Calcium- and myristoyl-dependent properties of guanylate cyclase-activating protein-1 and protein-2.
In visual transduction, guanylate cyclase-activating proteins (GCAPs) activate the membrane-bound guanylate cyclase 1 (ROS-GC1) to synthesize cGMP under conditions of low cytoplasmic [Ca2+]free. GCAPs are neuronal Ca2+-binding proteins with three functional EF-hands and a consensus site for N-terminal myristoylation. GCAP-1 and GCAP-2 regulated ROS-GC1 activities differently. The myristoyl group in GCAP-1 had a strong influence on the Ca2+-dependent regulation of ROS-GC1 (shift in IC50). In contrast, myristoylation of GCAP-2 did not change the cyclase activation profile (no shift in IC50). Thus, the myristoyl group controlled the Ca2+-sensitivity of GCAP-1, but not that of GCAP-2. The myristoyl group restricted the accessibility of one cysteine in GCAP-1 and GCAP-2 observed by measuring the time-dependent thiol reactivity of cysteines. This shielding effect was not relieved when Ca2+ was buffered by EGTA. We applied surface plasmon resonance (SPR) spectroscopy to monitor the Ca2+-dependent binding of myristoylated and nonmyristoylated GCAP-1 and GCAP-2 to immobilized phospholipid membranes. None of the GCAPs exhibited a Ca2+-myristoyl switch as observed for recoverin. Thus, the myristoyl group controls the Ca2+-sensitivity of GCAP-1 (not that of GCAP-2) by an allosteric mechanism, but this control step does not involve a myristoyl switch.
Hwang
Ji-Young
JY
Institut für Biologische Informationsverarbeitung 1, Forschungszentrum Jülich, D-52425 Jülich, Germany.
Koch
Karl-Wilhelm
KW
D000818:Animals; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation; D005136:Eye Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000737:chemistry / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D051604:Guanylate Cyclase-Activating Proteins; D008051:Lipid Bilayers; D013438:Sulfhydryl Compounds; C098768:guanylyl cyclase-activating protein, bovine; D019814:Myristic Acid; D004228:Dithionitrobenzoic Acid; D006162:Guanylate Cyclase; D003545:Cysteine; D002118:Calcium