Third calcium-modulated rod outer segment membrane guanylate cyclase transduction mechanism.
Ca2+-modulated rod outer segment membrane guanylate cyclase (ROS-GC1) has been cloned and reconstituted to show that it is regulated by two processes: one inhibitory, the other stimulatory. The inhibitory process is consistent with its linkage to phototransduction; the physiology of the stimulatory process is probably linked to neuronal transmission. In both regulatory processes, calcium modulation of the cyclase takes place through the calcium binding proteins; guanylate cyclase activating proteins (GCAP1 and GCAP2) in the case of the phototransduction process and calcium-dependent GCAP (CD-GCAP) in the case of the stimulatory process. The cyclase domains involved in the two processes are located at two different sites on the ROS-GC1 intracellular region. The GCAP1-modulated domain resides within the aa 447-730 segment of ROS-GC1 and the CD-GCAP-modulated domain resides within the aa 731-1054 segment. In the present study the GCAP2-dependent Ca2+ modulation of the cyclase activity has been reconstituted using recombinant forms of GCAP2 and ROS-GC1, and its mutants. The results indicate that consistent to phototransduction, GCAP2 at low Ca2+ concentration (10 nM) maximally stimulates the cyclase activity of the wild-type and its mutants: ext (deleted aa 8-408), kin (deleted aa 447-730) and hybrid consisting of the ext, transmembrane and kin domains of ANF-RGC and the C-terminal domain, aa 731-1054, of ROS-GC1. In all cases, it inhibits the cyclase activity with an IC50 of about 140 nM. A previous study has shown that under identical conditions the kin and the hybrid mutant are at best only minimally stimulated. Thus, the GCAP1 and GCAP2 signal transduction mechanisms are different, occurring through different modules of ROS-GC1. These findings also demonstrate that the intracellular region of ROS-GC1 is composed of multiple modules, each designed to mediate a particular calcium-specific signalling pathway.
Krishnan
A
A
Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford 08084, USA.
Goraczniak
R M
RM
Duda
T
T
Sharma
R K
RK
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D011956:Receptors, Cell Surface; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D014785:Vision, Ocular
D011956:Receptors, Cell Surface; C110715:guanylate cyclase 1; D006160:Guanosine Triphosphate; D006162:Guanylate Cyclase; D006152:Cyclic GMP; D002118:Calcium