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Ca2+-modulated ONE-GC odorant signal transduction
FEBS Letters 583 (2009) 1327–1330
journal homepage: www.FEBSLetters.org
Ca2+-modulated ONE-GC odorant signal transduction Teresa Duda *, Rameshwar K. Sharma * Research Divisions of Biochemistry and Molecular Biology, The Unit of Regulatory and Molecular Biology, Salus University, Elkins Park, PA 19027, United States
a r t i c l e
i n f o
Article history: Received 11 February 2009 Revised 14 March 2009 Accepted 17 March 2009 Available online 22 March 2009 Edited by Ned Mantei Keywords: Cyclic GMP ONE-GC membrane guanylate cyclase Odorant signal transduction
a b s t r a c t In a subset of olfactory epithelium the odorant receptor guanylate cyclase, ONE-GC, is a central transduction component of the cyclic GMP signaling pathway. The odorant binds to the extracellular domain and activates its intracellular catalytic domain to generate the odorant second messenger, cyclic GMP. The present study demonstrates that it is a two-step, Ca2+-independent and Ca2+-dependent, sequential process. In step one, the odorant, uroguanylin, binds ONE-GC and primes it for stimulation. In step two, Ca2+-bound neurocalcin d binds to the defined intracellular domain and saturates ONE-GC activity. A prototype model is proposed that depicts this signal transduction process. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The biochemical process by which an odorant signal generates an electric signal is termed odorant-transduction (reviewed in [1–3]). The site of odorant-transduction is the ciliated apical border of sensory neurons located in the olfactory neuroepithelium [3]. Besides the presence of the major cyclic AMP transduction pathway, a small population of olfactory receptor neurons (ORN) contains an odorant-responsive cyclic GMP signaling pathway [4–6]. These two pathways do not overlap and are operationally independent. The mechanisms of these pathways are also 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 [also known as GC-D (4)], which is both the odorant uroguanylin [6,7], green pepper [8], and possibly also CO2 [9] receptor, and a guanylate cyclase. Thus, on the lines of the prototype model of ANF-RGC membrane guanylate cyclase signal transduction [10], 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 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
* Corresponding authors. Fax: +1 215 780 3125 (R.K. Sharma). E-mail addresses: [email protected] (T. Duda), [email protected] (R.K. Sharma).
makes the cyclic GMP signal transduction pathway more direct and, theoretically faster. The immediate issue is: what is the mechanism of the uroguanylin signal transduction? How does the signal generate its second messenger, cyclic GMP; and, how cyclic GMP, in turn, generates the electric signal? Related to the first part of the issue, the available information is that ONE-GC in ORN is modulated through multiple modes within the physiological levels of [Ca2+]i. Its sensors, neurocalcin d [5,8], GCAP1 [11] and hippocalcin [12], operate within the [Ca2+]i of 500–800 nM and begin to detect the signals within 50–100 nM range. This signal transduction mechanism modulated by the Ca2+sensor neurocalcin d is unique [7]. Other Ca2+ sensors—GCAP1-, GCAP2- and S100B-linked ROS-GC transduction systems work through the ROS-GC domains that do not overlap with the core catalytic domain of the guanylate cyclase. However, the neurocalcin d signaling site in ONE-GC resides directly on its catalytic domain [7]. Given the sequential events that uroguanylin signal begins at the extracellular domain of ONE-GC [7], that there is further [Ca2+]i-dependent ONE-GC modulation [8], that it opens the cyclic GMP-gated channels [6] and finally depolarizes the ciliary membranes [6], these cascade of events need to be individually dissected to fully comprehend the odorant-transduction process. The present study is a step in this direction. It shows an intriguing [Ca2+]i-modulated transduction phenomenon for the production of cyclic GMP, the second messenger of the odorant. The mechanistic model for this phenomenon has been proposed.
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.03.036
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2. Materials and methods 2.1. Membrane preparations Rat olfactory neuroepithelium was homogenized in a buffer consisting of 250 mM sucrose, 10 mM Tris–HCl pH 7.4, 1 mM EGTA, containing a protease inhibitors cocktail, centrifuged at 1000 g and then at 100 000 g to pellet the membrane fraction. This fraction was suspended in 50 mM Tris–HCl pH 7.4/10 mM MgCl2 buffer and used for guanylate cyclase activity assay [5,7,8]. The membranes were devoid of endogenous neurocalcin d as verified by Western blot (data not shown). In preincubation experiments the homogenate was incubated on ice-bath for 10 min with 10 6 M uroguanylin; in parallel, mock preincubation (without uroguanylin) was carried out as a control. Following the incubation, the membrane fraction was prepared. Under these conditions no membrane vesicles are formed; the guanylate cyclase with some lipids attached to the transmembrane domain is present in the suspension. Identical protocol was used for COS-7 cells expressing ONE-GC, except that 5000 g pellet represented the membrane fraction. 3. Results and discussion 3.1. Uroguanylin signal transduction Background and strategy: ONE-GC is a cross-over member of the two subfamilies of membrane guanylate cyclases [7]. Because it is the receptor for uroguanylin, it belongs to the surface receptor subfamily; and because it is modulated by [Ca2+]i, it is also a member of the ROS-GC subfamily. Thus, three modes of its regulation were envisioned: (1) it behaves as a typical surface receptor guanylate cyclase; (2) it behaves as a typical ROS-GC guanylate cyclase; and (3) being a hybrid, it acquires attributes of both these subfamilies. These issues were analyzed through the following protocol. 3.1.1. The surface receptor guanylate cyclase model is partially applicable to ONE-GC The prototype surface receptor model is that of ANF-RGC [10,13,14]. In this model, uroguanylin will signal ONE-GC directly in a Ca2+-independent fashion.
To test this model, freshly prepared membranes of the olfactory neuroepithelium were individually exposed to 10 12 to 10 6 M uroguanylin in a Ca2+-free incubation buffer. Uroguanylin stimulated ONE-GC in a dose-dependent fashion with an EC50 of 20 pM, (Fig. 1A: closed circles). The stimulation was about 3.5-fold, and enzyme saturation occurred at 10 nM. To determine the influence of Ca2+ on the uroguanylin-modulated ONE-GC activity, parallel experiment was conducted in the presence of 10 lM Ca2+. The results obtained were almost identical (Fig. 1A: open circles) and in agreement with those obtained previously with the heterologous system of COS cells where the only signaling components were uroguanylin and ONE-GC [7]. These results demonstrate that uroguanylin signals ONE-GC, this signaling is Ca2+-independent and results in partial, three to fourfold, acceleration of ONE-GC activity. With the prior evidence that uroguanylin functions through the extracellular domain of ONE-GC [7] it is concluded that the ANF-RGC model is only partially applicable to ONE-GC because it is also modulated by [Ca2+]i. 3.1.2. The ROS-GC1 model also is partially applicable to ONE-GC The prototype ROS-GC1 signal transduction functions in two modes. In mode 1, operative in the rod/cone outer segments, Ca2+ through Ca2+-sensor GCAPs inhibits ROS-GC1 [15,16]. In mode 2, operative in the photoreceptor-bipolar synapse region and in the ganglion cell layer, elevations of [Ca2+]i are sensed by S100B and neurocalcin d and stimulate ROS-GC1 activity [17,18]. Thus, ROSGC1 is a bimodal Ca2+ signal transduction switch, both inhibited and stimulated by Ca2+. Prior evidence has indicated that ONE-GC transduction model does not follow the typical attributes of the ROS-GC1 model: it is only stimulated by Ca2+ signals. Rises in [Ca2+]i are sensed by neurocalcin d, GCAP1 and hippocalcin, and they, in turn, stimulate ONE-GC [5,11,12]. To assess applicability of the ROS-GC model to ONE-GC, freshly prepared membranes of the olfactory neuroepithelium were exposed to the incremental concentrations of neurocalcin d and 10 lM Ca2+. Neurocalcin d stimulated ONE-GC in a dose-dependent fashion with an EC50 of 0.7 lM (Fig. 1B). The overall stimulation was about 3.7-fold, and the enzyme saturation occurred at 2 lM. These results demonstrate that the ROS-GC1 model is also not fully applicable to the ONE-GC signal transduction. Importantly, the results also demonstrate that the Ca2+ signaling of ONE-GC is
Fig. 1. ONE-GC activity. Membranes of rat olfactory neuroepithelium were assayed for guanylate cyclase activity in the presence of (A) increasing concentrations of uroguanylin in the absence (closed circles) and presence (open circles) of 10 lM Ca2+ and (B) increasing concentrations of neurocalcin d and 10 lM Ca2+. The experiment was done in triplicate and repeated three times with separate membrane preparations. The results are mean ± S.E. from these experiments.
T. Duda, R.K. Sharma / FEBS Letters 583 (2009) 1327–1330
separate from uroguanylin signaling; and this step, like the uroguanylin step, stimulates ONE-GC only partially, 3.7-fold. 3.1.3. The odorant, uroguanylin, signal primes ONE-GC for the Ca2+ modulation of ONE-GC Intuitively, to bring the above biochemical evidences within the physiological perspective of odorant signal transduction in the production of its second messenger cyclic GMP, following sequence of events was envisioned. The Ca2+-indepndence of the uroguanylin signal matches the physiology of odorant signal transduction. Prior to its origin, in resting state the [Ca2+]i level in an ORN is within a range of 50– 100 nM. In this environment, the intracellular domain of ONE-GC is bound to neurocalcin d and this defines the basal state of ONEGC. The Ca2+-independent uroguanylin-dependent events cause primary activation of ONE-GC. And this activation is 3.5-fold. This level of cyclic GMP production may not be sufficient to explain the physiological cyclic GMP-dependent forward steps: opening of the cyclic GMP-gated channels, resulting in the inward flux of Ca2+ and depolarization of the ORN plasma membrane. To solve this puzzle, experimentation was initiated with the olfactory neuroepithelium homogenates. Freshly isolated rat olfactory neuroepithelium was homogenized under the Ca2+-depleted conditions (1 mM EGTA). The homogenate was first pre-incubated with 10 6 M uroguanylin and then the membranes were tested for the guanylate cyclase activity in presence of the increasing concentrations of neurocalcin d at 10 lM Ca2+. Control experiment was performed identically, except the homogenate was pre-incubated, without uroguanylin. The basal guanylate cyclase activity was 18 pmol cyclic GMP/ min/mg protein (Fig. 2). In the presence of increasing concentrations of neurocalcin d the ONE-GC activity of mock-preincubated membranes increased by about 3.5-fold (Fig. 2: open circles). The picture, however, was different for membranes pre-incubated with uroguanylin. Here the neurocalcin d dose-dependent Ca2+ signaling of ONE-GC resulted in more than 15-fold stimulation of ONE-GC activity, from 18 to 275 pmol cyclic GMP/min/mg protein (Fig. 2: closed circles); EC50 of neurocalcin d was 0.7 lM, it saturated the enzyme activity at 2 lM.
Fig. 2. Synergetic effect of uroguanylin and neurocalcin d on ONE-GC activity. Rat olfactory neuroepithelium was homogenized in the absence of Ca2+, preincubated with 10 6 M uroguanylin, the membrane fraction was prepared, and assayed for guanylate cyclase activity in the presence of 10 lM Ca2+ and increasing concentrations of neurocalcin d. Membranes from mock-preincubated homogenates were treated as controls. The experiment was done in triplicate and repeated three times with separate homogenates. The results are mean ± S.E. from these experiments.
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This indicates that uroguanylin signaling of ONE-GC in production of cyclic GMP is a two-step process. In the Ca2+-independent step it brings the ONE-GC’s catalytic module to a modest activity (below its full catalytic capacity) and also primes ONE-GC for the neurocalcin d/Ca2+-dependent step. Only after the last modulation occurs, ONE-GC achieves its full catalytic activity. The combined effects of uroguanylin and neurocalcin d far exceed the sum of their individual effects; they are not additive but synergetic. Does the two-step signal transduction besides uroguanylin, Ca2+ and ONE-GC involve additional components present in the olfactory neuroepithelium? To answer this question, the system of COS cells expressing ONE-GC was used. Membranes of these cells were preincubated with 10 6 M uroguanylin, washed and assayed for neurocalcin ddependent guanylate cyclase activity. The control consisted of membranes pre-incubated without uroguanylin. Like the pre-incubated neuroepithelial membranes, the neurocalcin d-modulated Ca2+ signaling of ONE-GC was also spectacular in the COS cell membranes (Fig. 3: closed circles). The kinetic parameters of ONE-GC activation were very similar in both systems. Neurocalcin d stimulated ONE-GC activity 12-fold with an EC50 of 0.7 lM and saturation at 2 lM. Without pre-incubation, the stimulation was only fourfold (Fig. 3: open circles). These results validate the two-step uroguanylin signal transduction model of ONE-GC. In addition they demonstrate that besides uroguanylin, Ca2+, and neurocalcin d no other olfactory transduction-specific component is involved in the ONE-GC transduction mechanism in production of the second messenger cyclic GMP. 3.1.4. The odorant uroguanyline signal transduction model In line with the present and past findings, an odorant uroguanylin signal transduction model in the production of its second messenger cyclic GMP is being presented. The model will serve as a useful template for future experimentation. A small population of ORNs contains a cyclic GMP signal transduction pathway [4–6]. This pathway resides at the apical region of the cilia [4–6]. Present in the region is ONE-GC membrane guanylate cyclase. Its outer domain is a receptor for uroguanylin [7]. In its inner domain, at the C-terminus, resides the catalytic domain
Fig. 3. Synergetic effect of uroguanylin and neurocalcin d on recombinant ONE-GC activity. Membranes of COS cells transiently expressing ONE-GC were preincubated with 10 6 M uroguanylin and assayed for guanylate cyclase activity in the presence of 10 lM Ca2+ and increasing concentrations of neurocalcin d. Mock-preincubated membranes were treated as controls. The experiment was done in triplicate and repeated three times. The results are mean ± S.E. of these experiments.
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model is unique because it is fundamentally different from the phototransduction and peptide hormone receptor signal transduction models. Acknowledgments The study was supported by NIH awards DC 005349 (R.K.S.) and HL 084584 (T.D.). References
Fig. 4. Two-step uroguanylin-neurocalcin d signal transduction model. Upper panel: Resting state (free [Ca2+] < 100 nM). ONE-GC is in its basal state, is bound to Ca2+free neurocalcin d (NCdelta) with low affinity, and maintains the steady-state cyclic GMP concentration. Calmodulin (CaM)-bound cyclic GMP-gated Ca2+ channel (CNGA3) is closed. Lower panel: ‘‘Step 1”, uroguanylin interacts with the receptor domain of ONE-GC causing its activation and primes ONE-GC. Cyclic GMP formed opens some of the CNGA3 channels increasing [Ca2+] to semi-micromolar range. ‘‘Step 2”, Ca2+-bound neurocalcin d fully interacts with uroguanylin-primed ONEGC, causing its full activation.
[7]. The M880-L921 segment of this domain is bound to Ca2+ sensor component neurocalcin d. In resting state, the ORN is in a 60– 100 nM range of [Ca2+]i [2] and ONE-GC is in its basal state. The odorant, uroguanylin, signal starts by its interaction with the receptor domain of ONE-GC [7]. It is processed through two sequential steps. In step one, ONE-GC is primed and activated minimally. In step 2, [Ca2+]i raises. With a K1/2 of 0.3–0.8 lM, Ca2+ binds to neurocalcin d facilitating its interaction with the ONEGC’s segment M880-L921 [7,8], signals full activation of ONE-GC and production of the odorant second messenger cyclic GMP. It is envisioned that the operation of step 2 starts with the generation of a small amount of cyclic GMP in step 1. This pool of cyclic GMP opens a limited number of the cyclic GMP-gated channels causing influx of Ca2+ in the ORN. Ca2+ binds to the neurocalcin d, which, then fully activates ONE-GC. It is noted that the ONE-GC downstream components, cyclic GMP-gated channel (CNGA3) and Ca2+ are physiologically linked. Fig. 4 depicts this model. The
[1] Buck, L.B. (2000) The molecular architecture of odor and pheromone sensing in mammals. Cell 100, 611–618. [2] Munger, S.D., Leinders-Zufall, T. and Zufall, F. (2008) Subsystem organization of the mammalian sense of smell. Annu. Rev. Physiol., 3.1–3.26. [3] Duda, T., Venkataraman, V. and Sharma, R.K. (2007) Constitution and operational principles of the retinal and odorant-linked neurocalcin ddependent Ca2+ modulated ROS-GC transduction machinery in: Neuronal Calcium Sensor Proteins (Philippov, P. and Koch, K.-W., Eds.), pp. 91–113, Nova Science Publishers Inc., Hauppauge, NY. [4] Juilfs, D.M., Fülle, H.J., Zhao, A.Z., Houslay, M.D., Garbers, D.L. and Beavo, J.A. (1997) A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway. Proc. Natl. Acad. Sci. USA 94, 3388–3395. [5] Duda, T., Jankowska, A., Venkataraman, V., Nagele, R.G. and Sharma, R.K. (2001) A novel calcium-regulated membrane guanylate cyclase transduction system in the olfactory neuroepithelium. Biochemistry 40, 12067–12077. [6] Leinders-Zufall, T., Cockerham, R.E., Michalakis, S., Biel, M., Garbers, D.L., Reed, R.R., Zufall, F. and Munger, S.D. (2007) Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium. Proc. Natl. Acad. Sci. USA 104, 14507–14512. [7] Duda, T. and Sharma, R.K. (2008) ONE-GC membrane guanylate cyclase, a trimodal odorant signal transducer. Biochem. Biophys. Res. Commun. 367, 440–445. [8] Duda, T., Fik-Rymarkiewicz, E., Venkataraman, V., Krishnan, A. and Sharma, R.K. (2004) Calcium-modulated ciliary membrane guanylate cyclase transduction machinery: constitution and operational principles. Mol. Cell. Biochem. 267, 107–122. [9] Sun, L., Wang, H., Hu, J., Han, J., Matsunami, H. and Luo, M. (2009) Guanylyl cyclase-D in the olfactory CO2 neurons is activated by bicarbonate. Proc. Natl. Acad. Sci. USA 106, 2041–2046. [10] Paul, A.K., Marala, R.B., Jaiswal, R.K. and Sharma, R.K. (1987) Coexistence of guanylate cyclase and atrial natriuretic factor receptor in a 180-kDa protein. Science 235, 1224–1226. [11] Duda, T., Krishnan, R. and Sharma, R.K. (2006) GCAP1: antithetical calcium sensor of ROS-GC transduction machinery. Calcium Binding Proteins 1, 102– 107. [12] Krishnan, A., Duda, T., Pertzev, A., Kobayashi, M., Takamatsu, K. and Sharma, R.K. (2009) Hippocalcin, new Ca2+ sensor of a ROS-GC subfamily member, ONE-GC, membrane guanylate cyclase transduction system. Mol. Cell. Biochem. [13] Sharma, R.K. (2002) Evolution of the membrane guanylate cyclase transduction system. Mol. Cell. Biochem. 230, 3–30. [14] Duda, T., Goraczniak, R.M. and Sharma, R.K. (1991) Site-directed mutational analysis of a membrane guanylate cyclase cDNA reveals the atrial natriuretic factor signaling site. Proc. Natl. Acad. Sci. USA 88, 7882–7886. [15] Koch, K.-W., Duda, T. and Sharma, R.K. (2002) Photoreceptor specific guanylate cyclases in vertebrate phototransduction. Mol. Cell. Biochem. 230, 97–106. [16] Dizhoor, A.M. and Hurley, J.B. (1999) Regulation of photoreceptor membrane guanylyl cyclases by guanylyl cyclase activator proteins. Methods 19, 521– 531. [17] Duda, T., Koch, K.-W., Venkataraman, V., Lange, C., Beyermann, M. and Sharma, R.K. (2002) Ca(2+) sensor S100beta-modulated sites of membrane guanylate cyclase in the photoreceptor-bipolar synapse. EMBO J. 21, 2547–2556. [18] Krishnan, A., Venkataraman, V., Fik-Rymarkiewicz, E., Duda, T. and Sharma, R.K. (2004) Structural, biochemical, and functional characterization of the calcium sensor neurocalcin delta in the inner retinal neurons and its linkage with the rod outer segment membrane guanylate cyclase transduction system. Biochemistry 43, 2708–2723.
journal homepage: www.FEBSLetters.org
Ca2+-modulated ONE-GC odorant signal transduction Teresa Duda *, Rameshwar K. Sharma * Research Divisions of Biochemistry and Molecular Biology, The Unit of Regulatory and Molecular Biology, Salus University, Elkins Park, PA 19027, United States
a r t i c l e
i n f o
Article history: Received 11 February 2009 Revised 14 March 2009 Accepted 17 March 2009 Available online 22 March 2009 Edited by Ned Mantei Keywords: Cyclic GMP ONE-GC membrane guanylate cyclase Odorant signal transduction
a b s t r a c t In a subset of olfactory epithelium the odorant receptor guanylate cyclase, ONE-GC, is a central transduction component of the cyclic GMP signaling pathway. The odorant binds to the extracellular domain and activates its intracellular catalytic domain to generate the odorant second messenger, cyclic GMP. The present study demonstrates that it is a two-step, Ca2+-independent and Ca2+-dependent, sequential process. In step one, the odorant, uroguanylin, binds ONE-GC and primes it for stimulation. In step two, Ca2+-bound neurocalcin d binds to the defined intracellular domain and saturates ONE-GC activity. A prototype model is proposed that depicts this signal transduction process. Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction The biochemical process by which an odorant signal generates an electric signal is termed odorant-transduction (reviewed in [1–3]). The site of odorant-transduction is the ciliated apical border of sensory neurons located in the olfactory neuroepithelium [3]. Besides the presence of the major cyclic AMP transduction pathway, a small population of olfactory receptor neurons (ORN) contains an odorant-responsive cyclic GMP signaling pathway [4–6]. These two pathways do not overlap and are operationally independent. The mechanisms of these pathways are also 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 [also known as GC-D (4)], which is both the odorant uroguanylin [6,7], green pepper [8], and possibly also CO2 [9] receptor, and a guanylate cyclase. Thus, on the lines of the prototype model of ANF-RGC membrane guanylate cyclase signal transduction [10], 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 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
* Corresponding authors. Fax: +1 215 780 3125 (R.K. Sharma). E-mail addresses: [email protected] (T. Duda), [email protected] (R.K. Sharma).
makes the cyclic GMP signal transduction pathway more direct and, theoretically faster. The immediate issue is: what is the mechanism of the uroguanylin signal transduction? How does the signal generate its second messenger, cyclic GMP; and, how cyclic GMP, in turn, generates the electric signal? Related to the first part of the issue, the available information is that ONE-GC in ORN is modulated through multiple modes within the physiological levels of [Ca2+]i. Its sensors, neurocalcin d [5,8], GCAP1 [11] and hippocalcin [12], operate within the [Ca2+]i of 500–800 nM and begin to detect the signals within 50–100 nM range. This signal transduction mechanism modulated by the Ca2+sensor neurocalcin d is unique [7]. Other Ca2+ sensors—GCAP1-, GCAP2- and S100B-linked ROS-GC transduction systems work through the ROS-GC domains that do not overlap with the core catalytic domain of the guanylate cyclase. However, the neurocalcin d signaling site in ONE-GC resides directly on its catalytic domain [7]. Given the sequential events that uroguanylin signal begins at the extracellular domain of ONE-GC [7], that there is further [Ca2+]i-dependent ONE-GC modulation [8], that it opens the cyclic GMP-gated channels [6] and finally depolarizes the ciliary membranes [6], these cascade of events need to be individually dissected to fully comprehend the odorant-transduction process. The present study is a step in this direction. It shows an intriguing [Ca2+]i-modulated transduction phenomenon for the production of cyclic GMP, the second messenger of the odorant. The mechanistic model for this phenomenon has been proposed.
0014-5793/$36.00 Ó 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2009.03.036
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2. Materials and methods 2.1. Membrane preparations Rat olfactory neuroepithelium was homogenized in a buffer consisting of 250 mM sucrose, 10 mM Tris–HCl pH 7.4, 1 mM EGTA, containing a protease inhibitors cocktail, centrifuged at 1000 g and then at 100 000 g to pellet the membrane fraction. This fraction was suspended in 50 mM Tris–HCl pH 7.4/10 mM MgCl2 buffer and used for guanylate cyclase activity assay [5,7,8]. The membranes were devoid of endogenous neurocalcin d as verified by Western blot (data not shown). In preincubation experiments the homogenate was incubated on ice-bath for 10 min with 10 6 M uroguanylin; in parallel, mock preincubation (without uroguanylin) was carried out as a control. Following the incubation, the membrane fraction was prepared. Under these conditions no membrane vesicles are formed; the guanylate cyclase with some lipids attached to the transmembrane domain is present in the suspension. Identical protocol was used for COS-7 cells expressing ONE-GC, except that 5000 g pellet represented the membrane fraction. 3. Results and discussion 3.1. Uroguanylin signal transduction Background and strategy: ONE-GC is a cross-over member of the two subfamilies of membrane guanylate cyclases [7]. Because it is the receptor for uroguanylin, it belongs to the surface receptor subfamily; and because it is modulated by [Ca2+]i, it is also a member of the ROS-GC subfamily. Thus, three modes of its regulation were envisioned: (1) it behaves as a typical surface receptor guanylate cyclase; (2) it behaves as a typical ROS-GC guanylate cyclase; and (3) being a hybrid, it acquires attributes of both these subfamilies. These issues were analyzed through the following protocol. 3.1.1. The surface receptor guanylate cyclase model is partially applicable to ONE-GC The prototype surface receptor model is that of ANF-RGC [10,13,14]. In this model, uroguanylin will signal ONE-GC directly in a Ca2+-independent fashion.
To test this model, freshly prepared membranes of the olfactory neuroepithelium were individually exposed to 10 12 to 10 6 M uroguanylin in a Ca2+-free incubation buffer. Uroguanylin stimulated ONE-GC in a dose-dependent fashion with an EC50 of 20 pM, (Fig. 1A: closed circles). The stimulation was about 3.5-fold, and enzyme saturation occurred at 10 nM. To determine the influence of Ca2+ on the uroguanylin-modulated ONE-GC activity, parallel experiment was conducted in the presence of 10 lM Ca2+. The results obtained were almost identical (Fig. 1A: open circles) and in agreement with those obtained previously with the heterologous system of COS cells where the only signaling components were uroguanylin and ONE-GC [7]. These results demonstrate that uroguanylin signals ONE-GC, this signaling is Ca2+-independent and results in partial, three to fourfold, acceleration of ONE-GC activity. With the prior evidence that uroguanylin functions through the extracellular domain of ONE-GC [7] it is concluded that the ANF-RGC model is only partially applicable to ONE-GC because it is also modulated by [Ca2+]i. 3.1.2. The ROS-GC1 model also is partially applicable to ONE-GC The prototype ROS-GC1 signal transduction functions in two modes. In mode 1, operative in the rod/cone outer segments, Ca2+ through Ca2+-sensor GCAPs inhibits ROS-GC1 [15,16]. In mode 2, operative in the photoreceptor-bipolar synapse region and in the ganglion cell layer, elevations of [Ca2+]i are sensed by S100B and neurocalcin d and stimulate ROS-GC1 activity [17,18]. Thus, ROSGC1 is a bimodal Ca2+ signal transduction switch, both inhibited and stimulated by Ca2+. Prior evidence has indicated that ONE-GC transduction model does not follow the typical attributes of the ROS-GC1 model: it is only stimulated by Ca2+ signals. Rises in [Ca2+]i are sensed by neurocalcin d, GCAP1 and hippocalcin, and they, in turn, stimulate ONE-GC [5,11,12]. To assess applicability of the ROS-GC model to ONE-GC, freshly prepared membranes of the olfactory neuroepithelium were exposed to the incremental concentrations of neurocalcin d and 10 lM Ca2+. Neurocalcin d stimulated ONE-GC in a dose-dependent fashion with an EC50 of 0.7 lM (Fig. 1B). The overall stimulation was about 3.7-fold, and the enzyme saturation occurred at 2 lM. These results demonstrate that the ROS-GC1 model is also not fully applicable to the ONE-GC signal transduction. Importantly, the results also demonstrate that the Ca2+ signaling of ONE-GC is
Fig. 1. ONE-GC activity. Membranes of rat olfactory neuroepithelium were assayed for guanylate cyclase activity in the presence of (A) increasing concentrations of uroguanylin in the absence (closed circles) and presence (open circles) of 10 lM Ca2+ and (B) increasing concentrations of neurocalcin d and 10 lM Ca2+. The experiment was done in triplicate and repeated three times with separate membrane preparations. The results are mean ± S.E. from these experiments.
T. Duda, R.K. Sharma / FEBS Letters 583 (2009) 1327–1330
separate from uroguanylin signaling; and this step, like the uroguanylin step, stimulates ONE-GC only partially, 3.7-fold. 3.1.3. The odorant, uroguanylin, signal primes ONE-GC for the Ca2+ modulation of ONE-GC Intuitively, to bring the above biochemical evidences within the physiological perspective of odorant signal transduction in the production of its second messenger cyclic GMP, following sequence of events was envisioned. The Ca2+-indepndence of the uroguanylin signal matches the physiology of odorant signal transduction. Prior to its origin, in resting state the [Ca2+]i level in an ORN is within a range of 50– 100 nM. In this environment, the intracellular domain of ONE-GC is bound to neurocalcin d and this defines the basal state of ONEGC. The Ca2+-independent uroguanylin-dependent events cause primary activation of ONE-GC. And this activation is 3.5-fold. This level of cyclic GMP production may not be sufficient to explain the physiological cyclic GMP-dependent forward steps: opening of the cyclic GMP-gated channels, resulting in the inward flux of Ca2+ and depolarization of the ORN plasma membrane. To solve this puzzle, experimentation was initiated with the olfactory neuroepithelium homogenates. Freshly isolated rat olfactory neuroepithelium was homogenized under the Ca2+-depleted conditions (1 mM EGTA). The homogenate was first pre-incubated with 10 6 M uroguanylin and then the membranes were tested for the guanylate cyclase activity in presence of the increasing concentrations of neurocalcin d at 10 lM Ca2+. Control experiment was performed identically, except the homogenate was pre-incubated, without uroguanylin. The basal guanylate cyclase activity was 18 pmol cyclic GMP/ min/mg protein (Fig. 2). In the presence of increasing concentrations of neurocalcin d the ONE-GC activity of mock-preincubated membranes increased by about 3.5-fold (Fig. 2: open circles). The picture, however, was different for membranes pre-incubated with uroguanylin. Here the neurocalcin d dose-dependent Ca2+ signaling of ONE-GC resulted in more than 15-fold stimulation of ONE-GC activity, from 18 to 275 pmol cyclic GMP/min/mg protein (Fig. 2: closed circles); EC50 of neurocalcin d was 0.7 lM, it saturated the enzyme activity at 2 lM.
Fig. 2. Synergetic effect of uroguanylin and neurocalcin d on ONE-GC activity. Rat olfactory neuroepithelium was homogenized in the absence of Ca2+, preincubated with 10 6 M uroguanylin, the membrane fraction was prepared, and assayed for guanylate cyclase activity in the presence of 10 lM Ca2+ and increasing concentrations of neurocalcin d. Membranes from mock-preincubated homogenates were treated as controls. The experiment was done in triplicate and repeated three times with separate homogenates. The results are mean ± S.E. from these experiments.
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This indicates that uroguanylin signaling of ONE-GC in production of cyclic GMP is a two-step process. In the Ca2+-independent step it brings the ONE-GC’s catalytic module to a modest activity (below its full catalytic capacity) and also primes ONE-GC for the neurocalcin d/Ca2+-dependent step. Only after the last modulation occurs, ONE-GC achieves its full catalytic activity. The combined effects of uroguanylin and neurocalcin d far exceed the sum of their individual effects; they are not additive but synergetic. Does the two-step signal transduction besides uroguanylin, Ca2+ and ONE-GC involve additional components present in the olfactory neuroepithelium? To answer this question, the system of COS cells expressing ONE-GC was used. Membranes of these cells were preincubated with 10 6 M uroguanylin, washed and assayed for neurocalcin ddependent guanylate cyclase activity. The control consisted of membranes pre-incubated without uroguanylin. Like the pre-incubated neuroepithelial membranes, the neurocalcin d-modulated Ca2+ signaling of ONE-GC was also spectacular in the COS cell membranes (Fig. 3: closed circles). The kinetic parameters of ONE-GC activation were very similar in both systems. Neurocalcin d stimulated ONE-GC activity 12-fold with an EC50 of 0.7 lM and saturation at 2 lM. Without pre-incubation, the stimulation was only fourfold (Fig. 3: open circles). These results validate the two-step uroguanylin signal transduction model of ONE-GC. In addition they demonstrate that besides uroguanylin, Ca2+, and neurocalcin d no other olfactory transduction-specific component is involved in the ONE-GC transduction mechanism in production of the second messenger cyclic GMP. 3.1.4. The odorant uroguanyline signal transduction model In line with the present and past findings, an odorant uroguanylin signal transduction model in the production of its second messenger cyclic GMP is being presented. The model will serve as a useful template for future experimentation. A small population of ORNs contains a cyclic GMP signal transduction pathway [4–6]. This pathway resides at the apical region of the cilia [4–6]. Present in the region is ONE-GC membrane guanylate cyclase. Its outer domain is a receptor for uroguanylin [7]. In its inner domain, at the C-terminus, resides the catalytic domain
Fig. 3. Synergetic effect of uroguanylin and neurocalcin d on recombinant ONE-GC activity. Membranes of COS cells transiently expressing ONE-GC were preincubated with 10 6 M uroguanylin and assayed for guanylate cyclase activity in the presence of 10 lM Ca2+ and increasing concentrations of neurocalcin d. Mock-preincubated membranes were treated as controls. The experiment was done in triplicate and repeated three times. The results are mean ± S.E. of these experiments.
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model is unique because it is fundamentally different from the phototransduction and peptide hormone receptor signal transduction models. Acknowledgments The study was supported by NIH awards DC 005349 (R.K.S.) and HL 084584 (T.D.). References
Fig. 4. Two-step uroguanylin-neurocalcin d signal transduction model. Upper panel: Resting state (free [Ca2+] < 100 nM). ONE-GC is in its basal state, is bound to Ca2+free neurocalcin d (NCdelta) with low affinity, and maintains the steady-state cyclic GMP concentration. Calmodulin (CaM)-bound cyclic GMP-gated Ca2+ channel (CNGA3) is closed. Lower panel: ‘‘Step 1”, uroguanylin interacts with the receptor domain of ONE-GC causing its activation and primes ONE-GC. Cyclic GMP formed opens some of the CNGA3 channels increasing [Ca2+] to semi-micromolar range. ‘‘Step 2”, Ca2+-bound neurocalcin d fully interacts with uroguanylin-primed ONEGC, causing its full activation.
[7]. The M880-L921 segment of this domain is bound to Ca2+ sensor component neurocalcin d. In resting state, the ORN is in a 60– 100 nM range of [Ca2+]i [2] and ONE-GC is in its basal state. The odorant, uroguanylin, signal starts by its interaction with the receptor domain of ONE-GC [7]. It is processed through two sequential steps. In step one, ONE-GC is primed and activated minimally. In step 2, [Ca2+]i raises. With a K1/2 of 0.3–0.8 lM, Ca2+ binds to neurocalcin d facilitating its interaction with the ONEGC’s segment M880-L921 [7,8], signals full activation of ONE-GC and production of the odorant second messenger cyclic GMP. It is envisioned that the operation of step 2 starts with the generation of a small amount of cyclic GMP in step 1. This pool of cyclic GMP opens a limited number of the cyclic GMP-gated channels causing influx of Ca2+ in the ORN. Ca2+ binds to the neurocalcin d, which, then fully activates ONE-GC. It is noted that the ONE-GC downstream components, cyclic GMP-gated channel (CNGA3) and Ca2+ are physiologically linked. Fig. 4 depicts this model. The
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