The role of calcium-binding sites in S-modulin function.
S-modulin controls rhodopsin phosphorylation in a calcium-dependent manner, and it has been suggested that it modulates the light sensitivity of the photoreceptor cell. S-modulin binds to the ROS membrane at high Ca2+ concentration, and N-terminal myristoylation is necessary for this property (the calcium-myristoyl switch). S-modulin has four EF-hand motifs, of which two (EF-2 and -3) are functional. Here, we report on the roles of EF-2 and -3 in S-modulin function (calcium binding, membrane association, and inhibition of rhodopsin phosphorylation) by site-directed mutants (E85M and E121M). Surprisingly, E121M, which has a mutation in EF-3, neither binds Ca2+ nor inhibits phosphorylation. In contrast, E85M binds one Ca2+ and has the same membrane affinity as wild-type S-modulin, but has lost the ability to inhibit rhodopsin phosphorylation. It is suggested that the binding of Ca2+ to EF-3 is probably required for EF-2 to be a functional Ca2+-binding site and to induce exposure of the myristoyl group; and that the binding of Ca2+ to EF-2 is important for the interaction with rhodopsin kinase.
Matsuda
S
S
Department of Earth and Space Science, Graduate School of Science, Osaka University, Osaka 560-0043, Japan.
Hisatomi
O
O
Ishino
T
T
Kobayashi
Y
Y
Tokunaga
F
F
D001665:Binding Sites / Q000502:physiology; D002118:Calcium / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D005136:Eye Proteins; D051606:G-Protein-Coupled Receptor Kinase 1; D051597:Hippocalcin; D008074:Lipoproteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D019814:Myristic Acid / Q000378:metabolism; D009419:Nerve Tissue Proteins; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D051603:Recoverin; D012243:Rhodopsin / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000502:physiology; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D005136:Eye Proteins; D008074:Lipoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D019814:Myristic Acid; D051603:Recoverin; D051597:Hippocalcin; D012243:Rhodopsin; D011494:Protein Kinases; D051606:G-Protein-Coupled Receptor Kinase 1; D002118:Calcium