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Endocrinology Vol. 142, No. 9 3749-3755
Copyright © 2001 by The Endocrine Society

ARTICLES

Distribution of Chimeric IGF Binding Protein (IGFBP)-3 and IGFBP-4 in the Rat Heart: Importance of C-Terminal Basic Region

K. L. Knudtson, M. Boes, A. Sandra, B. L. Dake, B. A. Booth and R. S. Bar

Department of Internal Medicine, Diabetes and Endocrinology Research Center, Veterans Administration Medical Center, The University of Iowa, Iowa City, Iowa 52246

Address all correspondence and requests for reprints to: Robert S. Bar, M.D., The University of Iowa, Department of Internal Medicine, ENDO-3E19 Veterans Administration Medical Center, Iowa City, Iowa 52246. E-mail: rbar{at}icva.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IGF binding proteins-3 and -4, whether given in the perfused rat heart or given iv in the intact animal, cross the microvascular endothelium of the heart and distribute in subendothelial tissues. IGF binding protein-3, like IGF-I/II, localizes in cardiac muscle, with lesser concentrations in CT elements. In contrast, IGFBP-4 preferentially localizes in CT. In this study, chimeric IGF binding proteins were prepared in which a basic 20-amino-acid C-terminal region of IGF binding protein-3 was switched with the homologous region of IGF binding protein-4, and vice-versa, to create IGF binding protein-34 and IGF binding protein-43. Perfused IGF binding protein-34 behaved like IGF binding protein-4, localizing in connective tissue elements, whereas IGF binding protein-43 now localized in cardiac muscle at concentrations identical to perfused IGF binding protein-3. To determine whether these small mutations altered the affinity of the chimera for cells, the ability of 125I-IGF binding protein-34 and 125I-IGF binding protein-43 to bind to microvascular endothelial cells was determined and compared with IGF binding protein-3. IGF binding protein-34 retained 15% of the binding capacity of IGF binding protein-3, whereas IGF binding protein-43 bound to microvessel endothelial cells with higher affinity and greater total binding than that of IGF binding protein-3. We conclude that small changes in the C-terminal basic domain of IGF binding protein-3 and the corresponding region of IGF binding protein-4 can alter their affinity for cultured cells and influence their tissue distribution in the rat heart.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IGF BINDING PROTEIN (IGFBP)-3, when perfused through the isolated, beating rat heart, crosses the microvascular endothelium and distributes primarily in cardiac muscle, with lesser association to connective tissue (CT) elements. Perfused IGFBP-4 also crosses the microvessel endothelium but, in contrast with IGFBP-3, preferentially associates with CT (1, 2). The ability of IGFBP-3 to bind to specific cells, such as endothelial cells, has been suggested to be related to a highly basic C-terminal region of the binding protein. Other IGFBPs, which include IGFBP-5 and IGFBP-6, have such a C-terminal segment, whereas the other three high-affinity IGFBPs do not and do not bind to endothelial cells (3). This basic C-terminal segment of IGFBP-3 has also been thought to play a primary role in IGFBP-3 associating with the acid labile subunit (4, 5), IGFBP-3 translocating to the nucleus (6), and initiating IGFBP-3 proteolysis by acting as a binding site for serine proteases (7, 8).

To determine the relevance of this region of IGFBP-3 to the localization of IGFBP-3 in cardiac muscle, recombinant technology was used to prepare chimeric IGFBP-4, in which a basic C-terminal 20-amino-acid portion of IGFBP-3 was substituted for the homologous region of IGFBP-4, to create IGFBP-43. Conversely, the homologous region of IGFBP-4 was substituted for the basic region of IGFBP-3, to produce IGFBP-34. In the present study, the distribution of perfused IGFBP-3, IGFBP-4, IGFBP-34, and IGFBP-43 in the heart has been evaluated using a modified Langendorff rat heart preparation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Microvessel endothelial cells were prepared from bovine heart adipose tissue and characterized as previously described (9). All in vitro binding studies were performed with confluent microvessel cells on 12-well Linbro trays (ICN Biomedicals, Inc., Aurora, OH).

Endothelial cell binding
For binding, 125I-IGFBP (2 x 104 cpm) was added to 12-well plates by itself or with unlabeled IGFBPs. After 90 min at 22 C, the entire monolayer was removed with 0.1 N NaOH and counted in a Beckman Coulter, Inc. (Fullerton, CA) {gamma}-counter. Data are expressed as mean ± SEM in 3 wells. Each experiment was performed 3–7 separate times.

For partition of binding between cells and extracellular matrix (ECM), 125I-IGFBP-3, 125I-IGFBP-34, or 125I-IGFBP-43 was incubated with endothelial monolayers for 90 min, as previously described (3). After incubation, the supernatant was removed and the monolayer washed twice with PBS to remove nonassociated counts. The cells were removed by a 3-min treatment of the monolayer with 0.5% TritonX-100, 0.02 M NH4OH in PBS, followed by one wash with PBS and all then counted as cell-associated binding. The ECM was subsequently removed with 0.1 N NaOH and the wells washed twice, with all counted as ECM binding.

Preparation of chimeric IGFBP-43 and IGFBP-34
The basic C-terminal 20 amino acids of IGFBP-3 (P3 region) and the homologous domain of IGFBP-4 (P4 region) were "swapped-out" to generate the IGFBP-43 and IGFBP-34 chimeras. This was achieved by first subcloning the genes encoding IGFBP-3 and IGFBP-4 between the EcoRI and XhoI restriction sites of the cloning vector, pSP73 (Promega Corp., Madison, WI). The restriction enzyme sites, MunI and SalI, were then introduced into the nucleic acid sequence flanking the region encoding the P3 region and the P4 region (Fig. 1Go) via site-directed mutagenesis using the QuickChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA). For IGFBP-3, the nucleotide sequence CAACTG was mutated to CAATTG to introduce a MunI restriction site 5-prime to the P3 region (Fig. 1Go). The nucleotide sequence GTGGAT was mutated to GTCGAC, introducing a SalI site 3-prime to the P3 region. Similarly, IGFBP-4 sequences CAACTG and GTGGAC were each mutated to introduce MunI and SalI sites, respectively. The introduction of the restriction sites did not alter the predicted amino acid sequence of IGFBP-3 or IGFBP-4. After DNA sequence and restriction digest analyses, to confirm that all four mutations were correctly introduced, the P3 and P4 regions between the MunI and SalI sites of IGFBP-3 and IGFBP-4, respectively, were switched by first digesting with these enzymes, then ligating them into the other IGFBP. The sequences of IGFBP-34 and IGFBP-43 constructs were confirmed by DNA sequence analysis. The genes encoding the chimeric binding proteins were then excised from the pSP73 cloning vector and ligated into the EcoRI/XhoI sites of the vector pBacPAK9 (CLONTECH Laboratories, Inc., Palo Alto, CA) for the expression and production of the chimeric IGFBPs.



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  		Figure 1. Sequence homology of the basic C-terminal region between IGFBP-3 and IGFBP-4. The double-underlined residues denote the P3 and P4 regions of IGFBP-3 and IGFBP-4, respectively. Homologous amino acids are shown in bold. Shaded bases denote the areas in which the MunI and SalI restriction enzyme sites were introduced. Single-underlined nucleotides represent the specific nucleic acid bases that were altered via site-directed mutagenesis. The dashed lines identify P3 and P4. The sequence YKKKQCRP is the purported heparin binding sequence of IGFBP-3.

 
Cotransfections and virus production
SF9 cells were cotransfected, according to the CLONTECH Laboratories, Inc. protocol, with BacPAK viral DNA and the pBacPAK9 construct for each binding protein. Binding protein production was confirmed by PEG assay and Western analysis using anti-IGFBP-3 and anti-IGFBP-4 sera obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). IGFBP-3 and IGFBP-34 reacted mostly with the anti-IGFBP-3 antibody and, to a lesser extent, with IGFBP-4 antibody; whereas IGFBP-4 and IGFBP-43 reacted only with the anti-IGFBP-4 antibody. Binding protein sizes were as expected. Viral stocks were expanded and used to inoculate High-Five cells (Invitrogen, Carlsbad, CA) in serum-free medium (Express Five SFM, Life Technologies, Inc., Gaithersburg, MD), for protein production.

Purification of recombinant binding proteins
High-Five insect cells were infected with recombinant baculovirus in the presence of a mammalian protease inhibitor cocktail (1:1000 dilution, Sigma, St. Louis, MO). At approximately 50 h, media from insect cell cultures were harvested and EDTA added to give a final concentration of 0.5 mM. Media were centrifuged to remove any cells or debris. Supernatant was loaded on an IGF-I affinity column, with each binding protein having a separate column. Columns were washed with HNT buffer (0.05 M HEPES, 0.05 M NaCl, 0.1 mM phenylmethylsulfonylfluoride, 20 µl/L, Tween 80, pH 7.5) containing 5 mM EDTA and the protease inhibitor cocktail (1:1000) and then with HNT containing 2.5 M NaCl and the protease inhibitors. Each column was washed with distilled water to remove salt and eluted with 0.5 M acetic acid. The acid eluate was dried in a Speed Vac (Savant, Hicksville, NY) and the binding protein(s) stored at -80 C.

Iodination of binding proteins
IGFBPs were iodinated using Iodo Beads (Pierce Chemical Co., Rockford, IL) then run over a desalting column. The iodinated binding proteins were purified on a Sephadex G100 column (Amersham Pharmacia Biotech, Piscataway, NJ). The Sephadex G100 fractions were analyzed on nonreducing SDS-PAGE, followed by autoradiography. All iodinated proteins were greater than 95% intact and migrated at the appropriate monomeric molecular weight. Specific activities averaged approximately 17 µCi/µg protein (range, 4–34 µCi/µg protein).

Heart perfusion
All studies involving rats were approved by both the University of Iowa Animal Care and Use Committee and the Veterans Administration Animal Care and Use Subcommittee. Adult male rats (Sprague Dawley, approximately 300 g; Harlan Sprague Dawley, Inc., Indianapolis, IN), were anesthetized with methoxyflurane, the chest cavities opened, and the hearts removed and suspended by a perfusion catheter placed in the aorta as previously described (1). The heart was perfused, in retrograde fashion, with perfusate flowing from aorta to coronary arteries to the microvessels and collected via a slit made in the right ventricle. Aerated buffer and IGFBP-containing solutions (pH 7.4, 37 C) were perfused using a peristaltic pump at sufficient rate and pressure to close the aortic valve leaflets and maintain a ventricular heart rate of 40–100 beats/min. Each heart was perfused for 5–10 min with buffer alone until a stable heart rate was established. Hearts were perfused with 125I-IGFBP-3, 125I-IGFBP-4, 125I-IGFBP-34, or 125I-IGFBP-43 at 2 x 106 cpm/ml for 1 min, which usually took 3.0 ml; <= 1% perfused radioactivity was retained in the heart. Hearts were then rapidly fixed for 1 min with 67 mM sodium phosphate buffer at pH 7.4, 4 C, containing 2.5% glutaraldehyde. Small (1 mm) portions of the heart were excised, washed with 0.1 M cacodylate buffer (pH 7.4), postfixed with buffered 1% osmium tetroxide (OsO4), dehydrated in a series of graded alcohols, and embedded in Epon. The two anatomic regions for sampling included an area 1–3 mm lateral to the terminal branch of the left anterior descending coronary artery and an area of the right atrium superior to the circumflex branch of the coronary artery. Paraffin sections (5 µm) were prepared and processed for light microscopic autoradiography as previously described (10). The density of background silver grains was subtracted from the density of silver grains over tissue, to give the specific number of silver grains per unit area. Areas were computed using an ocular grid. Morphometric analysis was carried out using the relative size of rat cardiac anatomic compartments. We, and others (1, 11, 12), have calculated the relative distribution of anatomic regions in the perfused rat heart. Connective tissue elements comprise approximately 4% of total area, myocytes approximately 84%, endothelium 3%, and capillary lumens 9%. The two subendothelial compartments that were analyzed for the studies included cardiac muscle (myocytes) and CT, which is composed of endomysium (fibroblasts and extracellular collagen) and basement membranes. Slides for radioautography were assigned random numbers so that scoring of 125I grains was done in a blinded fashion. 125I grains were assigned to cardiac muscle when the grain was entirely within the muscle fiber or myocyte and to CT when entirely within fibroblasts, collagen, or basement membrane compartments and having no overlap with myocytes or other adjacent cells or tissue. Data were expressed as the number of grains over each particular compartment divided by the total area that that compartment comprises.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IGFBPs used in heart perfusions and binding to cultured cells
Final preparation of IGFBP-3, -4, -34, and -43 was performed using IGF-I affinity columns. Materials used for studies were tested for purity (Fig. 2Go, Coomassie), binding to 125I-IGF-I and -II (Fig. 2Go, ligand binding), and reactivity with antisera to IGFBP-3 and IGFBP-4 (Fig. 2Go, antisera). Purity and integrity of iodinated binding proteins were also assessed by SDS-PAGE (see Materials and Methods).



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  		Figure 2. Purified preparations of IGFBP-3, -34, -4, and -43. Coomassie stain (left), 125I-IGF ligand blot (center), and immunoblot with antisera against IGFBP-3 and IGFBP-4 (right).

 
Heart perfusion of 125I-IGFBP-34 and 125I-IGFBP-43
Isolated beating rat hearts were perfused with 125I- IGFBP-3, 125I-IGFBP-43, 125I-IGFBP-34, or 125I-IGFBP-4, each at 2 x 106 cpm/ml. Each heart was perfused with 125I-IGFBP for 1 min, with the reactions stopped by perfusion of 2.5% glutaraldehyde. The hearts were then prepared for radioautographic analysis of 125I grains in cardiac muscle or CT elements (basement membranes, collagen, endomysium, fibroblasts). As previously demonstrated (2), IGFBP-3 primarily associated with cardiac muscle in a ratio of approximately 5:1, cardiac muscle:CT, whether measured as total or specific binding. IGFBP-4 localized preferentially to CT elements in a ratio of approximately 1:5 for total binding and 1:8 for specific binding, muscle:CT (Table 1Go). IGFBP-34 and IGFBP-43 distributed differently in the heart, relative to IGFBP-3 and IGFBP-4, respectively. IGFBP-34 behaved similarly to IGFBP-4, and IGFBP-43 was similar to IGFBP-3 (Table 1Go). IGFBP-34 had a muscle:CT ratio of 1:5, identical to the IGFBP-4 ratio. IGFBP-43 had a muscle:CT ratio of 4–5:1, similar to IGFBP-3 (5:1). The difference in tissue distribution was related to both a decrease in muscle grains and an increase in CT 125I grains, with IGFBP-4 and IGFBP-34 having 15–20 times the CT association of IGFBP-3 and IGFBP-43, respectively. Fig. 3Go is an example of representative radioautographs for 125I-IGFBP-3 (C), 125I-IGFBP-4 (A), 125I-IGFBP-43 (D), and 125I-IGFBP-34 (B). For 125I-IGFBP-3 (C) and 125I-IGFBP-43 (D), most of the grains are located in cardiac muscle; whereas 125I-IGFBP-4 (A) and 125I- IGFBP-34 (B) are localized to fibroblasts, basement membranes, and collagen fibers.


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  		Table 1. 125I Grains in cardiac muscle and connective tissue

 


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  		Figure 3. Autoradiographic sections of rat hearts perfused with 125I binding proteins. A, 125I-IGFBP-4. Section is through a cardiac arteriole. L is the vessel lumen. M is the muscularis layer of smooth muscle cells. The remainder of the field is CT adventitia containing fibroblasts (F) and CT. The adventitia is scattered with numerous silver grains. B, 125I-IGFPB-34. Numerous silver grains are localized to a CT septum in the tissue (open arrowheads). Grains are also located over cardiac muscle (arrow) and the capillary endothelium (closed arrowheads). C, 125I-IGFBP-3. Arrows point to silver grains localized over cardiac muscle tissue. Arrowhead shows grain at the level of the endothelial cell. D, 125I-IGFBP43. Lack of silver grains in CT septum (open arrowheads). Grains in muscle and endothelium. Magnification bar, 10 µm.

 
Endothelial cell binding of IGFBP-43 and IGFBP-34
Because of the apparent importance of the P3 region of IGFBP-3 in cell association of IGFBP-3 to cultured microvessel endothelial cells, we next determined whether the loss of the P3 region in IGFBP-34 or the substitution of the P3 region for the P4 region in IGFBP-43 affected the cell association of IGFBP-3 and IGFBP-4. 125I-IGFBP-43 and 125I-IGFBP-3 specifically bound to microvessel endothelial cells. Maximal specific binding to microvessel cells was 4.7–20.8% per well for 125I-IGFBP-3 and 125I-IGFBP-43. 125I-IGFBP-34 had 0.7–1.6% per well to cultured cells. 125 I-IGFBP-4 had no specific binding to endothelial cells.

Partition of 125I-IGFBP-43 monolayer binding to endothelial cells and ECM
125I-IGFBP-43 bound predominately to the cell surface of the endothelial cells, 72% binding to cells and 28% to ECM, a distribution of monolayer binding similar to previously reported studies with 125I-IGFBP-3 (2). In the same experiment with 125I-IGFBP-43, distribution of 125I-IGFBP-3 was 91% to endothelial cell surface and 9% to ECM (Fig. 4Go).



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  		Figure 4. Partition of binding of IGFBP-3, and -34 between cells and ECM. 125I binding proteins were incubated with endothelial cell monolayers, and cell-associated binding protein was removed by treatment with cells Triton X-100 and NH4OH. The ECM and associated binding protein was then removed by treatment with 0.1 M NaOH. Specific binding was calculated from total binding minus binding in the presence of 25 µg unlabeled homologous binding protein (n = 2) and expressed as specific binding ± SEM of three separate wells.

 
Competition-inhibition curves were performed with 125I-IGFBP-43 (Fig. 5AGo), 125I-IGFBP-3 (Fig. 5BGo), and 125I-IGFBP-34 (Fig. 5CGo) in the presence of unlabeled IGFBP-43, IGFBP-3, IGFBP-4, IGFBP-34, and P3, an 18-amino-acid synthetic peptide contained in the segment removed from IGFBP-3 to form IGFBP-34 and switched with the homologous region of IGFBP-4 to form IGFBP-43 (3). 125I-IGFBP-43 binding was inhibited by unlabeled IGFBP-43 in a dose-dependent manner. Binding was also inhibited by P3 and IGFBP-3 but with lower affinity than IGFBP-43. Fifty percent inhibition (ID50) of 125I-IGFBP-43 binding was caused by 1 µg/ml unlabeled IGFBP-43, approximately 2–5 µg/ml unlabeled P3, approximately 15 µg/ml unlabeled IGFBP-3, and minimally by IGFBP-4 and IGFBP-34. 125I-IGFBP-3 binding was inhibited by unlabeled IGFBP-3, P3, and IGFBP-43. ID50 of maximal binding of 125I-IGFBP-3 was caused by approximately 3 µg/ml IGFBP-3, 2 µg/ml P3, and approximately15 µg/ml IGFBP-43, with no inhibition by IGFBP-4 or IGFBP-34. In 3 detailed competition-inhibition studies, Scatchard analysis yielded straight-line plots with 125I-IGFBP-43 vs. unlabeled IGFBP-43, having greater maximum bound/free (B/F) (0.20, 0.13, 0.13) and average dissociation constant (Kd) = 5 x 10-8 M. For 125I-IGFBP-3 vs. unlabeled IGFBP-3, average Kd was 1.3 x 10-7 M and maximal B/F of 0.11, 0.05 and 0.06. One example of Scatchard analysis is shown in Fig. 6Go.



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  		Figure 5. Competition inhibition curves in microvessel endothelial cells: A, 125I-IGFBP-43; B, 125I-IGFBP-3; and C)125I-IGFBP-34. Binding data for 125I-IGFBP-43 (A) and 125I-IGFBP-3 (B) are given as maximal binding being 100%. For this experiment, 100% for A was 16.9% per well; and for B, 11.6% per well. Data with 125I-IGFBP-34 are given as actual binding per well because binding was so low. Each 125I-labeled ligand is competed against by unlabeled IGFBP-43 •, IGFBP-3 {circ}, IGFBP-4 {blacksquare}, IGFBP-34 {triangleup}, and P3 {blacktriangledown}.

 


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  		Figure 6. Scatchard analysis of 125I-IGFBP-3 and 125I-IGFBP-43 binding. For these Scatchard plots, Kd for IGFBP-3 ({circ}) is 1.3 x 10-7 M; and for IGFBP-43, 0.5 x 10-7 M.

 
Maximal specific binding of 125I-IGFBP-43 was usually greater than that of 125I-IGFBP-3, ranging from 5.9–20.8% per well (mean 12.1, n = 7) for 125I-IGFBP-43 and from 4.7–11.5% (mean, 6.6; n = 6) for 125I-IGFBP-3. ID50 for 125I-IGFBP-43 was always lower than for 125I-IGFBP-3. ID50 for 125I-IGFBP-43 was 1–3 µg/ml (n = 5), whereas the range for 125I-IGFBP-3 was 3–9 µg/ml (n = 4).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Perfused IGFBP-3 was shown to cross the microvessel endothelium and associate predominantly with subendothelial cardiac muscle and CT in a muscle:CT ratio of approximately 5:1. Perfused IGFBP-4 also crossed the endothelial boundary; but, in contrast to IGFBP-3, it preferentially associated with CT. IGFBP-34 behaved like IGFBP-4, and IGFBP-43 like IGFBP-3, even though each chimera was identical to IGFBP-3 or IGFBP-4 except for the switch of the 20-amino-acid segment containing the P3 or P4 region in IGFBP-34 and IGFBP-43. In cultured microvessel endothelial cells, IGFBP-43 bound to cells better than IGFBP-3, despite the fact that IGFBP-4 does not bind to endothelial cells. Similarly, IGFBP-34 lost much of its affinity for cultured endothelial cells, even though most of its structure is identical to IGFBP-3. These findings indicated that substitution of the small P3 region for P4 not only caused IGFBP-34 to lose most of its affinity for cultured endothelial cells but also directed it to CT elements in the perfused heart. The mechanism(s) that causes IGFBP-43 to lose its affinity for CT and increase its affinity for myocytes may be explained, in part, by the ability of IGFBP-43 to specifically bind to cells. In cultured endothelial cells, IGFBP-43 showed both increased maximal binding and higher affinity for cultured cells than IGFBP-3. Because muscle of the isolated heart represents the major compartment (84%), and CT a minor component (4%), total binding of IGFBP-43 to myocytes (muscle), should it mirror the binding seen with cultured endothelial cells, could partly explain the modest increase of IGFBP-43 per unit area of muscle and the marked decrease in CT association, again, per similar area of CT. However, until we define the CT element(s) that result in preferential localization of IGFBP-4 and IGFBP-34 in the perfused rat heart, the mechanisms that determine the tissue distribution of IGFBP-3, IGFBP-4, and the 2 chimera remain uncertain.

The present study also demonstrates that small mutations in the primary structure of IGFBP-3 and IGFBP-4 can result in major changes in tissue localization in the mutated IGFBP in the rat heart. The tissue distribution of IGFBP/IGF complexes was not studied but will be of obvious interest, given the recent data in which IGFBP-3/IGF-I complexes were given to humans, enabling safe delivery of larger quantities of IGF-I (13, 14, 15, 16, 17). The tissue localization of IGFBP mutants complexed with IGF-I may, a priori, not be totally predictable. For example, it has been shown that when IGFBP-4/IGF-I complexes are perfused in the rat heart, the complexes, like IGF-I alone, primarily associate with cardiac muscle; i.e. IGF-I, not IGFBP-4, seemed to dictate the movement of the complex (18, 19). However, when IGFBP-3/IGF-I complexes were infused in rats, the complex, like IGFBP-3, but not like IGF-I, localized in the glomeruli of the kidney (20). This raises the possibility that the distribution of IGFBP/IGF complexes may be organ- and tissue-specific and that the distribution of a given IGFBP-IGF complex can be modified based on specific mutations of an IGFBP. Carried to its most optimal clinical conclusion, there could be the ability to deliver IGF-I (or II) to selected tissues by having the IGF complexed to a specific or mutated IGFBP, in either case, with the IGFBP causing the IGF to be localized to the tissue of interest.

The mechanism(s) of binding of 125I-IGFBP-3 and 125I-IGFBP-43 to cultured microvessel endothelial cells is not totally explained by the data presented in this study. Although binding was saturable and specific, the Kd values of 0.5–1.3 x 10-7 M are several orders of magnitude higher than traditionally seen for classical plasma membrane receptors, as well as previous studies of IGFBP-3 cell binding, which typically have Kd values for their ligands of 10-9–10-10 M. Because of its unique position as the initial fixed surface exposed directly to circulating IGFBPs, vascular endothelium, should it have similar binding sites in vivo, could have particular relevance for IGF/IGBP action. The potential function of these "receptors" would take on added importance as well. Is the receptor similar to the 20–50-Kd receptors (or fragments) found on Hs578T breast cancer cells (21), the type V TGFß receptor (22), or proteoglycans-related binding sites, such as found for basic fibroblast growth factor (23) or glial cell line derived-neurotrophic factor (24)? Do the endothelial receptors for IGFBP-3 serve as a mechanism to bring IGF-I and II to the endothelial type I and type II IGF receptors or is this a mechanism for transendothelial passage of IGF-I/II? Each of these possibilities is not only plausible but can now be experimentally verified or refuted.


   Acknowledgments
 
This work was supported by funds from Veterans Affairs research and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-25421 and DK-25295.


   Footnotes
 
Abbreviations: B/F, Bound/free; CT, connective tissue; ECM, extracellular matrix; ID50, 50% inhibition; IGFBP, IGF binding protein; Kd, dissociation constant.

Received January 2, 2001.

Accepted for publication May 3, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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