Basic Fibroblast Growth Factor Binds Its Receptors, Is Internalized, and Stimulates DNA Synthesis in Balb/c3T3 Cells in the Absence of Heparan Sulfate

doi:10.1074/jbc.271.30.17949

Basic Fibroblast Growth Factor Binds Its Receptors, Is Internalized, and Stimulates DNA Synthesis in Balb/c3T3 Cells in the Absence of Heparan Sulfate*

From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118

^‡ To whom communication should be addressed:
Boston University School of Medicine, Room L912, 80 East Concord St., Boston, MA 02118.
Tel.: 617-638-4526; Fax: 617-638-5337.

Abstract

We have investigated the interaction of basic fibroblast growth factor (bFGF) with its receptors and heparan sulfate proteoglycans (HSPG). It has been suggested that in the absence of HSPG, cells are not able to bind bFGF or respond to treatment with bFGF. In our studies, Balb/c3T3 fibroblasts were treated with 50 mM sodium chlorate to completely inhibit (99%) sulfation of proteoglycans. We found that bFGF was able to bind, be internalized, and stimulate DNA synthesis in the absence of HSPG in a dose-dependent manner. bFGF bound to its receptors on chlorate-treated cells with a lower apparent affinity and no change in receptor number. To determine if this decreased affinity bFGF-receptor interaction is functional, we quantitatively analyzed bFGF internalization and stimulation of DNA synthesis in control and chlorate-treated cells. Endocytotic rate constants (k_e) for chlorate-treated and control cells were k_e = 0.078 ± 0.022 min⁻¹ and k_e = 0.043 ± 0.012 min⁻¹, respectively, suggesting that the process of bFGF internalization is not dramatically altered by HSPG. bFGF stimulated DNA synthesis to the same maximal level under both conditions, but chlorate-treated cells were significantly less responsive at low bFGF doses (∼10-fold increase in ED₅₀). The differences observed for control and chlorate-treated cells in the dose-response curves for stimulation of DNA synthesis and receptor binding correlated directly, suggesting that receptors are equally capable of eliciting a mitogenic signal under both conditions. It is unlikely that these results are due to residual HSPG since heparinase (I and III) digestion of chlorate-treated cells had little effect. Although the presence of HSPG on the cell surface increases the affinity of bFGF for its receptors, our observations suggest that HSPG are not “absolutely” required for binding, internalization, or stimulation of mitogenic activity.

Footnotes

↵* This study was supported by American Heart Association Grant-in-Aid 13-522-923, a Whitaker Foundation Biomedical Engineering Research grant, Grant-in-Aid GA93046 from the Fight For Sight Research Division of the National Society to Prevent Blindness, by Grant IRG-97 R from the American Cancer Society, and departmental grants from the Massachusetts Lions Eye Research Fund and Research to Prevent Blindness, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

bFGF

basic fibroblast growth factor

DMEM

Dulbecco's modified Eagle's medium

HSPG

heparan sulfate proteoglycans

K_d

equilibrium dissociation constant

k_e

endocytotic rate constant

k_on

association rate constant

k_off

dissociation rate constant

PBS

phosphate-buffered saline

R_s

Spearman rank correlation coefficient

PAGE

polyacrylamide gel electrophoresis.
- Received October 24, 1995.
- Revision received April 15, 1996.