PeaTAR1B: Characterization of a Second Type 1 Tyramine Receptor of the American Cockroach, Periplaneta americana

Abstract

The catecholamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. In insects; these neuroactive substances are functionally replaced by the phenolamines octopamine and tyramine. Phenolamines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Type 1 tyramine receptors are better activated by tyramine than by octopamine. In contrast; type 2 tyramine receptors are almost exclusively activated by tyramine. Functionally; activation of type 1 tyramine receptors leads to a decrease in the intracellular concentration of cAMP ([cAMP]_i) whereas type 2 tyramine receptors can mediate Ca²⁺ signals or both Ca²⁺ signals and effects on [cAMP]_i. Here; we report that the American cockroach (Periplaneta americana) expresses a second type 1 tyramine receptor (PeaTAR1B) in addition to PeaTAR1A (previously called PeaTYR1). When heterologously expressed in flpTM cells; activation of PeaTAR1B by tyramine leads to a concentration-dependent decrease in [cAMP]_i. Its activity can be blocked by a series of established antagonists. The functional characterization of two type 1 tyramine receptors from P. americana; PeaTAR1A and PeaTAR1B; which respond to tyramine by changing cAMP levels; is a major step towards understanding the actions of tyramine in cockroach physiology and behavior; particularly in comparison to the effects of octopamine.

Keywords: Ca2+ fluorimetry; G protein-coupled receptor; biogenic amines; cAMP; cell-based assay; cellular signaling; insect; octopamine; second messenger.

Figure 1

Structural characteristics of the deduced amino acid sequence of PeaTAR1B. (a) Hydrophobicity profile of PeaTAR1B. The profile was calculated according to the algorithm of Kyte and Doolittle [75] using a window size of 19 amino acids. Peaks with scores greater than 1.6 (dashed line) indicate possible transmembrane regions; (b) Prediction of transmembrane domains with TMHMM server v. 2.0 [76]. Putative transmembrane domains are indicated in red. Extracellular regions are shown as purple line, intracellular regions as blue line; (c) Color-coded (rainbow) 3D model of the receptor as predicted by Phyre2 [77]. The extracellular N-terminus (N) and the intracellular C-terminus (C) are labeled.

Figure 2

Amino acid sequence alignment of PeaTAR1B and orthologous receptors from Periplaneta americana (PeaTAR1A, CAQ48240.1), Drosophila melanogaster (DmTYR1; NP_524419.2), and Apis mellifera (AmTAR1, NP_001011594.1). Identical residues (≥75%) are shown as white letters against black, whereas conservatively substituted residues are shaded. Putative transmembrane domains (TM1–TM7) are indicated by gray bars. Potential N-glycosylation sites (▼), protein kinase A (PKA) phosphorylation sites (●), and protein kinase C (PKC) phosphorylation sites (●) of PeaTAR1B are indicated. The amino acid position is given on the right.

Figure 3

Bayesian phylogeny of insect biogenic amine receptors and trace amine-associated receptors (TAAR). Human rhodopsin (HsRHOD) and fly neuropeptide FMRF amide receptor (DmFMRF-R) were used as outgroup. Numbers at branches represent the posterior probabilities. Nodes with support values below 75% were collapsed. Receptor subclasses are highlighted by distinct colors and the respective ligands are given for each group. Accession numbers are listed behind the receptor’s name. Abbreviations of species in alphabetical order are: Aa, Amphibalanus amphitrite; Ai, Amphibalanus improvisus; Am, Apis mellifera; Bm, Bombyx mori; Cs, Chilo suppressalis; Dm, Drosophila melanogaster; Hs, Homo sapiens; Lm, Locusta migratoria; Pea, Periplaneta americana; Px, Papilio xuthus; Rp, Rhodnius prolixus; Tc, Tribolium castaneum.

Figure 4

Pharmacological characterization of the PeaTAR1B receptor. (a) Concentration-dependent effects of tyramine on intracellular cAMP levels in HEK 293 (flpTM) cells constitutively expressing the PeaTAR1B-HA receptor and in non-transfected cells. Relative fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained with 1 µM NKH 477 (=100%), a water-soluble forskolin analog. All measurements were performed in the presence of 100 µM isobutylmethylxanthine (IBMX). Data points represent the mean ± SD of eight values (PeaTAR1B-HA) or four values (non-transfected) from a typical experiment; (b) Effects of putative tyramine receptor antagonists on NKH 477-stimulated cAMP production in PeaTAR1B-HA expressing cells. Concentration series of the substances were applied in the presence of 1 µM NKH 477, 10 nM tyramine and 100 µM IBMX. Data represent the mean ± SD of four values from a typical experiment. Determinations for both (a,b) were independently repeated at least three times.

Figure 4

Pharmacological characterization of the PeaTAR1B receptor. (a) Concentration-dependent effects of tyramine on intracellular cAMP levels in HEK 293 (flpTM) cells constitutively expressing the PeaTAR1B-HA receptor and in non-transfected cells. Relative fluorescence (corresponding to the amount of cAMP) is given as the percentage of the value obtained with 1 µM NKH 477 (=100%), a water-soluble forskolin analog. All measurements were performed in the presence of 100 µM isobutylmethylxanthine (IBMX). Data points represent the mean ± SD of eight values (PeaTAR1B-HA) or four values (non-transfected) from a typical experiment; (b) Effects of putative tyramine receptor antagonists on NKH 477-stimulated cAMP production in PeaTAR1B-HA expressing cells. Concentration series of the substances were applied in the presence of 1 µM NKH 477, 10 nM tyramine and 100 µM IBMX. Data represent the mean ± SD of four values from a typical experiment. Determinations for both (a,b) were independently repeated at least three times.