The overall aim of this proposal is to determine the role of guanylyl cycle activating protein (GCAP) in olfactory signal transduction. GCAP is a calcium(CA/2+)-dependent regulator of retinal guanylyl cyclase (GC) activity in photoreceptors. In low Ca/2+, GCAP is activated to enhance GC and restore cGMP levels in rod outer segments. Our preliminary studies demonstrate that GCAP is expressed in olfactory receptor neurons (ORNs) and regulates particulate guanylyl cyclase (pGC) in a Ca/2+- dependent manner. We have recently identified two odorant-responsive pGCs. We hypothesize that GCAP regulates olfactory pGC in a Ca/2+- dependent manner. There are three objectives in this proposal.
Aim 1 : Identify Olfactory GCAP (oGCAP). Pilot studies using heterologous retinal GCAPs 1 and 2 studies have provided a framework for understanding GCAP function in olfaction (Moon et al., 2998). We will complete cloning studies to obtain oGCAP for homologous studies. Thus far, we know oGCAP is at least 80% related to retinal GCAP1, and that it is identified by anti-GCAP1 were used to identify clones from a rat olfactory cDNA library. We will complete sequencing and determine its distribution by Northern blot.
Aim 2 : Determine the Distribution of GCAP Insight into the function of a protein is gained from knowledge of its distribution and regeneration. pGC and GCAP are co-expressed in normal adult olfactory cilia. Immunohistochemical studies will be extended to embryonic and lesioned (olfactory bulbectomy (OBx) and chemical ablation of epithelium) models to evaluate a possible role in development and regeneration (in the presence and absence of target).
Aim 3 : Characterize the Regulation of Olfactory pGC by GCAP. The effect of oGCAP on the kinetic properties is the first step in understanding the role of OGCAP. Isolated rat olfactory cilia, shown by us to contain high levels of pGC (Moon et al., 1998), and a purified preparation of olfactory pGC are used. We will determine the effect of Ca/2+ and the effect of the presence/absence of GCAP on the K/m and V/max of the olfactory pGCs. These experiments will then serve as the basis for investigating the role of GCAP in the odorant-stimulated cGMP response.
