Hippocalcin, new Ca(2+) sensor of a ROS-GC subfamily member, ONE-GC, membrane guanylate cyclase transduction system.

Hippocalcin is a member of the neuronal Ca(2+) sensor protein family. Among its many biochemical functions, its established physiological function is that via neuronal apoptosis inhibitory protein it protects the neurons from Ca(2+)-induced cell death. The precise biochemical mechanism/s, through which hippocalcin functions, is not clear. In the present study, a new mechanism by which it functions is defined. The bovine form of hippocalcin (BovHpca) native to the hippocampus has been purified, sequenced, cloned, and studied. The findings show that there is the evolutionary conservation of its structure. It is a Ca(2+)-sensor of a variant form of the ROS-GC subfamily of membrane guanylate cyclases, ONE-GC. It senses physiological increments of Ca(2+) with a K(1/2) of 0.5 microM and stimulates ONE-GC or ONE-GC-like membrane guanylate cyclase. The Hpca-modulated ONE-GC-like transduction system exists in the hippocampal neurons. And hippocalcin-modulated ONE-GC transduction system exists in the olfactory receptor neuroepithelium. The Hpca-gene knock out studies demonstrate that the portion of this is about 30% of the total membrane guanylate cyclase transduction system. The findings establish Hpca as a new Ca(2+) sensor modulator of the ROS-GC membrane guanylate cyclase transduction subfamily. They support the concept on universality of the presence and operation of the ROS-GC transduction system in the sensory and sensory-linked neurons. They validate that the ROS-GC transduction system exists in multiple forms. And they provide an additional mechanism by which ROS-GC subfamily acts as a transducer of the Ca(2+) signals originating in the neurons.

High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate.

Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca2+-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca2+/myristoyl switch that allows their reversible membrane association in response to Ca2+ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K+ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] with high affinity (K(d) 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P2. Co-expression of hippocalcin-(1-14)-ECFP (enhanced cyan fluorescent protein) or NCS-1-ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase delta1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P2 than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P2.