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First published online December 14, 2007
Journal of Experimental Biology 211, 92-105 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012450
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Conservation of structure, signaling and pharmacology between two serotonin receptor subtypes from decapod crustaceans, Panulirus interruptus and Procambarus clarkii

Nadja Spitzer*, Donald H. Edwards and Deborah J. Baro{dagger}

Department of Biology, Georgia State University, Atlanta, GA 30302, USA


Figure 1
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  		Fig. 1. 5-HT1{alpha}Pan can be stably expressed in an inducible tissue culture system. Western blot with anti-5-HT1{alpha}Crust showing expression of 5-HT1{alpha}Pan in the lobster nervous system (Lns) and in stably transfected 293-TR-5-HT1{alpha}Pan cells after induction with tetracycline for the indicated times (24, 12, 8, 6 h) and in non-induced cells (NI).

 

Figure 2
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  		Fig. 2. 5-HT2β receptors contain key structural elements typical of the 5-HT receptor superfamily and are conserved among arthropods. Predicted protein sequences of 5-HT2β from Panulirus and Procambarus are aligned with their ortholog from Drosophila and the human 5-HT2C receptor. Residues identical to the 5-HT2βPro sequence are indicated with thin lined boxes. Transmembrane domains are indicated with black bars above the sequence and the reference residue for numbering in each is shaded (see Results). Black ovals surround amino acids important for 5-HT ligand binding. Grey boxes surround areas important for G protein coupling or activation. Asterisks above the sequences indicate consensus sites for N-linked glycosylations in crustacean sequences. A black dot indicates the evolutionary change from DRY to DRF in crustacean sequences (see Results).

 

Figure 3
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  		Fig. 3. 5-HT1{alpha} receptors contain key structural elements typical of the 5-HT receptor superfamily and are conserved among arthropods. Predicted protein sequences of 5-HT1{alpha} from Panulirus and Procambarus are aligned with their orthologs from Drosophila, Heliothis and Papilio and the human 5-HT1A receptor. Residues identical to the 5-HT1{alpha}Pro sequence are boxed with thin lines. Transmembrane domains are indicated with black bars above the sequence and the reference residue for numbering in each is shaded (see Results). Black ovals surround amino acids important for 5-HT ligand binding. Grey boxes surround areas important for G protein coupling or activation. Asterisks above the sequences indicate consensus sites for N-linked glycosylations in crustacean sequences.

 

Figure 4
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  		Fig. 4. 5-HT is the only biogenic amine that acts as a potent agonist at 5-HT2βPan and 5-HT2βPro. (A) Non-transfected parental HEK cells do not show significant IP responses to any of the amines tested. (B) IP release in response to biogenic amines (10–3 mol l–1) in cells transiently expressing 5-HT2βPan. Cells expressing 5-HT2βPan demonstrate a greater than sixfold increase in IP release in response to 5-HT and a smaller but significant increase in response to dopamine and tyramine. Values are means ± s.e.m., N=3, **P<0.001 and *P<0.05 versus no drug control. (C) Dose–response curves of 5-HT2βPan to biogenic amines in IP assay. 5-HT2βPan responds to 5-HT (squares) with an EC50 of 52 nmol l–1. Dopamine (DA; circles) and tyramine (Tyr; triangles) activate the receptors only at very high concentrations. Non-transfected HEK cells do not respond to 5-HT (crosses). Values are means ± s.e.m., N=3. (D) IP release in response to biogenic amines (10–3 mol l–1) in cells transiently expressing 5-HT2βPro. IP release is increased more than fourfold in response to 1 mmol l–1 5-HT in cells expressing 5-HT2βPro. A smaller but significant increase in response to dopamine is observed. Values are means ± s.e.m., N=3, **P<0.001 vs no drug control. (E) Dose–response curves of 5-HT2βPro to biogenic amines in IP assay. 5-HT2βPro responds to 5-HT (squares) with an EC50 of 270 nmol l–1. Dopamine (circles) activates the receptor only at very high concentrations. Non-transfected HEK cells do not respond to 5-HT (crosses). Values are means ± s.e.m., N=3.

 

Figure 5
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  		Fig. 5. 5-HT is the only biogenic amine that acts as a potent agonist at 5-HT1{alpha}Pan and 5-HT1{alpha}Pro. (A) Inhibition of forskolin-stimulated cAMP accumulation in response to biogenic amines (10–3 mol l–1) in cells induced to express 5-HT1{alpha}Pan. Non-induced (NI) cells do not significantly respond to biogenic amines (left). Induced cells show supersensitivity and accumulate significantly higher levels of cAMP in response to forskolin only (grey bars). In induced cells adenylyl cyclase is significantly inhibited in response to 1 mmol l–1 5-HT but no other biogenic amines (right). Values are means ± s.e.m., N=3, #P<0.05 vs non-induced, **P<0.001 vs forskolin only. (B) Dose–response curve of 5-HT1{alpha}Pan (squares) to 5-HT. The EC50 of 5-HT at 5-HT1{alpha}Pan is 8.4 nmol l–1. Non-induced cells do not show significant changes in forskolin-stimulated cAMP accumulation in response to 5-HT (crosses). Values are means ± s.e.m., N=3. (C) Inhibition of forskolin-stimulated cAMP accumulation in response to amines (10–3 mol l–1) in cells induced to express 5-HT1{alpha}Pro. Adenylyl cyclase in non-induced cells is activated by dopamine, octopamine and histamine (left). Induced cells accumulate significantly higher levels of cAMP in response to forskolin only (grey bars). In induced cells adenylyl cyclase is significantly inhibited in response to 1 mmol l–1 5-HT and tyramine but no other biogenic amines (right). As observed in non-induced cells, histamine elicits an increase in cAMP levels in cells expressing 5-HT1{alpha}Pro. Values are means ± s.e.m. N=3, #P<0.001 vs non-induced, **P<0.001 and *P<0.05 vs forskolin only. (D) Non-induced (NI) cells show a positive response in cAMP levels at high concentrations of 5-HT (crosses). Owing to this background activity, the dose–response curve of 5-HT1{alpha}Pro (black squares) to 5-HT is biphasic. When the activity of non-induced cells in response to 5-HT is subtracted from the response of induced cells (5-HT–NI; grey squares), a sigmoidal dose–response curve is obtained. This curve has an EC50 for 5-HT at 5-HT1{alpha}Pro of 31 nmol l–1. Even at high concentrations tyramine has a minimal effect on 5-HT1{alpha}Pro (triangles). Values are means ± s.e.m., N=3.

 

Figure 6
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  		Fig. 6. Specific drugs have differential agonist activity at 5-HT2β and 5-HT1{alpha}. (A) Release of IP in cells expressing 5-HT2βPro (right) in response to various putative agonist drugs (10–3 mol l–1, except methysergide, 10–5 mol l–1). Non-transfected HEK cells (left) show no IP release in response to the drugs. Values are means ± s.e.m., N=3, ***P<0.001 and *P<0.05 vs no drug. (B) Inhibition of forskolin-stimulated cAMP production in response to the same drugs in cells induced to express 5-HT1{alpha}Pro. Values are means ± s.e.m., N=3, #P<0.05 vs non-induced, **P<0.001 and {dagger}P<0.05 vs forskolin only.

 

Figure 7
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  		Fig. 7. Example dose–response curves for agonists and antagonists at 5-HT2βPro and 5-HT1{alpha}Pro. (A) Cells expressing 5-HT2βPro respond with a dose-dependent increase in IP levels in response to 5-HT (10–3 mol l–1; black squares) and two agonists with different potencies and efficacies, 2-methyl-5-HT (black circles) and 5-XT (black triangles). The 5-HT-indexed IP response is blocked by increasing concentrations of two antagonists, methiothepin (grey inverted triangles) and cinanserin (grey diamonds). Values are means ± s.e.m., N=3. (B) Dose-dependent responses to 5-HT (squares) and two agonists, mCPP (circles) and methysergide (triangles) with different potencies and efficacies in cells induced to express 5-HT1aPro. Values are means ± s.e.m., N=3.

 

Figure 8
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  		Fig. 8. Identification of antagonists of 5-HT2β and 5-HT1{alpha}. (A) Putative antagonists have no effect on the parental HEK cell line (left). Several of the antagonists (10–5 mol l–1) block 5-HT (1 mmol l–1)-stimulated increases in IP levels in cells expressing 5-HT2βPro (right). Values are means ± s.e.m., N=3, *P<0.05 vs 5-HT only. (B) Antagonist drugs have no significant effect on non-induced 5-HT1{alpha}Pro cells (left). None of these drugs (10 mmol l–1) blocks inhibition of adenylyl cyclase by 10–5 mol l–1 5-HT in cells induced to express 5-HT1{alpha}Pro (right). Some putative antagonists actually increase the efficacy of the 5-HT effect. Values are means ± s.e.m., N=3, *P<0.05 vs 5-HT only.

 

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© The Company of Biologists Ltd 2008