Distinct ONE-GC transduction modes and motifs of the odorants: Uroguanylin and CO2

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Abstract

In a subset of the olfactory sensory neurons ONE-GC$ membrane guanylate cyclase is a central component of two odorant-dependent cyclic GMP signaling pathways. These odorants are uroguanylin and CO2. The present study was designed to decipher the biochemical and molecular differences between these two odorant signaling mechanisms. The study shows (1) in contrast to uroguanylin, CO2 transduction mechanism is Ca2+-independent. (2) CO2 transduction site, like that of uroguanylin-neurocalcin δ, resides in the core catalytic domain, aa 880–1028, of ONE-GC. (3) The site, however, does not overlap the signature neurocalcin δ signal transduction domain, 908LSEPIE913. Finally, (4) this study negates the prevailing concept that CO2 uniquely signals ONE-GC activity (Sun et al. [19]; Guo et al. [21]). It demonstrates that it also signals the activation of photoreceptor membrane guanylate cyclase ROS-GC1. These results show an additional new transduction mechanism of the membrane guanylate cyclases and broaden our understanding of the molecular mechanisms by which different odorants using a single guanylate cyclase can regulate diverse cyclic GMP signaling pathways.

Introduction

The odorant signal begins at the ciliated apical border of the olfactory sensory neurons located in the main olfactory epithelium. To be transmitted to the defined cortical neurons of the brain where the final perception of SMELL is completed, in the first step the odorant generates an electrical signal. The biochemical term for this process is odorant-transduction [1], [2], [3], [4]. In this process the odorant signal is first converted to its second messenger and, then, the second messenger transforms the signal into an electrical signal. This, then, through action potentials becomes a means of signal transmission and the final perception of SMELL in the cortical layers of the brain.

The major second messenger of the odorant signal is cyclic AMP [5], [6], [7], [1], [8]. In the incremental development of the field, beginning in 1995 [9], [10], [11], it has now been finally established that cyclic GMP is also the second messenger of the odorant signal [2], [3], [4]. This signaling pathway resides in a small population of the olfactory receptor neurons (ORNs) and is independent of the cyclic AMP signaling pathway [10], [11]. The pathway begins with the ONE-GC membrane guanylate cyclase (also named GC-D [9]) which is co-present with the cyclic GMP-dependent components, cyclic GMP-specific cyclic nucleotide-gated channel subunit, CNGA3, and a cyclic GMP-dependent phosphodiesterase, PDE2 [10], [11] thus, it appears to be linked with them for its down stream functions.

It is important to understand that the modi operandi of these two pathways are radically different. In contrast to the cyclic AMP, cyclic GMP pathway does not function through the GTP-binding protein, Golf. It directly originates from ONE-GC, which is both the receptor for the odorants uroguanylin [12], [13] and green pepper [14], [15], and also the transducer through its guanylate cyclase activity. Thus, on the lines of the prototype model of ANF-RGC membrane guanylate cyclase signal transduction [16], coexistence of the uroguanylin receptor and guanylate cyclase activities on a single transmembrane spanning polypeptide chain demonstrates that the mechanism of signal transduction involving mediation by the second messenger, cyclic GMP, is different from the adenylate cyclase system. The single polypeptide chain of ONE-GC contains both the information for signal recognition and its translation into a second messenger. This makes the cyclic GMP signal transduction pathway more direct and, theoretically faster.

ONE-GC in addition to being a direct odorant receptor and transducer possesses an additional intriguing feature. Indirectly, through carbonic anhydrase enzyme, it senses atmospheric CO2 and gets accelerated in its production of cyclic GMP [17], [18]. Fragmentary evidence suggests that the direct and the indirect odorant signal transduction mechanisms of ONE-GC are different [17], [18]. This presentation investigates this problem and demonstrates the differences at the functional and structural levels.

Section snippets

Materials and methods

ONE-GC tm-catd mutant. Construction of the mutant is described in [13] and the mutant is schematically presented in Fig. 1.

ONE-GCΔ908LSEPIE913 mutant. Deletion of the 908LSEPIE913 motif from the ONE-GC sequence was done using Quick Change mutagenesis kit (Stratagene) and mutagenic primers: forward primer 5′-GGCTTTACCACCATCTCAGCCGTGTGGTGGGCTTCCTCAATGAT-3′; reverse primer 5′-ATCATTGAGGAAGCCCACCACGGCTGAGATGGTGGTAAAGCC-3′. The deletion was verified by sequencing. The mutant is schematically

Bicarbonate stimulates ONE-GC in a Ca2+-independent manner

Bicarbonate, the mediator of the odorant atmospheric CO2, stimulates ONE-GC activity [17], [18], [21]. The stimulation is through the intracellular domain of ONE-GC [18], [21]. This situation is similar to the uroguanylin-neurocalcin δ-modulated Ca2+-signaling step of the ONE-GC activation, which also occurs at the intracellular domain of ONE-GC [22]. To determine whether the bicarbonate stimulation of ONE-GC requires Ca2+, ONE-GC was expressed in the heterologous COS cell system and their

Discussion

This study contributes to our understanding of the emerging field of membrane guanylate cyclase linked with the odorant signaling. It demonstrates that bicarbonate, a mediator of the odorant atmospheric CO2 signaling of ONE-GC is independent of Ca2+. This makes ONE-GC a unique member of the membrane guanylate cyclase family because it is the only one that can, through distinct modes, transduce multiple, at least three, odorant signals into the production of their second messenger cyclic GMP.

Acknowledgment

This study was supported by NIH awards DC 005349 (R.K.S.) and HL 084584 (T.D.).

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