Length analyses of mammalian G-protein-coupled receptors.

G-protein-coupled receptors (GPCRs) play a crucial role in mediating effects of extracellular messengers in a wide variety of biological systems, comprising the largest gene superfamily at least in mammals. Mammalian GPCRs are broadly classified into three families based on pharmacological properties and sequence similarities. These sequence similarities are largely confined to the seven transmembrane domains, and much less in the extracellular and intracellular loops and terminals (LTs). Together with the fact that the LTs vary considerably in length and sequence, the LT length of GPCRs has not been studied systematically. Here we have applied a statistical analysis to the length of the LTs of a wide variety of mammalian GPCRs in order to examine the existence of any trends in molecular architecture among a known mammalian GPCR population. Tree diagrams constructed by cluster analyses, using eight length factors in a given GPCR, revealed possible length relations among GPCRs and defined at least three groups. Most samples in Group J (joined) and Group M (minor) had an exceptionally long N-terminal and I3 loop, respectively; and other samples were considered as Group O (other/original). This length-based classification largely coincided with the conventional sequence- and pharmacology-based classification, suggesting that the LT length contains some biological information when analysed at the population level. Principle component analyses suggested the existence of inherent length differences between loops and terminals as well as between extracellular and intracellular LTs. Wilcoxon rank transformation tests unveiled statistically significant differences between Group O and Group J, not only in the N-terminal and I3 loop, but also in the E3 loop. Correlation analyses identified an E1-I2 length-correlation in Group O and Group J and an N-E3 length-correlation in Group J. Taken together, these results suggest a possible functional importance of LT length in the GPCR superfamily.

Segregated expression of neurestin in the developing olfactory bulb.

Neurestin is a putative transmembrane protein whose expression is developmentally regulated in neurons. Here we examined neurestin expression pattern in mitral/tufted cells in the developing rat olfactory bulb. In the main olfactory bulb, neurestin expression was segregated in the dorso-rostral area and in the ventro-caudal area, but not in between. In the accessory olfactory bulb, neurestin expression was found only in the far caudal area. This area did not completely correspond to a caudal half of the vomeronasal nerve and glomerular layers positive for a G-protein Go alpha. These spatio-temporal expression patterns suggest that neurestin functions as a target recognition molecule that specifies zonal projection patterns of olfactory and vomeronasal sensory neurons.

Neurestin: putative transmembrane molecule implicated in neuronal development.

We have cloned a novel cDNA encoding a putative transmembrane protein, neurestin, from the rat olfactory bulb. Neurestin was identified based on a sequence similar to that of the second extracellular loops of odorant receptors in the cysteine-rich CC box located immediately after EGF-like motifs. Neurestin shows homology to a neuregulin gene product, human gamma-heregulin, a Drosophila receptor-type pair-rule gene product, Odd Oz (Odz) / Ten(m), and Ten(a), suggesting a possible function in synapse formation and morphogenesis. Recently, a mouse neurestin homolog has independently been cloned as DOC4 from the NIH-3T3 cell line. Northern blot analysis showed that neurestin is highly expressed in the brain and also in other tissues at much lower levels. In situ hybridization studies showed that neurestin is expressed in many types of neurons, including pyramidal cells in the cerebral cortex and tufted cells in the olfactory bulb during development. In adults, neurestin is mainly expressed in olfactory and hippocampal granule cells, which are known to be generated throughout adulthood. Nonetheless, in adults the expression of neurestin was experimentally induced in external tufted cells during regeneration of olfactory sensory neurons. These results suggest a role for neurestin in neuronal development and regeneration in the central nervous system.

Functional expression of a mammalian odorant receptor.

Candidate mammalian odorant receptors were first cloned some 6 years ago. The physiological function of these receptors in initiating transduction in olfactory receptor neurons remains to be established. Here, a recombinant adenovirus was used to drive expression of a particular receptor gene in an increased number of sensory neurons in the rat olfactory epithelium. Electrophysiological recording showed that increased expression of a single gene led to greater sensitivity to a small subset of odorants.

Color-pattern modifications of butterfly wings induced by transfusion and oxyanions.

The color-pattern determination of butterfly wings was studied, focusing on the cold-shock-induced color-pattern modifications of a species of butterfly, Vanessa (Cynthia) cardui (Lepidoptera: Nymphalidae). It was shown that the modification property could be transferred to the noncold-shocked individuals by the transfusion of hemolymph taken from the cold-shocked individuals, suggesting the existence of an unknown diffusible factor or hormone, induced or activated by the cold shock. The involvement of a receptor tyrosine kinase for the color-pattern modifications was tested by the simple application of some oxyanions such as sodium tungstate, sodium molybdate, and molybdic acid to pupae, since these oxyanions have been known to up-regulate the process of phosphorylation via receptor tyrosine kinases in general. It was shown that they could modify the wing color-pattern in a way very similar to the cold shock. Moreover, the topical applications of sodium tungstate or molybdic acid induced large ectopic black spots on the treated pupal wings. Among the treatment methods, the sodium tungstate treatment was by far more effective than the cold shock treatment itself. Taken together, these data suggest that an unknown cold-shock hormone activates the process of phosphorylation via a receptor tyrosine kinase necessary for the color-pattern development.

Adenovirus-mediated gene transfer in olfactory neurons in vivo.

We used recombinant adenoviruses as a means of expressing exogenous genes in olfactory neurons in vivo. A replication incompetent adenovirus (type 5, Ad5) carrying the reporter gene lacZ, which codes for the enzyme beta-galactosidase (beta-Gal), was applied in solution to the olfactory epithelia of rats. The expression of lacZ was controlled by the cytomegalovirus immediate-early promoter/enhancer. beta-Gal expression was observed 1 day postinfection and was maximal at 3-10 days, although it could be detected for at least 21 days postinfection. Expression patterns were heterogeneous, ranging from a few percent to over 25% of the cells in different regions of both turbinate and septal epithelium. Staining was stronger in the olfactory versus respiratory epithelia. In olfactory epithelium staining was almost entirely restricted to olfactory neurons. beta-Gal staining was also observed in the olfactory axons so that nerve bundles could be traced to their targets in the glomerular layer of the olfactory bulb. Intense staining of some glomeruli was evident as long as 21 days postinfection. There was no evidence of cell loss or tissue damage due to viral infection. These results demonstrate that it is possible to use recombinant Ad5 for expressing foreign genes in olfactory neurons. This technique could be used in olfactory neurons to increase expression levels of olfactory specific genes, including the odor receptor, putative guidance and growth molecules, or elements of the transduction cascade, in order to elucidate their biological functions in vivo.