The use of gene deletion by homologous recombination to determine gene or protein function has wide application in vertebrate neurobiology. An ideal complement to gene deletion would be subsequent gene replacement to demonstrate re-acquisition of function. Here we used an adenoviral vector to replace the olfactory marker protein (OMP) gene in olfactory receptor neurons of adult OMP-null mice and demonstrated the subsequent re-acquisition of function. Our results show that short-term expression of OMP restores the kinetics of electrophysiological responses of OMP-null mice to those of the control phenotype. This adenoviral-mediated rescue of the OMP-null phenotype is consistent with involvement of OMP in olfactory transduction.
The olfactory cyclic nucleotide-gated channel subunit 1 (OCNC1) is required for signal transduction in olfactory receptor cells. To further investigate the role of this channel in the olfactory system, the biochemical and morphological consequences of targeted disruption of OCNC1 were investigated in adult mice. Null as compared to wild-type mice had smaller olfactory bulbs, suggesting compromised development of the central target of the receptor cells. Ectopic olfactory marker protein (OMP)-stained fibers localized to the external plexiform layer reflected the relative immaturity of the olfactory bulb in the null mice. The olfactory epithelium of the knock-out mouse was thinner and showed lower expression of olfactory marker protein and growth-associated protein 43, indicating decreases in both generation and maturation of receptor cells. Tyrosine hydroxylase (TH) expression in the olfactory bulb, examined as a reflection of afferent activity, was reduced in the majority of periglomerular neurons but retained in atypical or "necklace" glomeruli localized to posterior aspects of the olfactory bulb. Double label studies demonstrated that the remaining TH-immunostained neurons received their innervation from a subset of receptor cells previously shown to express a phosphodiesterase that differs from that found in most receptor cells. These data indicate that expression of OCNC1 is required for normal development of the olfactory epithelium and olfactory bulb. The robust expression of TH in some periglomerular cells in the OCNC1-null mice suggests that receptor cells innervating these glomeruli may use an alternate signal transduction pathway.
Olfactory marker protein (OMP) participates in the olfactory signal transduction pathway. This is evident from the behavioral and electrophysiological deficits of OMP-null mice, which can be reversed by intranasal infection of olfactory sensory neurons with an OMP-expressing adenovirus. Bex, brain expressed X-linked protein, has been identified as a protein that interacts with OMP. We have now further characterized the interaction of OMP and Bex1/2 by in vitro binding assays and by immuno-coprecipitation experiments. OMP is a 19 kDa protein but these immunoprecipitation studies have revealed the unexpected presence of a 38 kDa band in addition to the expected 19 kDa band. Furthermore, the 38 kDa form was preferentially co-immunoprecipitated with Bex from cell extracts. In-gel tryptic digestion, mass spectrometry, and two-dimensional gel electrophoresis indicate that the 38 kDa protein behaves as a covalently cross-linked OMP-homodimer. The 38 kDa band was also identified in western blots of olfactory epithelium demonstrating its presence in vivo. The stabilities and subcellular localizations of the OMP-monomer and -dimer were studied in transfected cells. These results demonstrated that the OMP-dimer is much less stable than the monomer, and that while the monomer is present both in the nuclear and cytosolic compartments, the dimer is preferentially located in a Triton X-100 insoluble cytoskeletal fraction. These novel observations led us to hypothesize that regulation of the level of the rapidly turning-over OMP-dimer and its interaction with Bex1/2 is critical for OMP function in sensory transduction.
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