage of collagen 01 chains. The molecular weights are: (a chains) 95,000; (al-CB7) 24,800; (al-CB8) 24,800; (al- CB3) 13,800 daltons. All collagen molecules and peptides are type I and of rodent origin.

Clq was purified from human serum and provided by I. Gigli (New York University Medical Center).

binding of the ligand. Ligand so warmed to 37°C was still resistant to limited digestion with pepsin, indicating that the warming had not caused sepa- ration of the three chains of the triple helix. The warming requirement was not a consequence of the radioiodination of the molecule because unla- beled LRSC also required preincubation at 37°C to compete effectively for binding sites in the inhibi- tion assays. I conclude that warming of the ligand to the physiologic temperature made binding de- terminants accessible by causing some relaxation of triple helical packing.

There is no evidence that noncollagenous mole- cules can compete for binding sites in the assay. Binding of 1*51-LRSC occurs in the presence of 5 mg/ml of bovine serum albumin. A globular pep- tide isolated from the carboxy terminal end of human type I procollagen (Sherr, Taubman and Goldberg, 1973) does not compete significantly in the binding assay when added at a concentration of 25 /*g/ml. As shown below, cold-insoluble glob- ulin from plasma does not displace lZ51-LRSC from fibroblasts.

From these data, I conclude that the binding reaction is highly specific for collagen. The binding

Collagen Binding to Fibroblasts 269

sites reside in the helical portions of the al and a2 chains, but the binding specificity is critically de- termined by primary structure rather than by gen- eral helical conformation or interchain interac- tions.

Binding as a Function of Cell Concentration, Ligand Concentration and Temperature To determine the relation between cell numbers and the extent of collagen binding, 3T3 cultures containing from 4 x 104-9 x lo5 cells were incu- bated with a constant amount of 1251-LRSC. The data of Figure 6 show that binding increased with increasing cell numbers, but not in a direct propor- tion. This result indicates that binding sites were in excess even at the lowest cell numbers tested. Accordingly, in confluent cultures (2 x lo6 cells), the binding sites should be in great excess and binding should be directly proportional to the amount of ‘251-LRSC added over a rather wide range. The experiment summarized in Figure 7 shows that this is the case. Confluent cultures received increasing amounts of the radiolabeled ligand up to a maximum of 850 ng (2 x lo6 cpm). Binding is observed to be directly proportional to the amount of ligand added over a range of O-425 w

Binding assays were performed at 4”, 26” and 37”C, and the results are presented in Figure 8. The lowest rate of binding was observed at 4°C. The persistence of binding at this low temperature indicates that the ligand can be retained by the cells in the absence of pinocytosis. The rates of

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Ml NUTES Figure 6. Binding of 1Z51-LRSC to 3T3 Fibroblasts as a Function of Cell Numbers

Cultures containing the indicated cell numbers were incubated with 38 ng of ‘*51-LRSC (127,000 cpm) at 26°C.

binding at 37” and 26°C were equal for the first IO min of incubation, but thereafter cells at 37°C bound less of the radiolabeled ligand than cells at

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Figure 7. Binding of ‘251-LRSC to 3T3 Fibroblasts as a Function of Nanograms of Labeled Ligand Added

Confluent cultures (2 x lo6 cells) received varying amounts of radiolabeled LRSC (spec. act. 2500 cpm/ng). The cultures were incubated for 30 min at 26”C, and the bound radioactivity was measured and expressed in nanograms

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Figure 8. Effect of Temperature on Rates of Binding of ‘251-LFiSC to 3T3 Fibroblasts

Confluent cultures (2 x lo6 cells) were incubated with 47 ng of ‘251-LRSC (113,300 cpm) at the indicated temperatures, and the bound radioactivities were measured at intervals. Dotted lines indicate nonspecific binding in cultures which also received 100 pg of unlabeled collagen.

Cell 270

26°C. This result prompted a study of the tempera- that the binding sites do not discriminate between ture dependence of the rate of dissociation of the intact collagen molecules and the constituent otl collagen-acceptor complex. Confluent cell layers and cu2 chains. As a further test of this result, jz51- which had been incubated for 60 min with lz51- cul chains were prepared and a binding-inhibition LRSC were washed and then incubated at 37” or experiment analogous to that of Figures 4 and 10 26°C in fresh buffer; the radioactivity remaining was performed. In this instance, a constant amount bound to the cell layers was measured at intervals, of lz51-al was mixed with increasing amounts of and the results are presented in Figure 9. The unlabeled crl or a2 chains, respectively. With both semilogarithmic plots of bound radioactivity versus types of CY chain addition, the Scatchard plots (not time gave straight lines, indicating that the rates of shown) gave straight lines whose x and y intercepts dissociation are first-order processes at both tem- were not significantly different. The abscissa1 inter- peratures. The rate of dissociation, however, was cepts were 1.8 picomoles for al and 1.9 picomoles much higher at 37”C, so that by 60 min a culture at for ~2. The values for K, were 1.3 x 10-I’ M for al this temperature had lost about 3 times more of the and 1.5 x 10-I’ M for ~2. These values are in bound radioactive ligand than a culture at 26°C. reasonable agreement with those obtained from The half-life of the ligand-acceptor complex is 46 the binding-inhibition experiment in Figure 10, in min at 37°C and approximately 2.5 hr at 26°C. which the collagen molecule was the ligand. Ac- Accordingly, the finding of less binding at 37” than cordingly, I conclude that there is a homogeneous at 26°C (as shown in Figure 8) is ascribed to an set of binding sites on the fibroblast which interact equilibrium favoring greater dissociation of the with either the al or a2 chains of the type I complex at the higher temperature. collagen molecule.

Scatchard Analysis of the Binding Data Binding data were taken from the experiment of Figure 4, in which increasing amounts of unlabeled collagen were mixed with a constant amount of ‘251-LRSC and incubated with cells for 2 hr to achieve binding equilibrium. The Scatchard plot (Scatchard, 1949) of Figure 10 shows a linear fit to the data points indicating a homogeneous class of binding sites. The abscissa1 intercept of the line falls at 1.6 picomoles, a value which calculates to approximately 500,000 binding sites per fibroblast. The equilibrium dissociation constant (K,) calcu- lated from the slope of the line is 1 .2 x 10-l’ M.

The competition experiments in Table 2 indicate

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MINUTES Figure 9. Semilogarithmic Plot of the Dissociation of ‘251-LRSC Bound to 3T3 Fibroblasts as a Function of Time at 37” and 26°C

Confluent cultures (2 x IO6 cells) were each incubated with 57 ng of ‘251-LRSC (138,000 cpm) for 60 min at 26°C. The cell layers were washed, overlaid with buffer at either 37” or 26°C and then incubated at the respective temperatures for the indicated inter- vals. The bound radioactivities were measured as described.

Evidence for Binding of Collagen to a Structural Component of the Plasma Membrane The monolayers were well washed before binding assays were performed, but it was possible that “51-LRSC was binding to collagen, proteoglycans or hyaluronate on the fibroblast surface rather than to a component of the plasma membrane.

To determine whether surface collagen was re- sponsible for the binding, washed 3T3 monolayers were incubated with and without 80 units of active bacterial collagenase for 60 min at 37°C before the monolayers were incubated with ‘251-LRSC in stan- dard assays. The collagenase-treated and control

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B (PICOMOLESI Figure 10. Plot of the Ratio of Bound (B) to Free (F) Ligand versus Bound Ligand

The data are from the experiment of Figure 4.

Collagen Binding to Fibroblasts 271

cells gave typical curves for radioactivity bound versus time, and the curves were similar with re- spect to the amounts of ligand bound and the rates of binding (data not shown). I therefore conclude that the described binding of lz51-LRSC is not due to an interaction with surface collagen.

When binding buffer containing ‘Y-LRSC (35 rig/ml) was incubated at 37°C for 2 hr and then centrifuged at 27,000 x g for 20 min, none of the radioactivity sedimented. This result rules against the possibility that the ligand aggregated and pre- cipitated onto the cells during the binding assays.

Confluent 3T3 cultures that had been treated with chondroitinase ABC (0.4 U at pH 7.0; 60 min, 37°C) or with testicular hyaluronidase (800 U at pH 6.0; 60 min, 37”C), respectively, were compared with control cultures in the standard binding assay. The enzyme treatments did not alter the binding characteristics of the cells (data not shown), and thus surface proteoglycans or hyaluronate do not seem to be required for the binding reaction.

Fibronectin (LETS protein) is a surface glycopro- tein of fibroblasts which is released to the extracel- lular space. An almost identical molecule isolated from plasma is called cold-insoluble globulin (CIG). Both fibronectin and CIG have been shown to bind to collagen gels and to mediate the attachment of cells to such gels (Klebe, 1974; Pearlstein, 1976; Kleinman, McGoodwin and Klebe, 1976; Engvall and Ruoslahti, 1977; Engvall, Ruoslahti and Miller, 1978). It was therefore appropriate to determine whether the soluble 1251-LRSC ligand was binding to fibronectin on the fibroblast surface. Treatment of 3T3 monolayers with 5 pg/ml of crystalline trypsin for 20 min at 37°C detaches all the cells and is sufficient to remove identifiable fibronectin from the cell surface. As shown in Figure 11, such trypsinized fibroblasts were still able to bind lz51- LRSC specifically, although the level of binding was about 6 fold less than that given by an equal number of intact cells in monolayer culture. This result suggested that a less trypsin-sensitive mole- cule than fibronectin was responsible for binding of the ligand. The putative role of fibronectin was also assessed by incubating fibroblasts with anti- serum to CIG and then measuring the binding of 1251-LRSC to the cells. Table 3 shows that pretreat- ment with the antiserum did not alter the fibro- blasts’ capacity for binding the ligand. In another experiment, CIG was tested for its ability to dis- place lz51-LRSC bound to fibroblasts. Figure 12 shows that the normal rate of dissociation of ‘Y- LRSC was not accelerated by the addition of 50 pg of CIG, whereas the addition of 50 pg of unlabeled collagen caused the rate of dissociation to approx- imately double. Taken together, the data favor the view that the binding of 1251-LRSC to fibroblasts

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Figure 11. Binding of ‘251-LRSC to Trypsin-Treated 3T3 Fibro- blasts in Suspension

Confluent cultures were incubated for 20 min at 37°C with 5 pg1 ml of crystalline trypsin. The detached cells were uniformly dispersed by gentle agitation, soybean trypsin inhibitor was added to a concentration of 50 yglml and the cells were collected by centrifugation. The cells were resuspended in binding buffer, and IO6 cells and 43 ng of ‘V-LRSC (121,000 cpm) were added to individual wells of Linbro plates. The plates were incubated at 26°C with gentle shaking. At intervals, the cells were collected by centrifugation and washed, and the bound radioactivities were measured (O---O). Nonspecific binding to cells incubated with 100 pg of unlabeled collagen (O---O).

Table 3. Binding of 1*51-LRSC to Fibroblasts after Treatment with Anti-CIG Serum

Serum Additions ‘=I-LRSC Bound (cpm)

(1) None 11,013

(2) Normal rabbit serum 9,885

(3) Rabbit anti-human CIG serum 10,191

Confluent 3T3 cultures were incubated for 30 min at 37°C with binding buffer or one quarter dilutions in binding buffer of normal rabbit serum or anti-human CIG serum, respectively. The cell layers were then washed with binding buffer and incubated for 30 min at 26°C with 55 ng (129,000 cpm) of 1251-LRSC, and bound radioactivity was measured as described.

The antiserum cross-reacts with mouse CIG and mouse fibro- nectin. A one quarter dilution of the antiserum blocks CIG-me- diated attachment of rodent fibroblasts to collagen gels, and binds to the surface of rodent fibroblasts in immunofluorescence assays (Pearlstein and Gold, 1978). The antiserum was provided by E. Pearlstein (New York University Medical Center).

does not involve an interaction with fibronectin. It is worth emphasizing that in the system under study, the binding is reversible and that the ligand is the soluble and native collagen molecule. In contrast, the fibronectin-mediated cell attachment assays reported by other investigators (see above) depend upon an irreversible reaction between col- lagen and fibronectin and upon the attachment of

Cell 272

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MINUTES Figure 12. Dissociation of Bound ‘251-LRSC after the Addition of CIG or Collagen

Confluent 3T3 cultures were incubated for 45 min at 26°C with 33 ng (126,000 cpm) of ‘251-LRSC. The cultures were washed and overlaid with binding buffer (O-O), or with binding buffer containing 50 pg of CIG (O-O) or 50 pg of collagen (A-A), respectively. Incubation at 26°C was continued, and at intervals, the radioactivity bound to each culture was determined as de- scribed.

The CIG was purified from human plasma and supplied by D. B. Rifkind (New York University Medical Center).

cells to insoluble gels of denatured collagen. The demonstration that al(I)-CB7 peptides is an effec- tive inhibitor in both assay systems (Kleinman et al,, 1976; see Table 2) does not necessarily weaken the argument that fibronectin is not required for the binding of soluble lZ51-LRSC to the fibroblast surface.

Phospholipase C hydrolyzes plasma membrane phospholipids without significantly changing the conformation of. membrane protein (Glaser et al., 1970). Cuatrecasas (1971) reported that digestion of fat cells, fat cell membranes and liver cell mem- branes with the enzyme caused an increase in the binding of insulin to receptors in these structures. Given these precedents, I examined the binding of ‘*51-LRSC to fibroblasts which had been treated with phospholipase C. As shown in Figure 13, the enzyme-treated cells specifically bound about 40% more ligand than did control cells. This result supports the conclusion that the collagen ligand binds to a protein in the plasma membrane.

Discussion

The reported experiments demonstrate that soluble 1251-labeled type I collagen binds to a component of the plasma membrane of fibroblasts, and the data define the specificity, kinetics and equilibrium con- stants of the binding reaction.

Although the function or functions of the binding complex are not known, there is some basis for

_c____-- -_-- -cl -_-- -4 I I I I

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MINUTES Figure 13. Binding of ‘251-LRSC to 3T3 Fibroblasts Treated with Phospholipase C

Confluent cultures were incubated for 50 min at 37°C without (O-O) or with 10 pg/ml (A-A) or 50 pg/ml (0-O) of phospholipase C in binding buffer. After the incubation, the cultures were washed with binding buffer and standard binding assays were performed with each culture receiving 40 ng of 1251- LRSC (131,600 cpm). The dotted line represents nonspecific binding in cultures receiving 100 pg of unlabeled collagen.

considering its participation in the secretion and processing of procollagen, in the initiation and control of fibrillogenesis and in the expression of controls governing collagen synthesis.

Procollagen is transported to the ceil surface in secretory vesicles, and secretion occurs when the vesicles fuse with the plasma membrane and dis- charge their contents. The binding sites in the helical portion of procollagen could hold the mol- ecule at the fibroblast surface after secretion. If the procollagen peptidases were also held or concen- trated on the same surface, the processing of the precursor would be optimized. The finding that type I procollagen is processed much more rapidly and efficiently in the cell layer than in the medium of fibroblast cultures (Taubman and Goldberg, 1976) is consistent with this proposal.

Models for collagen fibrillogenesis in vivo have been influenced by the observation that typical cross-striated fibrils precipitate when buffered so- lutions of pure collagen are warmed to 37°C (Gross, 1956). Accordingly, the assumption has been made that collagen fibrils are formed in vivo when colla- gen molecules in solution in the extracellular space undergo a spontaneous one-step polymerization. A detailed kinetic analysis by Wood (1960), however, demonstrated that collagen aggregation in vitro is not a one-step transition from monomer to polv-

Collagen Binding to Fibroblasts 273

mer. An initial lag phase is characterized by the formation of microaggregates termed “nuclei,” and this is followed by a phase during which the nuclei grow to form large fibrils. The analysis indicated that the concentration and shape of the nuclei critically determined the rate of precipitation and the ultimate size of the fibril. Trelstad, Hayashi and Gross (1976) have provided ultrastructural evi- dence that collagen polymerization in vitro pro- ceeds through stepwise stages of subassembly, and they argue that fibril formation in vivo occurs by a similar mechanism. I propose that the initial stages of nuclei formation and subassembly occur in vivo while the procollagen or collagen molecules are bound to the plasma membrane of the fibro- blast. Clustering of membrane binding sites would bring bound molecules into apposition to facilitate assembly, and membrane movements could also determine the orientation of aggregates. It is as- sumed that once the subassemblies reach a critical size, they detach from the membrane. This model has the attraction of providing a link between cell movement and the size and orientation of the developing fibrils. The large number of high affinity binding sites per fibroblast and the slow rate of spontaneous dissociation of collagen from these sites are compatible with the proposal that the binding complex participates in fibrillogenesis.

Electron microscopic studies of fibroblasts indi- cate that fibrillar assembly of collagen begins after secretion. In certain other specialized cell types, however, there is morphologic evidence that colla- gen fibrils can form within Golgi and secretory vacuoles (Trelstad, 1971 ; Weinstock and LeBlond, 1974). These fibrillar packets are probably formed from procollagen molecules, and perhaps assem- bly occurs in these instances because binding sites are present and accessible on the internal ,surfaces of the vesicles.

If collagen binding to the fibroblast surface acti- vated systems which controlled collagen synthesis and secretion, a feedback loop would be available for coordinating the amount of collagen in the extracellular space with the metabolic activities of the fibroblast. There is a precedent for surface- bound collagen altering the metabolic state of a cell in the case of the collagen-platelet interaction. It has been shown that binding of soluble type I al chains to the surface of platelets will trigger aggre- gation and the platelet release reaction (Katzman, Kang and Beachey, 1973; Kang, Beachey and Katz- man, 1974; Chiang, Beachey and Kang, 1977). It is of interest that these studies identified glucosylga- lactosylhydroxylysine as a critical determinant for the binding. This residue is present in crl-CB5 and absent from al-CB7, and only the former peptide effectively blocks the binding of the al chain to platelets. In contrast, al-CB7 effectively blocks the

binding of collagen to fibroblasts (see Table 2) and thus the binding specificities may be quite different for fibroblasts and platelets.

In many ligand-cell surface receptor interactions, adenylate cyclase is activated and the metabolic effects are presumed to be mediated by the in- creased levels of cyclic AMP. The effects of cyclic AMP on collagen synthesis are not yet clearly defined, however; increased levels of the cyclic nucleotide have been reported to both increase and decrease collagen synthesis in culture systems (Hsie, Jones and Puck, 1971; Manner and Kuleba, 1974; Peterkofsky and Prather, 1974; Baum et al., 1978).

It is clear that additional experimental evidence is needed to define the functions subserved by collagen bound to the plasma membrane of the fibroblast. It will be of interest to determine whether all procollagen and collagen types bind to the fibroblast, and whether different specificities and binding sites are required for each of these interactions. Studies of binding reactions with fi- broblasts from patients with certain inherited dis- orders of collagen metabolism may provide clues to the role of the complexes in fibrillogenesis and control of collagen synthesis. With respect to con- trol mechanisms, binding studies with keloid fibro- blasts and malignant fibroblasts may also be in- formative.

Experimental Procedures

Radioiodination of Collagen The purified lathyritic rat skin collagen was provided by G. Martin (National Institute of Dental Research, Bethesda, Maryland). 50 pg of collagen were labeled with 1 mCi of carrier-free Na’? (Amersham-Searle) by the chloramine T method (Greenwood, Hunter and Glover, 1963). The iodination was performed in a Tris buffer system, and the iodinated protein was eluted from a Sephadex G-25 column with a 0.05 M Tris-HCI buffer (pH 7.5) containing 0.15 M NaCl and 1 mg/ml of bovine serum albumin. A comparable procedure was used to radioiodinate LYI chains. Stock solutions of radiolabeled and unlabeled collagen were stored at 4°C.

Cells Fibroblast lines and strains were maintained in Dulbecco’s modi- fied Eagle’s medium (DMEM) made 10% in calf or fetal calf serum.

Binding Studies Aliquots of the stock solutions of radiolabeled and unlabeled collagen were incubated at 37% for 30 min before use in the binding assays. Cells used for binding assays were grown in 60 x 15 mm Lux plastic petri dishes. The cell layers were washed three times with 4 ml of binding buffer [DMEM containing 0.1 M HEPES (pH 7.2) and 5 mg/ml of bovine serum albumin]. Binding assays were performed by incubating the washed cell layers with 2 ml of binding buffer containing the indicated amounts of jz51- LRSC. Nonspecific binding to cells was measured in replicate cultures which received equal amounts of ‘251-LRSC and 100 pg of unlabeled LRSC. Incubations were generally at room tempera- ture (26°C) except where noted. After incubation, the plates were chilled, the binding buffer was rapidly removed by vacuum aspi- ration and the cell layers were washed three times with 4 ml of

Cell 274

cold binding buffer. The cell layers were then lysed with 1.5 ml of 0.01 M Tris-HCI buffer (pH 7.2) containing 0.5% SDS and 0.001 M EDTA, and the radioactivities of the lysates were measured in a gamma spectrometer. In every instance, dishes without cells were carried through the incubations and lysing procedure to correct for binding of ‘251-LRSC to plastic.

Binding assays were also performed with suspensions of fibro- blasts. The cells were detached from plates with trypsin or versene and resuspended in 1.5 ml of binding buffer containing the indicated amounts of 1*51-LRSC. The suspensions were incu- bated with gentle shaking in the wells of Linbro plates. After incubation, the cell suspensions were chilled and centrifuged in a Beckman microfuge for 15 sec. The pellets were washed three times with cold binding buffer and the radioactivities of the pellets were measured in a gamma spectrometer.

Polyacrylamide Gels Electrophoresis in 5% polyacrylamide gels was performed as previously described (Goldberg, Epstein and Sherr, 1972). Ra- dioactivities in gel slices were measured in a gamma spectrome- ter.

Enzymes The following enzymes were used in these experiments: bacterial collagenase form III (Advance Biofactors, Lynbrook, New York); chondroitinase ABC (Miles): testicular hyaluronidase (Worthing- ton; chromatographically purified); ttypsin (Worthington; 2X crys- tallized); and phospholipase C (Worthington; from Cl. perfrin- gens, partially purified).

Acknowledgments

This study was supported by grants from the NIH and the Bear Foundation. I thank Sheila Heitner for her expert assistance, Joan Berman for her help with the preliminary experiments and Dr. Efrat Kessler for her helpful discussions.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received October 3, 1978; revised November 9.1978

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