Abstract
Abstract: :
Purpose: Calcium-binding guanylyl cyclase activating protein 2 (GCAP-2) mediates calcium feedback in photoreceptors by activating or inhibiting retinal membrane guanylyl cyclase, retGC, as a function of free intracellular Ca2+ concentrations. Main regulatory domains in GCAP-2 have been mapped, and reversible Ca2+-sensitive dimerization of GCAP-2 has been demonstrated, the three-dimensional structure of Ca-bound GCAP-2 has been determined (except for the C-terminal portion of the protein) [1-3]. However, the exact structural changes in GCAP-2 caused by the Ca-switch remain undetermined. In order to follow possible structural changes related to the C-terminal part of the protein we used phosphorylation near the C-terminus of the molecule. Methods: Wild type GCAP-2 and its various mutants were expressed in E. coli and phosphorylated by the retinal extract or by purified cyclic nucleotide-dependent protein kinases (CNDPK) in vitro at different Ca2+ concentrations followed by SDS PAAGE and autoradiography. Results: In the presence of retinal extract GCAP-2 undergoes both phosphorylation in a cyclic nucleotide-dependent manner and rapid dephosphorylation by protein phosphates PP2C. GCAP-2 mutants in which C-terminal CNDPK phosphorylation site was either deleted or inactivated by a point mutation did not undergo phosphorylation. Incorporation of radioactive phosphate in GCAP-2 was enhanced in the presence of EGTA and inhibited up to 5-fold in the presence of 1 µM Ca. Contrary to this, Ca2+ had no effect on phosphorylation of constitutively active mutant of GCAP-2 in which Ca2+-binding within the EF-hand loops was prevented by point mutations. Similar pattern of phosphorylation was observed in vitro using purified PKGI and PKGII. Inactivation of this phosphorylation site had little effect on retGC activation by purified expressed GCAP-2 in vitro. Conclusions: (1) GCAP-2 undergoes Ca-sensitive phosphorylation by retinal CNDPK and dephosphorylation by PP2C. (2) Consistently with the previous observations, the very C-terminus of GCAP-2, either phosphorylated or non-phosphorylated, has little effect on the GCAP-2 interaction with the cyclase. (3) The C-terminus in Ca-free GCAP-2 becomes exposed, and Ca2+ binding promotes structural changes that constrain its C-terminus within the protein structure. References: [1] Olshevskaya et al., J Biol Chem 274:10823-32, 1999, [2] Ames JB, et al., J Biol Chem 274:19329-37, 1999, [3] Olshevskaya E.V. et al., J Biol Chem 274:10823-32, 1999
Keywords: 515 phosphorylation • 527 protein structure/function • 334 calcium