Changes in biological activity and folding of guanylate cyclase-activating protein 1 as a function of calcium.
Guanylate cyclase-activating protein 1 (GCAP1), a photoreceptor-specific Ca2+-binding protein, activates retinal guanylate cyclase 1 (GC1) during the recovery phase of phototransduction. In contrast to other Ca2+-binding proteins from the calmodulin superfamily, the Ca2+-free form of GCAP1 stimulates the effector enzyme. In this study, we analyzed the Ca2+-dependent changes in GCAP1 structure by limited proteolysis and mutagenesis in order to understand the mechanism of Ca2+-sensitive modulation of GC1 activity. The change from a Ca2+-bound to a Ca2+-free form of GCAP1 increased susceptibility of Ca2+-free GCAP1 to proteolysis by trypsin. Sequencing data revealed that in the Ca2+-bound form, only the N-terminus (myristoylated Gly2-Lys9) and C-terminus (171-205 fragment) of GCAP1 are removed by trypsin, while in the Ca2+-free form, GCAP1 is readily degraded to small fragments. Successive inactivation of each of the functional EF loops by site-directed mutagenesis showed that only EF3 and EF4 contribute to a Ca2+-dependent inactivation of GCAP1. GCAP1(E75D,E111D,E155D) mutant did not bind Ca2+ and stimulated GC1 in a [Ca2+]-independent manner. GCAP1 and GCAP2, but not S-100beta, a high [Ca2+]free activator of GC1, competed with the triple mutant at high [Ca2+]free, inhibiting GC1 with similar IC50's. These competition results are consistent with comparable affinities between GC1 and GCAPs. Our data suggest that GCAP1 undergoes major conformational changes during Ca2+ binding and that EF3 and EF4 motifs are responsible for changes in the GCAP1 structure that converts this protein from the activator to the inhibitor of GC1.
Rudnicka-Nawrot
M
M
Department of Ophthalmology, School of Medicine, University of Washington, Seattle 98195, USA.
Surgucheva
I
I
Hulmes
J D
JD
Haeseleer
F
F
Sokal
I
I
Crabb
J W
JW
Baehr
W
W
Palczewski
K
K
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D002417:Cattle; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010786:Photoreceptor Cells / Q000201:enzymology; D017510:Protein Folding; D009418:S100 Proteins / Q000494:pharmacology; D014357:Trypsin
D002135:Calcium-Binding Proteins; D051604:Guanylate Cyclase-Activating Proteins; D009418:S100 Proteins; D014357:Trypsin; D006162:Guanylate Cyclase; D002118:Calcium