MEMBRANE TARGETING CALCIUM SENSORS IN VISION

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Intracellular calcium controls the excitability and desensitization of retinal rod and cone cells. Recoverin and guanylate cyclase activating proteins (GCAP-1 and GCAP-2) are Ca2+ sensing proteins in the retina that mediate Ca2+-dependent signaling in vision. Recoverin and GCAPs are new members of the EF-hand superfamily that includes calmodulin and troponin C. They contain a myristoyl or related fatty acyl group at their amino-terminus. Myristoylated, but not unmyristoylated, recoverin binds to cellular membranes at high Ca2+ concentration, but it does not bind to membranes at low Ca2+. Conversely, GCAPs bind membranes at low Ca2+ but do not bind membranes at high Ca2+. The recently determined structure of recoverin in solution indicates that Ca2+ induces extrusion of the amino-terminal myristoyl group to the exterior where it can interact with membrane targets. What structural determinants promote membrane localization of GCAPs? Does the amino-terminal myristoyl group insert directly into the lipid bilayer? The structures of Ca2+- bound GCAP-2, recoverin bound to membranes, and recoverin bound to its physiological target, rhodopsin kinase will be studied by nuclear magnetic resonance (NMR), x-ray crystallography, and spin-label electron spin resonance (ESR). Recoverin and GCAPs belong to a rapidly growing family of myristoylated calcium sensors. More than ten homologs of recoverin are found in the central nervous system, and may couple calcium cascades and G- protein cascades. The recoverin family is also found in invertebrates. Yeast contains a homolog that is 46 percent identical to mammalian recoverin. Structure and genetic studies of the yeast protein will be carried out to elucidate new physiological targets. The structures determined by this research will reveal how covalently attached myristoyl groups work in concert with Ca2+ binding sites to guide this family of proteins to membrane bound targets. These structures may also reveal insights into the membrane-targeting mechanisms of many other biologically important myristoylated proteins such as Src, oncogenic Ras, myristoylated alanine rich C-kinase substrates (MARCKS) and ADP ribosylation factors (ARFs).