Regulatory function of the C-terminal segment of guanylate cyclase-activating protein 2

Abstract

Neuronal responses to Ca2+-signals are provided by EF-hand-type neuronal Ca2+-sensor (NCS) proteins, which have similar core domains containing Ca2+-binding and target-recognizing sites. NCS proteins vary in functional specificity, probably depending on the structure and conformation of their non-conserved C-terminal segments. Here, we investigated the role of the C-terminal segment in guanylate cyclase activating protein-2, GCAP2, an NCS protein controlling the Ca2+-dependent regulation of photoreceptor guanylate cyclases. We obtained two chimeric proteins by exchanging C-terminal segments between GCAP2 and its photoreceptor homolog recoverin, a Ca2+-sensor controlling rhodopsin kinase (RK) activity. The exchange affected neither the structural integrity of GCAP2 and recoverin nor the Ca2+-sensitivity of GCAP2. Intrinsic fluorescence, circular dichroism, biochemical studies and hydrophobic dye probing revealed Ca2+-dependent conformational transition of the C-terminal segment of GCAP2 occurring in the molecular environment of both proteins. In Ca2+-GCAP2, the C-terminal segment was constrained and its replacement provided the protein with approximately two-fold inhibitory activity towards RK, suggesting that the segment contributes to specific target recognition by interfering with RK-binding. Upon Ca2+-release, it became less constrained and more available for phosphorylation by cyclic nucleotide-dependent protein kinase. The transition from the Ca2+-bound to the apo-state exposed hydrophobic sites in GCAP2, and was associated with its activating function without affecting its dimerization. The released C-terminal segment participated further in photoreceptor membrane binding making it sensitive to phosphorylation. Thus, the C-terminal segment in GCAP2 confers target selectivity, facilitates membrane binding and provides sensitivity of the membrane localization of the protein to phosphorylation by signaling kinases.

Keywords: Guanylate cyclase-activating protein 2 (GCAP2); Neuronal calcium sensor (NCS) protein; Photoreceptors; Recoverin; Retinal guanylate cyclase (ROS-GC1); Rhodopsin kinase (RK, GRK1).