INACTIVATION OF EF-HANDS MAKES GCAP-2 (P24) A CONSTITUTIVE ACTIVATOR OF PHOTORECEPTOR GUANYLYL CYCLASE BY PREVENTING A CA2-INDUCED ACTIVATOR-TO-INHIBITOR TRANSITION()

Guanylyl cyclase activator proteins GCAP-1 and GCAP-2 (Dizhoor et al, 1995, Gorczyca et al,, 1995) are members of a recently identified subc lass of EF-hand type Ca2+-binding proteins that respond to Ca2+ differ ently than any other known members of the EF-hand superfamily. GCAPs a cquire an activating conformation only in their Ca2+-free form. Free C a2+ concentrations corresponding to levels in dark-adapted vertebrate photoreceptors inhibit the ability of GCAPs to activate photoreceptor guanylyl cyclases (RetGCs). We studied the effects of mutations that b lock binding of Ca2+ to the EF-hands of GCAP-2. Unlike other EF-hand p roteins, which fail to activate their target when their EF-hands are i nactivated by mutations, GCAP-2 with any single EF-hand inactivated re mains active and is 3-6 times less sensitive to the inhibitory effect off Ca2+. Inactivation of any two or all three EF-hands produces activ e forms of GGAP-2 that are insensitive to inhibition by physiological intracellular concentrations of Ca2+. Unexpectedly we also found that activation of RetGCs by a Ca2+-insensitive mutant is inhibited by Ca2-loaded wild type GCAP-2. We propose the following, 1) GCAP-2 can exis t in two extreme functional forms: an apo form that activates RetGCs a nd a Ca2+-loaded form that blocks activation of RetGCs, 2) All three E F-hands of GCAP-2 contribute to the inhibitory effect of Ca2+. 3) Inac tivation of two off three EF-hands is sufficient to shift the ''activa tor-inhibitor'' transition outside the physiological range of intracel lular free Ca2+.