Alternative Splicing Determines the Binding of Platelet-Derived Growth Factor (PDGF-AA) to Glycosaminoglycans ² Florentyna Lustig,* ,‡ Johan Hoebeke, | Gunnel O ¨ stergren-Lunde `n, ‡ Florence Velge-Roussel, | Go ¨ran Bondjers, ‡ Urban Olsson, ‡ Ulla Ru ¨etschi, § and Gunnar Fager ‡ The Wallenberg Laboratory for CardioVascular Research, Sahlgrenska UniVersity Hospital, S-413 45 Go ¨ teborg, Sweden, The Department of Clinical Chemistry, UniVersity of Go ¨ teborg and Sahlgren’s Hospital, S-400 33 Go ¨ teborg, Sweden, and CJF93/09 INSERM, Faculte des Sciences Pharmaceutiques, UniVersite ´ Francois Rabelais, F-37200 Tours Cedex, France ReceiVed January 18, 1996; ReVised Manuscript ReceiVed July 2, 1996 X ABSTRACT: We have shown previously that the platelet-derived growth factor (PDGF) and a synthetic oligopeptide, corresponding to the basic carboxyl-terminal amino acid extension of the long PDGF-A isoform, bind to heparin. Here, we have expressed the long (rA 125 ) and the short (rA 109 ) variants of PDGF A-chains in Escherichia coli and produced the functional homodimers. Surface plasmon resonance analyses showed that while the dimeric rA 125 bound with high affinity to low molecular weight heparin, the rA 109 , lacking the basic extension, did not. This strongly indicated that high affinity binding is due to the carboxyl-terminal extension. Investigations of kinetics and thermodynamics suggested an allosteric binding mechanism. Thus, dimeric rA 125 contains two equivalent binding sites. Following low affinity binding of heparin to one binding site, the dimer undergoes a conformational change, increasing the affinity for heparin about 40 times. This positive cooperativity requires the basic amino acid extension in both monomers of the dimeric PDGF molecule. Thermodynamics of the reaction, showing an entropy-driven endothermic process, suggest the involvement of hydrophobic interactions in this rearrangement. Three amino acids in the basic carboxyl-terminal extension were essential for the interaction: the basic residues Arg 111 and Lys 116 , and the polar Thr 125 . We also found that other glycosaminoglycan species, corresponding to those produced by human arterial smooth muscle cells, bound to dimeric rA 125 and that heparan sulfate showed the highest affinity. The accumulation of smooth muscle cells (SMCs) 1 in the arterial intima is a well-recognized feature of atherosclerosis, especially prominent in transplant atherosclerosis and restenosis after balloon angioplasty or coronary bypass grafting (Ross et al., 1990). The increase in arterial SMC (ASMC) mass is due to migration from the media as well as proliferation within the intima/media and has been associated with activity of the platelet-derived growth factor (PDGF) (Ross, 1981; Schwartz et al., 1986; Thyberg et al., 1990). However, the proliferation of these cells is also under the influence of several other cytokines including fibroblast growth factors (FGFs) (Lindner & Reidy, 1991), interleukins, and R-thrombin (Bobik & Campbell, 1993; Fager, 1995). PDGF is a disulfide-linked dimer composed of two poly- peptide chains, denoted A and B and represented in ViVo by all three chimeras; PDGF-AA, PDGF-AB, and PDGF-BB (Heldin et al., 1986; Hart et al., 1990). There are two isoforms of the PDGF-A chain. This is a result of translation of two PDGF A-chain transcripts, arising from alternative usage of exon 6 coding for a basic amino acid sequence. Thus, only the longer A-chain isoform contains a sequence with a high proportion of lysine and arginine residues in its carboxyl-terminal extension. The conservation of A- and B-chains of PDGF can be associated with the existence of distinct R and  cell-surface PDGF receptors and their recognition pattern. Several attempts have been made to explain the biological role of the carboxyl-terminal extension of the long A-chain. Ac- cording to Maher et al. (1989), this basic sequence is responsible for targeting a nonsecreted form of the A-chain to the nucleus. On the other hand, increasing evidence suggests an involvement of the basic carboxyl-terminal in binding of PDGF to glycosaminoglycans (GAGs) (Fager et al., 1992a,b; Raines & Ross, 1992; Khachigian & Chester- man, 1992). Heparin-like GAGs bind PDGF and inhibit its mitogenic effect on human ASMC (hASMC) in Vitro (Fager et al., 1992a). An oligopeptide (Oligo-108-124), corre- sponding to the exon 6-derived amino acid residues 108- 124 of the long PDGF A-chain isoform, bound to heparin- Sepharose and competed with PDGF for binding to heparin. By interfering with the binding of PDGF to heparin, the ² This study was supported by grants from the Swedish Medical Research Council (Project No. 4531) and the Swedish Heart Lung Foundation (Project No. 51044). * Author to whom correspondence should be addressed. ‡ The Wallenberg Laboratory for Cardiovascular Research, University of Go ¨teborg, Sweden. § The Department of Clinical Chemistry, University of Go ¨teborg, Sweden. | Faculte des Sciences Pharmaceutiques, Universite ´ Francois Rabe- lais, Tours Cedex, France. X Abstract published in AdVance ACS Abstracts, August 15, 1996. 1 Abbreviations: PDGF, platelet-derived growth factor; rA109, short recombinant PDGF A-chain; rA125, long recombinant PDGF A-chain; nA125, native PDGF A-chain; aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; AT-III, antithrombin III; SMC, smooth muscle cells; hASMC, human arterial smooth muscle cells; LMW-heparin, low molecular weight heparin (Mr ) 3000); GAG(s), glycosaminoglycan(s); PG(s), proteoglycan(s); HS, heparan sulfate; DS, dermatan sulfate; CS, chondroitin sulfate; UF, unretained fraction obtained after subfractionation of fresh human serum on heparin- Sepharose column; LDL, low density lipoproteins; PMSF, phenyl- methanesulfonyl fluoride; Gdn-HCl, guanidine hydrochloride; BSA, bovine serum albumin; SPR, surface plasmon resonance. 12077 Biochemistry 1996, 35, 12077-12085 S0006-2960(96)00118-3 CCC: $12.00 © 1996 American Chemical Society