Calcium-sensitive interaction between calmodulin and modified forms of ratbrain neurogranin/RC3

Neurogranin (NC) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca2+ concentration and to modifications by protein kinase C (PKC) and ox idants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incor poration of four glutathiones per NG. Circular dichroism (CD) showed that m odification of NG by phosphorylation, oxidation forming intramolecular disu lfides, or glutathiolation did not affect the alpha-helical content of this protein. Mutation of the four Cys residues [Cys(-)-NG] to Gly and Ser did not affect the alpha-helical content either. Interaction of CaM with the re duced (red)-, glutathiolated (GS)-, or Cys(-)-NG in the Ca2+-free solution resulted in an increase in the cx-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosp horylated NG (PO4-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation eq uilibrium: their affinities were Cys(-)-NG > red-NG, CS-NG > ox-NC > PO4-NG . Unlike Cys(-)-, red-, and GS-NG, neither ox- nor PO4-NG bound to a CaM-af finity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracell ular level of CaM. These results indicate that modification of NG to form i ntramolecular disulfides outside the IQ domain provides an alternative mech anism fur regulation of its binding affinity to CaM.