FORMATION OF MITOGENICALLY ACTIVE PDGF-B DIMER DOES NOT REQUIRE INTERCHAIN DISULFIDE BONDS

Platelet-derived growth factor (PDGF), a major mitogen for mesenchymal cells, is a disulfide bonded dimer of two subunit polypeptides named A and B. All of the three possible dimeric forms, i.e. AA, BB, and AB, exist in nature. The dimeric structure has been presumed to be necess ary for biological activity, since reduction of the dimer results in l oss of activity and simultaneous conversion to monomeric form as deter mined by SDS-gel electrophoresis. However, reduction of the native mol ecule destroys intrachain, as well as interchain, disulfide bonds, and it is possible that the former rather than the latter are critical fo r proper conformation of the active protein. We show here that PDGF-B polypeptides in which all 8 cysteines or the 2nd, 4th, 5th, and 8th cy steines have been mutated to serines fail to form covalent dimers and possess dramatically less mitogenic activity than native PDGF-BB. Anot her mutant, PDGF-B(C2,4S), in which just the 2 cysteines involved in i nterchain disulfides were converted to serine, ran as a monomer on SDS -polyacrylamide gels as expected. Somewhat unexpectedly, however, the mitogenic activity of the PDGF-B(C2,4S) analog was similar to the acti vity of wild-type PDGF-BB disulfide-bonded dimer under physiological c onditions. The activity of the analog was more sensitive to the effect of low pH than was the activity of wild-type PDGF-BB. Molecular weigh t analysis utilizing light scattering and sedimentation equilibrium de monstrated that the PDGF-B(C2,4S) analog exists as a noncovalent dimer at pH 4-7 but dissociates to a monomer at pH 2.5. Disulfide analysis of the mutant protein demonstrated that the intrachain disulfide bonds are the same as those formed in wild-type PDGF-BB homodimers. We conc lude that proper formation of intrachain disulfide bonds is critical t o maintaining the correct conformation of PDGF monomers, but that appr opriately folded monomers can associate into active noncovalent dimers in the absence of interchain disulfide bonds. Interchain disulfide bo nds thus appear to increase the stability of the PDGF dimer rather tha n being crucial to its existence.