Ca2+-dependent conformational changes in guanylyl cyclase-activating protein 2 (GCAP-2) revealed by site-specific phosphorylation and partial proteolysis

Abstract

Guanylyl cyclase-activating proteins (GCAPs) are calcium sensor proteins of the EF-hand superfamily that inhibit retinal photoreceptor membrane guanylyl cyclase (retGC) in the dark when they bind Ca2+ but activate retGC when Ca2+ dissociates from GCAPs in response to light stimulus. We addressed the difference in exposure of GCAP-2 structure to protein kinase and a protease as indicators of conformational change caused by binding and release of Ca2+. We have found that unlike its homolog, GCAP-1, the C terminus of GCAP-2 undergoes phosphorylation by cyclic nucleotide-dependent protein kinases (CNDPK) present in the retinal extract and rapid dephosphorylation by the protein phosphatase PP2C present in the retina. Inactivation of the CNDPK phosphorylation site in GCAP-2 by substitutions S201G or S201D, as well as phosphorylation or thiophosphorylation of Ser 201, had little effect on the ability of GCAP-2 to regulate retGC in reconstituted membranes in vitro. At the same time, Ca2+ strongly inhibited phosphorylation of the wild-type GCAP-2 by retinal CNDPK but did not affect phosphorylation of a constitutively active Ca2+-insensitive GCAP-2 mutant. Partial digestion of purified GCAP-2 with Glu-C protease revealed at least two sites that become exposed or constrained in a Ca 2+-sensitive manner. The Ca2+-dependent conformational changes in GCAP-2 affect the areas around Glu62 residue in the entering helix of EF-hand 2, the areas proximal to the exiting helix of EF-hand 3, and Glu136-Glu 138 between EF-hand 3 and EF-hand 4. These changes also cause the release of the C-terminal Ser201 from the constraint caused by the Ca2+-bound conformation.