ONE-GC membrane guanylate cyclase, a trimodal odorant signal transducer

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Biochemical and Biophysical Research Communications 367 (2008) 440–445 www.elsevier.com/locate/ybbrc

ONE-GC membrane guanylate cyclase, a trimodal odorant signal transducer Teresa Duda *, Rameshwar K. Sharma * Research Divisions of Biochemistry and Molecular Biology, The Unit of Regulatory and Molecular Biology, Pennsylvania College of Optometry, Elkins Park, PA 19027, USA Received 18 December 2007 Available online 4 January 2008

Abstract The Ca2+-modulated ONE-GC membrane guanylate cyclase is a central component of the cyclic GMP signaling in odorant transduction. It is a single transmembrane spanning modular protein. Its intracellular region contains Ca2+ sensor recognition domains linked to GCAP1 and to neurocalcin d, and a catalytic module. These domains sense increments in free Ca2+ and stimulate the catalytic module. The present study makes three significant mechanistic advancements. First, to date no ligand for the extracellular (ext) domain is known, for this reason ONE-GC has been deemed as an orphan receptor. The present study identifies its ligand. Uroguanylin stimulates ONEGC through its ext domain. Second, so far no ligand is known that directly stimulates the catalytic module of any membrane guanylate cyclase. The presented evidence shows that in the presence of the semimicromolar range of free Ca2+, neurocalcin binds to the catalytic module and stimulates ONE-GC. Thus, ONE-GC has trimodal regulation, two occurring intracellularly and one extracellularly. Third, guanylin, a urine odorant, does not directly stimulate ONE-GC. This challenges the proposed hypothesis that the guanylin odorant signal occurs via ONE-GC [T. Leinders-Zufall, R.E. Cockerham, S. Michalakis, M. Biel, D.L. Garbers, R.R. Reed, F. Zufall, S.D. Munger, Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium, Proc. Natl. Acad. Sci. USA. 104 (2007) 14507–14512]. Ó 2007 Elsevier Inc. All rights reserved. Keywords: ONE-GC membrane guanylate cyclase; Uroguanylin signaling; Neurocalcin d signaling; Odorant transduction

Odorant transduction is a biochemical process by which an odorant signal generates an electric signal reviewed in, [1–4]. The transduction process is initiated at the ciliated apical border of sensory neurons located in the olfactory neuroepithelium [Fig. 3 of Ref. 5]. The consensus is that the major mechanism involved in this transduction process is that of cyclic AMP signaling pathway reviewed in, [6–10]. The recent evidence indicates that besides the cyclic AMP signal transduction pathway, a small population of olfactory receptor neurons contains an odorant-responsive membrane guanylate cyclase-linked cyclic GMP signaling pathway [11–14, reviewed in, 5,15]. This pathway is inde*

Corresponding authors. Fax: +1 215 780 3125. E-mail addresses: [email protected] (T. Duda), [email protected] (R.K. Sharma). 0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.12.153

pendent of the cyclic AMP signaling pathway. The pathway begins with the ONE-GC membrane guanylate cyclase (also named GC-D [11]) and it appears to be linked down-stream with at least two recognized cyclic GMP-processing signaling components, cyclic GMP-specific cyclic nucleotide-gated channel subunit, CNGA3, and a cyclic GMP-stimulated phosphodiesterase, PDE2 [12,14,16]. There are three proposed mechanisms of the ONE-GC transduction. Two are Ca2+-modulated [13,17] and one is typical for the peptide hormone signal [14]. In the first two, ONE-GC is modulated indirectly by the Ca2+ impulses occurring within the cilia. The rise in Ca2+ is detected by myr-neurocalcin d and/or by GCAP1, which at the membrane, bind to their defined intracellular domains of ONE-GC and stimulate it [13,17]. These mechanisms envision two-step odorant-dependent stimulation of

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ONE-GC. In step one, odorant causes a rise in Ca2+ level; in step two, Ca2+ stimulates ONE-GC [13,17, reviewed in, 5]. The neurocalcin d-modulated Ca2+ signaling mechanism has been studied in more detail and has successfully passed through the five criteria set forth where ONE-GC qualifies as a genuine odorant transducer. (1) In its native state, ONE-GC is odorant-responsive; (2) the response is rapid, occurring in seconds; (3) ONE-GC resides in the membrane portion of the cilia, the site of odorant transduction; (4) the physiological levels of Ca2+ mimic the odorant response; (5) ONE-GC coexists with its Ca2+ sensor component, myristoylated neurocalcin d. In the third mechanism, the odorant directly binds to the extracellular receptor domain of ONE-GC and stimulates its catalytic module, which is located in the intracellular region at C-terminal site of the cyclase. However, to date, no odorant, or any other ligand, directly interacting with ONE-GC has been found and for this reason, this cyclase has been dubbed as an orphan receptor [11], and the mechanism lacks any evidence. However, a recent patch clamp study with the genetically modified ONE-GC and CNGA 3 mouse model systems indicate that the urine odorants, guanylin and uroguanylin, through ONE-GC cause ‘‘excitory cyclic GMP-dependent signaling action potential firing” [14]. The biochemical features of this signal transduction mechanism are not known. The present study demonstrates a novel direct uroguanylin signaling ONE-GC transduction mechanism and also an intriguing aspect of the myr-neurocalcin d-modulation of the Ca2+ signaling of ONE-GC. Together with the

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earlier evidence that ONE-GC in the ciliary membranes is also modulated by the GCAP1-modulated Ca2+ signaling pathway [17], the findings provide three modes by which ONE-GC can act as an odorant transducer. Materials and methods Materials. Uroguanylin (human) and guanylin (rat) were purchased from Bachem, myristoylated neurocalcin d was expressed and purified according to the laboratory protocol [18]. ONE-GC mutants. The ONE-GC mutants used in this study with their domain structures are schematically represented in Fig. 1. The transmembrane-bound tm-catd mutant (deleted aa regions 75–466, 521–879, and 1029–1110) was prepared by introducing TGA STOP codon into the ONE-GC D75–466 & 521–823 mutant [13] and by introducing Sma1 restriction site at positions 879 and 880, Sma1 digestion and religation of the remaining part. Mutagenesis was performed using Quick Change mutagenesis kit. The constructs were sequenced to confirm their identities. The cDNA of the soluble catd (aa 880–1028) was amplified from ONEGC by PCR and cloned into pET30aLIC vector. The resulting expression construct was used for transformation of E. coli BL21-Codon-Plus-RIL cells for protein expression. The protein was purified on Ni–NTA affinity column. Expression and guanylate cyclase activity assay. COS-7 cells were transfected with the wild-type (wt) ONE-GC cDNA or its deletion mutants through standard procedures [19]. After sixty hours the cells were washed with 50 mM Tris–HCl (pH 7.5)/10 mM buffer, homogenized, centrifuged at 5000g and washed several times with the same buffer. The resulting pellet represented crude membranes. The guanylate cyclase activity was assessed according to the previous protocols [20–23]. Peptide competition experiments. Membranes of COS cells expressing wt ONE-GC were incubated with 2 lM neurocalcin d, 10 lM CaCl2, and increasing concentrations of peptides for 10 min at 37 °C and assessed for the guanylate cyclase activity.

Fig. 1. Schematic representation of ONE-GC and its expression constructs used in the study. The following abbreviations denote the predicted domains: ls, leader sequence; ext, extracellular domain; tm, transmembrane domain; jmd, juxtamembrane domain; khd, kinase homology domain; dd, putative dimerization domain; catd, cyclase catalytic domain; cte, C-terminal extension. Specific basal guanylate cyclase activity of ONE-GC and of the mutants expressed in COS or E.coli cells is given in the right-hand column [A]. The neurocalcin d (NCd) binding and transduction site (aa 880–921) within the catd is shown in the last panel.

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Surface plasmon resonance (SPR) measurements. These measurements were done according to the established laboratory protocols [21]. Binding analyses were performed at 25 °C, using BIAcore X system. Myristoylated neurocalcin d [150 ng/ll in 50 mM sodium acetate (pH 4.0)] was immobilized on the CM5 sensor chip via primary amino group. The amount of immobilized protein was 0.4 ng/mm2. An independent flow cell on the same sensor was subjected to a ‘‘blank immobilization” (no protein) and used as a control flow cell. The buffer for the mobile phase was 10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM CaCl2 and 0.005% Surfactant P20. The purified ONE-GC catd was diluted to the concentrations varying from 0 to 20 lM in the same buffer and injected into the flow cell. The flow rate was maintained at 10 ll/min. The binding was observed as an increase in RU units. The specific binding was calculated by subtraction of the binding at the control cell from that at the experimental cell. The curves were fitted according to a 1:1 Langmuir model. The binding parameters were calculated using the BIAevaluation 3.2 software.

Results and discussion The peptide uroguanylin but not guanylin stimulates ONEGC

5 (fold stimulation)

Guanylate cyclase activity

Original cloning of ONE-GC from total rat olfactory cDNA library showed that it was a membrane guanylate cyclase [11]. Because it had no known extracellular ligand, it was named an orphan receptor guanylate cyclase [11]. To date this picture has not changed and the guanylate cyclase remains an orphan receptor. A recent patch clamp study has indicated that ONE-GC is the signal transducer of two urine odorant constituents, uroguanylin and guanylin [14]. The signal transduction mechanisms of these odorants are not known. To determine whether these urine odorant peptides are effective ligands of ONE-GC, the membranes of COS cells transiently expressing ONE-GC were individually exposed to 10 12 to 10 8 M guanylin or uroguanylin. Uroguanylin stimulated ONE-GC activity (Fig. 2A). The overall stimulation was in excess of 4-fold and it was in a dose-depen-

4 3

Uroguanylin signals through the ext domain of ONE-GC To determine whether uroguanylin signal transduction occurs through the external or the intracellular domain of ONE-GC, ONE-GCDext mutant (deleted extracellular domain of ONE-GC) was used. The basal guanylate cyclase activity of the mutant expressed in COS cells was almost identical to that of wt ONE-GC [Fig. 1: compare 49 with 48 pmol cGMP 1 min 1(mg prot 1)], indicating that deletion of the extracellular domain has no effect on the tertiary structure of the protein. The COS cell membranes were incubated with a series of uroguanylin concentrations. All concentrations of uroguanylin were ineffective in stimulating ONE-GCDext (Fig. 2: closed triangles). These results demonstrate that uroguanylin signals ONE-GC activation through its interaction with the extracellular domain of ONE-GC. Thus, uroguanylin is the extracellular ligand of ONE-GC and ONE-GC is no longer an orphan receptor membrane guanylate cyclase. It now qualifies as a classical member of the surface receptor membrane guanylate cyclase family along with other family members—ANF-RGC, the receptor of atrial natriuretic factor (ANF), CNP-RGC, the receptor of type C natriuretic peptide (CNP) and STa-RGC the receptor of enterotoxin, guanylin and uroguanylin. Two interesting facets of this finding are that uroguanylin becomes the ligand of two surface receptors: STa-RGC and ONE-GC, and yet, unlike STa-RGC, guanylin a close structural analog of uroguanylin is not the ligand of ONE-GC. Furthermore, these biochemical findings rationalize the transduction mechanism involved in the gene-targeting-patch clamp studies which show that uroguanylin through ONE-GC functions as an odorant [14]. The mechanism is that uroguanylin binds to the receptor portion of ONE-GC and signals activation of its intracellular catalytic domain.

EC50 =20 pM

2

The catalytic domain of ONE-GC is the direct transduction and the binding site of the Ca2+-bound myr-neurocalcin d

1 0 10-13

dent fashion. EC50 value of uroguanylin was 20 pM and saturation of the guanylate cyclase occurred at 500 pM (Fig. 2, closed circles). In contrast, guanylin was totally ineffective in stimulating ONE-GC (Fig. 2). These results demonstrate that uroguanylin but not guanylin is a direct ligand of ONE-GC. They, however, do not reveal whether the signaling occurs through extracellular or intracellular domain of ONE-GC.

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Peptide [M] Fig. 2. Effect of guanylin and uroguanylin on ONE-GC activity. Membranes of COS cells expressing wt-ONE-GC (-s- and -d-) or its Dext mutant (-N-) were assayed for guanylate cyclase activity in the presence of indicated concentrations of guanylin (-s-) or uroguanylin (-dand -N-). The experiment was done in triplicate and repeated three times. The results presented are means ± SD of these experiments.

Basal activity Based on the cues that the photoreceptor ROS-GC1 constitutes the pivotal component of phototransduction and this Ca2+-modulated transduction mechanism extends in and beyond the retinal layers, the search of a membrane guanylate cyclase possibly linked with odorant transduction was initiated [5,15]. A membrane guanylate cyclase from the olfactory neuroepithelium cDNA was cloned and named olfactory neuroepithelial membrane

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guanylate cyclase (ONE-GC) to show its source of origin [13]. Its structure was found to be identical to the previously cloned GC-D, which, as indicated earlier, had been dubbed as an orphan receptor [11]. ONE-GC in its native conditions within the olfactory neuroepithelium binds and is activated by myr-neurocalcin d in a semimicromolar free Ca2+ range [13,21]. Further biochemical and immunological studies demonstrate that myr-neurocalcin d and ONE-GC co-exist at the site of the apical region of the ciliary membranes [13,21]. The neurocalcin d-modulated domain in ONE-GC stretches between the amino acid residues 824–1028 [21]. The odorant signal is transduced into cyclic GMP by the myr-neurocalin d-modulated Ca2+ signaling of ONE-GC [21]. To qualify as a genuine transducer of the odorant signal, myr-neurocalcin d/ONE-GC transduction was subjected to multiple test criteria. All were met establishing the participation of the cyclic GMP signaling system in odorant signal transduction [21]. The neurocalcin d-modulated segment, aa824–1028, of ONE-GC is composed of the partial domain of kinase homology (khd), and of the complete dimerization domain (dd) and catalytic domain (catd). The catd boundary stretches from aa 880 to 1028. This domain is almost completely conserved in all members of the membrane guanylate cyclase family (85–90% structural homology among ROS-GC subfamily members). To date no regulatory peptide ligand directly interacting with it is known. Intuitively, the authors felt that this might be the case with neurocalcin d. To test this possibility two ONE-GC mutants were constructed and tested (Fig. 1). In one, tm-catd, the catalytic domain (aa 880–1028) was accompanied by the transmembrane (tm) domain; and, in the other, it was solely the soluble catd. The rational for the tm-bound mutant was to assess the role of tm domain in controlling the cat module activity. The tm bound mutant was expressed in COS cells and the soluble cat mutant expressed in bacteria and purified. Analysis of the membrane bound and the soluble catd mutants show that both have intrinsic catalytic activity, indicating that mutations have not adversely affected their tertiary structures. In fact, tm-catd mutant almost has identical basal guanylate cyclase activity to that of the wtONE-GC (Fig. 1). The 10-fold drop in the basal activity of the soluble cat domain is due to the absence of its tm domain. These findings reveal a new biochemical property of the catalytic module of a membrane guanylate cyclase. To date the consensus opinion is that the isolated form of the catalytic module of any membrane guanylate cyclase is not active. To be active it requires the co-presence of its putative dd component (Fig. 1). This study revises that thinking and establishes that dd is not required for expression of the basal activity of ONEGC. It will become clear latter that similar is the case with the regulatory activity of ONE-GC.

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The conclusion on preservation of the structural integrity of the mutants is further supported by analysis of the regulatory activities of the mutants, elaborated below. Neurocalcin d transduction site The membranes of COS cells expressing tm-catd mutant were used to determine whether the isolated catalytic domain of ONE-GC directly responds to the Ca2+-bound neurocalcin d. The subsequent were reconstitution experiments where the sole signal transduction components were the isolated catd and recombinant myr-neurocalcin d. The control in each case consisted of the COS cell membranes expressing wt-ONE-GC. The membranes were incubated with increasing concentrations of neurocalcin d in presence of the 10 lM Ca2+. Membranes expressing wt-ONE-GC were treated identically. Both the tm-catd mutant and the wt-ONE-GC responded almost identically to the increasing concentrations of neurocalcin d and showed a neurocalcin d dosedependent stimulation (Fig. 3A). The maximal stimulation was 8 fold over the basal value and was achieved at 2 lM neurocalcin d. The EC50 value for the neurocalcin was 0.8 lM. The reconstitution experiment with the catd soluble construct demonstrated that in the presence of 10 lM free Ca2+, neurocalcin d stimulated its activity in a dose-dependent fashion (Fig. 3A). The maximal stimulation was about 3.75-fold over the basal value and was achieved at 2 lM neurocalcin d. The EC50 value for neurocalcin d was 0.8 lM. These results demonstrate that except for their Vmax activities, the kinetic parameters of the catalytic module in two environments, transmembrane-bound and in isolation, are identical. The difference in its saturation levels is ascribed to its transmembrane component. To assess the Ca2+ requirement for the above reconstitution experiment, catd was incubated with the incremental (0.01–100 lM) concentrations of Ca2+ in the presence of the saturating concentration of neurocalcin d (4 lM). Like wt ONE-GC, the neurocalcin d-dependent catd activity was Ca2+ dependent (Fig. 3B). The Ca2+ K1/2 value in both cases was comparable, 0.75 and 0.9 lM, respectively. These results demonstrate that the isolated catalytic module of ONE-GC is intrinsically active, is directly modulated by neurocalcin d, the modulation is Ca2+-dependent and the putative dd component is not needed for its basal and regulated activity. Neurocalcin d-binding kinetics To determine the kinetics of neurocalcin d binding to the catd of ONE-GC SPR spectroscopy was used. Myristoylated neurocalcin d was immobilized on a sensor chip and incremental concentrations of catd soluble fragment were supplied in the mobile phase. To determine the half-maximal binding (EC50), the experimental RU values at equilibrium were plotted as a function of catd concentration (Fig. 1A in Supplemental material). Half-maximal binding was at 0.4 lM. Scatchard analysis of the binding data

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B 10

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EC50 =0.75 μM

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Fig. 3. (A) Effect of neurocalcin d on wt-ONE-GC, tm-catd mutant and soluble catd construct. Wt-ONE-GC and its tm-catd mutant were expressed in COS cells and the soluble catd fragment was expressed in E. coli as described in ‘‘Materials and methods”. Membranes of COS cells and the purified catd were individually assayed for cyclase activity in the presence of indicated concentrations of neurocalcin d and 10 lM Ca2+. The experiment was carried out in triplicate and repeated three times. The results presented are means ± SD of these experiments. (B) Ca2+-dependence of neurocalcin d stimulated activity of ONE-GC and its catd construct. The experiment was performed as described in (A) except that neurocalcin d concentration of 4 lM was constant and Ca2+ concentrations were as indicated. The experiment was done in triplicate and repeated two times. Means ± SD of these experiments are presented.

Peptide competition experiments map aa 880–921 segment of ONE-GC as the core binding and the transduction site of neurocalcin d

resulted in the KD value of 0.43 lM (Fig. 1B in Supplemental material) and the calculated value using the BIAevaluation 3.2 software was 0.38 lM. These three binding values are comparable, and they are also in good agreement with the 0.8 lM EC50 value of neurocalcin d for ONE-GC activation. Other binding kinetic parameters are as follows: kon (association rate constant) equal to 6.1  104 M 1 s 1, koff (dissociation constant) equal to 4.2  10 3 s 1, KA (equilibrium association constant) equal to 2  106 M 1. These Ca2+-dependent binding and transduction parameters between neurocalcin d and the catalytic module prove that the catalytic module by itself houses the complete neurocalcin d signal transduction site.

To determine the core neurocalcin d signal transduction site in ONE-GC, its aa 880–1028 segment was scanned through the peptide competition experiments. A library of 22-amino acids peptides overlapping by 2 amino acids covering the aa 880–1028 segment was synthesized and scanned for their inhibitory effects on the Ca2+-dependent neurocalcin d timulation of the wt-ONE-GC expressed in the COS cell membranes. The membranes were incubated with 0.2 and 0.5 mM concentrations of each peptide in

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Fig. 4. Peptide competition. (A) Inhibition of ONE-GC activity as a function of the peptide concentration. Membranes of COS cells expressing ONE-GC were incubated with the indicated concentrations of the peptide in the presence of 2 lM neurocalcin d and 10 lM Ca2+. Experiments were carried out in triplicate and repeated twice. The data shown are from one representative experiment. (B) Effect of excess of neurocalcin d. Incremental concentrations of neurocalcin d were added to the reaction mixture containing 0.2 mM peptide and 2 lM neurocalcin d and assayed as in (A).

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the presence of 2 lM concentration of neurocalcin d and 10 lM Ca2+. Only two peptides, aa880–901 and aa900– 921, inhibited the neurocalcin d-stimulated cyclase activity and the inhibition was almost complete. Peptide 900 VGFTTISALSEPIEVVGFLNDL921 was the most effective (Fig. 4A: closed triangles). It caused 95% inhibition; its IC50 value was 60 lM (Fig. 4A); Peptide 880 MGTTVEPEYFDQVTIYFSDIVG901 caused 90% inhibition, with an EC50 of 0.1 mM (Fig. 4A: closed circles). These results were validated by the reconstitution experiments: excess of neurocalcin d resulted in reversal of the inhibition by both peptides (Fig. 4B). These results prove that M880-L921 region of ONE-GC is the Ca2+dependent neurocalcin binding and the transduction site of ONE-GC. Acknowledgments The study was supported by NIH awards DC 005349 (R.K.S.) and HL 070015 (T.D.). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.bbrc.2007.12.153. References [1] F. Zufall, T. Leinders-Zufall, The cellular and molecular basis of odor adaptation, Chem. Senses 25 (2000) 473–481. [2] T. Nakamura, Cellular and molecular constituents of olfactory sensation in vertebrates, Comp. Biochem. Physiol. A Mol. Integr. Physiol. 126 (2000) 17–32. [3] A. Menini, Calcium signalling and regulation in olfactory neurons, Curr. Opin. Neurobiol. 9 (1999) 419–426. [4] D. Schild, D. Restrepo, Transduction mechanisms in vertebrate olfactory receptor cells, Physiol. Rev. 78 (1998) 429–466. [5] T. Duda, V. Venkataraman, R.K. Sharma, Constitution and operational principles of the retinal and odorant-linked neurocalcin d-dependent Ca2+ modulated ROS-GC transduction machinery, in: P. Philippov, K.-W. Koch (Eds.), Neuronal Calcium Sensor Proteins, Nova Science Publishers, Inc., Hauppauge, NY, 2007, pp. 91–113. [6] L. Belluscio, G.H. Gold, A. Nemes, R. Axel, Mice deficient in G(olf) are anosmic, Neuron 20 (1998) 69–81. [7] H. Breer, Olfactory receptors: molecular basis for recognition and discrimination of odors, Anal. Bioanal. Chem. 377 (2003) 427–433. [8] L.B. Buck, Unraveling chemosensory diversity, Cell 83 (1995) 349–352.

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