Expression of a Bombyx mori Tyramine Receptor in HEK-293 Cells

Chapter 17

Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch017

Expression of a BombyxmoriTyramine Receptor in HEK-293 Cells and Action of a Formamidine Insecticide 1

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1

Yoshihisa Ozoe , Hiroto Ohta , Idumi Nagai , and Toshihiko Utsumi 2

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Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690-8504, Japan Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan 2

B96Bom was cloned from the silkworm Bombyx mori and used for stable expression in HEK-293 cells. The expressed B96Bom receptor led to an attenuation of forskolin-stimulated intracellular cAMP production in response to tyramine. The attenuation was abolished by yohimbine. Octopamine was less effective than tyramine. Tyramine inhibited [ H]tyramine binding to the B96Bom receptor more potently than octopamine and dopamine. The acaricide/insecticide chlordimeform and its metabolite demethylchlordimeform inhibited [ H]tyramine binding but did not show significant agonist activity. The stable expression system might prove useful for the discovery of novel ligands and bioactive compounds. 3

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© 2005 American Chemical Society Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

183


184 Biogenic amines play important roles as neurotransmitters, neuromodulators, and neurohormones, and are implicated in a variety of physiological events from energy metabolism and muscle contraction to learning and memory {1,2). Octopamine (Figure 1) is one of the well-studied biogenic amines in invertebrates {3,4). In the 1970s and 1980s, the octopamine receptor was extensively studied as a target of formamidine acaricides/insecticides such as chlordimeform and amitraz (5). On the other hand, tyramine (Figure 1) was regarded as a mere precursor of the biosynthesis of octopamine, because the βhydroxylation of tyramine results in octopamine {3,4). Recently, however, evidence has been accumulating that tyramine also functions as a chemical messenger in insects {6,7). The biological functions of octopamine and tyramine are generally to increase and decrease intracellular levels of cAMP, respectively, by interacting with distinct seven-transmembrane, G protein-coupled, adenylate cyclase-linked receptors (2). In view of a close structural similarity of these two amines, we are interested in examining how their receptors differentiate the two amines and their analogues. In this article, we describe the cloning and expression of an insect tyramine receptor, and the actions of chlordimeform and a demethylated analogue (demethylchlordimeform) (Figure 1) on the tyramine receptor.

Octopamine

Tyramine

CH

3

b H

Chlordimeform

3

Dopamine

CH

3

CH3

Demethylchlordimeform

Figure 1. Structures of biogenic amines andformamidines.

Molecular Cloning of B96Bom from the Silkworm As lepidopteran insects are particularly sensitive to octopaminergic insecticides (5), we decided to clone B96Bom (Accession No. X95607), the sequence of which had been deposited in the EMBL/GenBank/DDB J database as encoding an octopamine receptor of the silkworm Bombyx mori but the functional properties of the encoding product of which were not reported {8). The PCR-amplified full-length cDNAfromthe heads of thefifth-instarsilkworm larvae (the Kinshu-Showa strain) was ligated into a pT7Blue vector and sequenced by standard techniques. B96Bom was found to code 479 amino acids

Clark and Ohkawa; New Discoveries in Agrochemicals ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

185 (Figure 2). The deduced amino acid sequence is the same as that in the database, except four amino acids (see the caption) (Accession No. AB162828). The difference is probably due to the difference of the strains used. The sequence reveals seven transmembrane domains characteristic of G protein-coupled receptors, as well as conserved amino acid residues; e.g., Asp 134 and Ser222, which are thought to interact with the amino group and the p-hydroxy group of biogenic amines, respectively, from analogy to adrenergic receptors. Downloaded by UNIV OF MASSACHUSETTS AMHERST on May 29, 2018 | https://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch017

MGQAATHDANNYTSINYTEIYDVIEDEKDVCAVADEPKYPSS FGISLAVP EWEAICTAIILTMI11S T W G N I L V I L S VFTYKPLRIVQNFFIVS LAVAD LTVAILVLPLNVAYSILGQWVFGIYVCKMWLTCDIMCCTSSILNLCAIAL DRYWAITDPINYAQKRTLERVLFMIGIVWILSLVISSPPLLGWNDWPEVF Ε PDTPCRLTSQPGFVIFSSSGSFYIPLVIMTWYFEIYLATKKRLRDRAK ATKISTISSGRNKYETKESDPNDQDSVSSDANPNEHQGSTRLVAENEKKH

R T R K L T P K K K P K R R YWS K D D K S H N K L 1 1 Ρ I L S N E N S V T D I G E N L E N R N T S SESNSKETHEDNMIΕITEAAPVKIQKRPKQNQTNAVYQFIEEKQRISLTR ERRAARTLGIIMGVFWCWLPFFVIYLVIPFCVSCCLSNKFINFITWLGY VNSALNPLIYTIFNMDFRRAFKKLLFIKC

Figure 2. Amino acid sequence of the B96Bom receptor. The seven putative transmembrane domains are underlined. Amino acid residues in bold are thought to function in binding tyramine. Dots on four letters refer to amino acid residues differentfromthose reported by von Nickisch-Rosenegk et al (8); the observed replacements are N38K, I39Y, C4JS, andG289S.

Functional Properties of the B96Bom Receptor Expressed in a Cell Line B96Bom was subcloned into the expression vector pcDNA3 and introduced into a human embryonic kidney cell line (HEK-293) using LipofectAmine (P). HEK-293 cells stably expressing the B96Bom receptor (B96Bom/HEK-293 cells) were selected in the presence of the antibiotic G-418. First of all, we examined the effects of biogenic amines on cAMP production in B96Bom/HEK293 cells. However, neither octopamine nor tyramine elicited an increase in intracellular cAMP levels over basal levels (P). Therefore, we next examined the



186 effects of biogenic amines on cAMP production stimulated by forskolin, a direct activator of adenylate cyclase. In response to octopamine and tyramine, the B96Bom receptor led to an attenuation of forskolin-stimulated cAMP production in a dose-dependent manner (Figure 3). Both amines apparently exhibited a maximum attenuation level of approximately 40%. However, tyramine was found to be at least two orders of magnitude more effective than octopamine, when the concentrations needed to reach the maximum attenuation level were compared. Furthermore, the tyramine receptor antagonist yohimbine abolished the tyramine-induced attenuation in the low tyramine concentration range. The effect of yohimbine was not observed in the high tyramine concentration range, indicating that yohimbine is a competitive antagonist for the B96Bom receptor. ~m- Tyramine Octopamine

log [octopamine (M)]

^

^

-o-

+Yohimbine

log [tyramine (M)]

Figure 3. Dose-response curves of octopamine and tyramine in attenuating forskolin-stimulated cAMP production in B96Bom/HEK-293 cells. Forskolin (10 μΜ^ΞίυηηΙαίβά cAMP levels are shown as 100%. Yohimbine (10 μΜ) was tested as an antagonist. Data represent means ± SE of at least four independent experiments. *p < 0.01 vs control; **p < 0.05 vs control (paired t test); ***p < 0.01 vs tyramine-attenuated levels (unpaired t test). (Reproduced with permissionfromreference 9. Copyright 2003 Blackwell.)

Affinity of Biogenic Amines for the B96Bom Receptor We investigated how the B96Bom receptor discriminates between octopamine, tyramine, and dopamine, which are different from each other by the presence or the position of only one hydroxy group. Figure 4 displays the doseresponse curves for the inhibitory effects of the three amines on the binding of [ H]tyramine to the B96Bom receptor. Although all three biogenic amines 3


187 3

inhibited the binding of [ H]tyramine in a dose-dependent manner, the most potent amine was tyramine with an IC50 of approximately 5 nM. Both octopamine and dopamine were two orders of magnitude less potent than tyramine. Thus, the B96Bom receptor clearly discriminates tyramine from the other two amines. The hydroxy groups at the m-position and the β-position of phenylethylamines are detrimental in their binding to the B96Bom receptor. No specific binding of [ H]octopamine to the membranes of HEK-293 cells expressing the B96Bom receptor was observed under the same conditions. Taken together with the results of the functional assay described above, these findings indicate that the B96Bom receptor is not an octopamine receptor but a tyramine receptor negatively coupled to the effector adenylate cyclase.


3

100

• Tyramine • Octopamine • Dopamine

-11 -TO -9 -8 -7 -6 -5 -4 -3 log [amine (M)]

Figure 4. Dose-response curves of octopamine, tyramine, and dopamine in inhibiting specific f HJ tyramine (3 nM) binding to the membranes of B96Bom/HEK-293 cells. Data represent means ± SD of at least two or three experiments, each done in duplicate. The specific to total binding ratio was 0.94 under the conditions used. The ICsoS of octopamine, tyramine, and dopamine were estimated to be 1.4 μΜ, 5.2 nM, and 1.7 μΜ, respectively. (Reproduced with permissionfromreference 9. Copyright 2003 Blackwell) 3

Actions of a Formamidine Insecticide on the B. mori Tyramine Receptor Chlordimeform is a formamidine acaricide/insecticide or a pestistatic chemical (10). Demethylchlordimeform is a biologically active form of chlordimeform and is known as a partial agonist of octopamine receptors (11-13). We examined if the B. mori tyramine receptor recognizes the formamidines as ligands. Figure 5 shows the dose-response curves of the formamidines in inhibiting the binding of [ H]tyramine to the B. mori tyramine receptor. The 3


188 results of the binding assay indicate that the formamidines are capable of interacting with the tyramine receptor. The IC of chlordimeform estimated in this assay is one order of magnitude smaller than that reported in the inhibition of the specific binding of [ H]octopamine to the homogenates of Drosophila heads and those of firefly light organs (14,15). The IC of demethylchlordimeform is one order of magnitude smaller than that reported in the inhibition of [ H]yohimbine binding to a cloned Drosophila tyramine receptor (16) but two orders of magnitude larger than that in the inhibition of specific [ H]octopamine binding to the homogenates of firefly light organs (15). The conclusion that demethylchlordimeform interacts with tyramine receptors with a lower affinity than with octopamine receptors is also the conclusion obtained with membranes from the brain of the locust (/ 7). 50

3

50


3

3

• Chlordimeform O Demethylchlordimeform

-10 -9 -8 -7 -6 -5 -4 -3 log [comp (M)]

Figure 5. Dose-response curves of chlordimeform and demethylchlordimeform in inhibiting specific [ H]tyramine (3 nM) binding to the membranes of B96Bom/HEK-293 cells. Data represent means ± SD of two to three experiments, each done in duplicate. The IC s of chlordimeform and demethylchlordimeform were determined to be 0.30 μΜ and 0.35 μΜ, respectively. 3

50

We next performed a functional assay to see whether the formamidines act as agonists in the tyramine receptor. Figure 6 shows the effects of demethylchlordimeform on forskolin-stimulated cAMP production in normal HEK-293 cells and HEK-293 cells stably expressing die B. mori tyramine receptor. Demethylchlordimeform at 100 μΜ attenuated forskolin-stimulated cAMP production by approximately 20 % in HEK-293 cells expressing the tyramine receptor. However, the attenuation was observed in normal HEK-293 cells as well. These findings indicate that demethylchlordimeform does not function as an agonist for the tyramine receptor, although the compound is


189 capable of interacting with the receptor. Chlordimeform had no effects on both cell lines (data not shown). It remains to be solved whether the formamidines act as antagonists in the tyramine receptor. We are investigating the structureactivity relationships of synthetic amine analogues and the ligand recognition of mutated tyramine receptors.


Demethylchlordimeform Tyramine

1140 -

140 h

g 120

I ioo 3

------

80

80 60 -

CO 3

.

I

ο u.

t*

3

Ε

Ε

20 .

Untransfected HEK-293 cells 1

-9

-8

L

1,

40

1

1

-7 -6 -5 -4 log [comp (M)]

B96Bom/ HEK-293 cells .

ο

-9

ι

-8

,

ι ι I I, -7 -6 -5 -4 log [comp (M)]

Figure 6. Effects of demethylchlordimeform on forskolin-stimulated cAMP production in normal HEK-293 cells and HEK-293 cells stably expressing a B. mori tyramine receptor. Forskolin (10 μΜ)-5ί^^αίβά cAMP levels are shown as 100%. *p < 0.01 vs control; **p< 0.05 vs control (paired t test). Data represent means ± SE of two tofiveexperiments, except data at 0.1 μΜ and 1 μΜΐη normal HEK-293 cells (n=l).

Conclusion Most of reported data, including those of our studies, indicate that the invertebrate octopamine and tyramine receptors convey the activation and inactivation signals for adenylate cyclase, respectively (2,9,18). It is generally difficult to discover compounds acting selectively for either the octopamine or tyramine receptor by conventional cAMP assays using membrane homogenates from nervous tissues, because membrane homogenates might contain both receptors that have the opposite effects on cAMP production (18). The use of stable expression systems of cloned biogenic amine receptors would facilitate the discovery of specific receptor ligands as well as novel insect bioregulators.


190

Acknowledgments


We would like to thank Ms. F. Ozoe (Shimane University) for helpful advice on molecular biological experiments. This study was supported in part by a grant (to H.O.) from Heart Co. Ltd., Japan.

References 1. 2. 3. 4. 5.

Evans, P. D. Adv. Insect Physiol. 1980, 15, 317-473. Blenau, W.; Baumann, A. Arch. Insect Biochem. Physiol. 2001, 48, 13-38. Robertson, Η. Α.; Juorio, Α. V. Int. Rev. Neurobiol. 1976, 19, 173-224. Roeder, T. Prog. Neurobiol. 1999, 59, 533-561. Hollingworth, R. M.; Johnstone, E. M.; Wright, N . In Pesticide Synthesis Through Rational Approaches; Magee, P. S.; Kohn, G. K.; Menn, J. J., Eds.; ACS Symposium Series No. 255; American Chemical Society: Washington, D.C., 1984; pp 103-125. 6. Nagaya, Y . ; Kutsukake, M.; Chigusa, S. I.; Komatsu, A. Neurosci. Lett. 2002, 329, 324-328. 7. Blumenthal, Ε. M. Am. J. Physiol. Cell Physiol. 2003, 284, C718-C728. 8. von Nickisch-Rosenegk, E.; Krieger, J.; Kubick, S.; Laage, R.; Strobel, J.; Strotmann, J.; Breer, H. Insect Biochem. Mol. Biol. 1996, 26, 817-827. 9. Ohta, H.; Utsumi, T.; Ozoe, Y. Insect Mol. Biol. 2003, 12, 217-223. 10. Hollingworth, R. M. Environ. Health Perspect. 1976, 14, 57-69. 11. Hollingworth, R. M.; Murdock, L. L. Science 1980, 208, 74-76. 12. Evans, P. D.; Gee, J. D. Nature 1980, 287, 60-62. 13. Nathanson J. Α.; Hunnicutt, E. J. Mol. Pharmacol. 1981, 20, 68-75. 14. Dudai, Y.; Zvi, S. Comp. Biochem.Physiol.1984, 77C, 145-151. 15. Hashemzadeh, H.; Hollingworth, R. M.; Voliva, A. Life Sci. 1985, 37, 433440. 16. Robb, S.; Cheek, T. R.; Hannan, F. L.; Hall, L. M.; Midgley, J. M.; Evans, P. D. EMBO J. 1994, 13, 1325-1330. 17. Hiripi, L.; Nagy, L.; Hollingworth, R. M. Acta Biol. Hung. 1999, 50, 81-87. 18. Khan, Μ. Α. Α.; Nakane, T.; Ohta, H.; Ozoe, Y. Arch. Insect Biochem. Physiol. 2003, 52, 7-16.


Expression of a Bombyx mori Tyramine Receptor in HEK-293 Cells

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