A DIVALENT METAL-ION BINDING-SITE IN THE KINASE INSERT DOMAIN OF THE ALPHA-PLATELET-DERIVED GROWTH-FACTOR RECEPTOR REGULATES ITS ASSOCIATION WITH SH2 DOMAINS

TO investigate the effects of metal ion binding to the alpha-PDGFR kin ase insert domain, a PCR product representing amino acid residues 691- 795 (104 amino acids) was bacterially expressed and purified. Secondar y structure prediction and circular dichroism spectroscopy indicated t his domain to be a mixed alpha+beta protein with a large coil/turn con tribution. This 16 kDa, soluble, nonphosphorylated domain bound to Ca- 45(2+) and Zn-65(2+) through a common shared site. Of the unlabeled di valent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn 2+ > Mg2+, Ba2+ in competing for Ca-45(2+) binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed s ignificantly, and this changed conformation was resistant to subtilisi n proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using pu rified baculovirus-expressed alpha-PDGFR showed a marked increase in b inding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (K-D = 0.5 mu M), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presenc e of the KI domain. The nonphosphorylated KI domain was effective in c ompeting with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microin jection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI do main has a correctly folded three-dimensional structure compatible wit h biological activity. Together these findings indicate that the recom binant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this bindi ng is modulated by the presence of a novel divalent metal ion binding site within its structure.