title: 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.
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Evgeni Yu
authors.initials: EY
authors.lastname: Zernii
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Ilya I
authors.initials: II
authors.lastname: Grigoriev
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Aliya A
authors.initials: AA
authors.lastname: Nazipova
authors.affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, Oldenburg, 26111 Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Scholten
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Tatiana V
authors.initials: TV
authors.lastname: Kolpakova
authors.affiliation: Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Dmitry V
authors.initials: DV
authors.lastname: Zinchenko
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Kazakov
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Ivan I
authors.initials: II
authors.lastname: Senin
authors.affiliation: Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region, 142290 Russia.
authors.forename: Sergei E
authors.initials: SE
authors.lastname: Permyakov
authors.affiliation: Department of Life Sciences and Reproduction, Section of Biological Chemistry and Center for BioMedical Computing, University of Verona, Verona, 37134 Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Dell'Orco
authors.affiliation: Department of Cell Signaling, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992 Russia.
authors.forename: Pavel P
authors.initials: PP
authors.lastname: Philippov
authors.affiliation: Department of Neurosciences, Biochemistry Group, University of Oldenburg, Oldenburg, 26111 Germany. Electronic address: karl.w.koch@uni-oldenburg.de.
authors.forename: Karl-W
authors.initials: KW
authors.lastname: Koch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002417:Cattle; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006162:Guanylate Cyclase / Q000235:genetics / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051603:Recoverin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D051604:Guanylate Cyclase-Activating Proteins; C067574:RCV1 protein, Bos taurus; D011993:Recombinant Fusion Proteins; D051603:Recoverin; D051606:G-Protein-Coupled Receptor Kinase 1; D006162:Guanylate Cyclase; D002118:Calcium
keywords: Guanylate cyclase-activating protein 2 (GCAP2); Neuronal calcium sensor (NCS) protein; Photoreceptors; Recoverin; Retinal guanylate cyclase (ROS-GC1); Rhodopsin kinase (RK, GRK1)