Activation of retinal guanylyl cyclase-1 by Ca2+-binding proteins involves its dimerization.
Retinal guanylyl cyclase-1 (retGC-1), a key enzyme in phototransduction, is activated by guanylyl cyclase-activating proteins (GCAPs) if [Ca2+] is less than 300 nM. The activation is believed to be essential for the recovery of photoreceptors to the dark state; however, the molecular mechanism of the activation is unknown. Here, we report that dimerization of retGC-1 is involved in its activation by GCAPs. The GC activity and the formation of a 210-kDa cross-linked product of retGC-1 were monitored in bovine rod outer segment homogenates, GCAPs-free bovine rod outer segment membranes and recombinant bovine retGC-1 expressed in COS-7 cells. In addition to recombinant bovine GCAPs, constitutively active mutants of GCAPs that activate retGC-1 in a [Ca2+]-independent manner and bovine brain S100b that activates retGC-1 in the presence of approximately 10 microM [Ca2+] were used to investigate whether these activations take place through a similar mechanism, and whether [Ca2+] is directly involved in the dimerization. We found that a monomeric form of retGC-1 ( approximately 110 kDa) was mainly observed whenever GC activity was at basal or low levels. However, the 210-kDa product was increased whenever the GC activity was stimulated by any Ca2+-binding proteins used. We also found that [Ca2+] did not directly regulate the formation of the 210-kDa product. The 210-kDa product was detected in a purified GC preparation and did not contain GCAPs even when the formation of the 210-kDa product was stimulated by GCAPs. These data strongly suggest that the 210-kDa cross-linked product is a homodimer of retGC-1. We conclude that inactive retGC-1 is predominantly a monomeric form, and that dimerization of retGC-1 may be an essential step for its activation by active forms of GCAPs.
Yu
H
H
Kresge Eye Institute, Wayne State University, School of Medicine, Detroit, Michigan 48201, USA.
Olshevskaya
E
E
Duda
T
T
Seno
K
K
Hayashi
F
F
Sharma
R K
RK
Dizhoor
A M
AM
Yamazaki
A
A
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D002417:Cattle; D003432:Cross-Linking Reagents; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins; D009154:Mutation; D009414:Nerve Growth Factors / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012160:Retina / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014162:Transfection; D014785:Vision, Ocular
D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; D051604:Guanylate Cyclase-Activating Proteins; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D006162:Guanylate Cyclase; D002118:Calcium