Factors that Determine Ca super(2+) Sensitivity of Photoreceptor Guanylyl Cyclase. Kinetic Analysis of the Interaction between the Ca super(2+)-Bound and the Ca super(2+)-Free Guanylyl Cyclase Activating Proteins (GCAPs) and Recombinant Photoreceptor Guanylyl Cyclase 1 (RetGC-1)

• Published in: Biochemistry (Easton) 2004-11, Vol.43 (43), p.13796-13804
• Main Authors: Peshenko, I V, Moiseyev, G P, Olshevskaya, E V, Dizhoor, A M
• Format: Article
• Language: English
• ISSN: 0006-2960
• DOI: 10.1021/bi048943m

We explored the possibility that, in the regulation of an effector enzyme by a Ca super(2+)-sensor protein, the actual Ca super(2+) sensitivity of the effector enzyme can be determined not only by the affinity of the Ca super(2+)sensor protein for Ca super(2+) but also by the relative affinities of its Ca super(2+)-bound versus Ca super(2+)-free form for the effector enzyme. As a model, we used Ca super(2+)-sensitive activation of photoreceptor guanylyl cyclase (RetGC-1) by guanylyl cyclase activating proteins (GCAPs). A substitution Arg super(838)Ser in RetGC-1 found in human patients with cone-rod dystrophy is known to shift the Ca super(2+) sensitivity of RetGC-1 regulation by GCAP-1 to a higher Ca super(2+) range. We find that at physiological concentrations of Mg super(2+) this mutation increases the free Ca super(2+) concentration required for half-maximal inhibition of the cyclase from 0.27 to 0.61 mu M. Similar to rod outer segment cyclase, Ca super(2+) sensitivity of recombinant RetGC-1 is strongly affected by Mg super(2+), but the shift in Ca super(2+) sensitivity for the R838S mutant relative to the wild type is Mg super(2+)-independent. We determined the apparent affinity of the wild-type and the mutant RetGC-1 for both Ca super(2+)-bound and Ca super(2+)-free GCAP-1 and found that the net shift in Ca super(2+) sensitivity of the R838S RetGC-1 observed in vitro can arise predominantly from the change in the affinity of the mutant cyclase for the Ca super(2+)-free versus Ca super(2+)-loaded GCAP-1. Our findings confirm that the dynamic range for RetGC regulation by Ca super(2+)/GCAP is determined by both the affinity of GCAP for Ca super(2+) and relative affinities of the effector enzyme for the Ca super(2+)-free versus Ca super(2+)-loaded GCAP.